RU2772585C2 - SC-β CELLS AND COMPOSITIONS AND METHODS FOR THEIR CREATION - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to methods for differentiating PDX1-positive pancreatic precursor cells to NKX6.1-positive pancreatic precursor cells using a culture medium. In this case, the culture medium contains KGF, SANT1 and retinoic acid (RA) and does not contain a protein kinase C (PKC) activator or does not contain an inhibitor of BMP signal path.

EFFECT: obtaining SC-β cells and compositions.

11 cl, 17 dwg, 6 tbl, 1 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority in U.S. Provisional Application No. 61/833,898, filed June 11, 2013, and U.S. Provisional Application No. 61/972,212, filed March 28, 2014, the entire contents of which are incorporated herein by links.

BACKGROUND OF THE INVENTION

[2] Current research only produces abnormally functioning insulin-expressing cells that do not secrete adequate amounts of insulin in response to successive changes in glucose levels, or pancreatic progenitor cells that can mature into functioning insulin-expressing cells after only 3 months after transplantation into the host mouse, (Cheng et. al., 2012; D'Amour et al., 2005; D'Amour et al., 2006; Kroon et al., 2008; Nostra et al., 2011; Rezania et al., 2012; Schulz et al., 2012; Xie et al., 2013). Unlike normal islet cells or dispersed adult β-cells, which release high levels of insulin in response to high glucose levels in a glucose-stimulated insulin secretion (GSIS) assay and can do so repeatedly, insulin-expressing cells obtained by existing methods from hPSC are not able to properly secrete insulin in response to the addition of different concentrations of glucose. Accordingly, there is a need for a method to obtain cells from hPSCs that exhibit the phenotype of normal islet cells or adult β-cells.

SUMMARY OF THE INVENTION

[3] In some aspects, the invention provides a stem cell-derived β-letter (SC-β).

[4] In some embodiments, the cell is mature. In some embodiments, the cell exhibits an in vitro glucose-stimulated insulin secretion (GSIS) response. In some embodiments, the cell demonstrates an iv vivo GSIS response. In some embodiments, the cell exhibits in vitro and in vivo glucose-stimulated insulin secretion (GSIS) responses. In some embodiments, the cell exhibits a GSIS response to at least one glucose stimulation. In some embodiments, the cell demonstrates a GSIS response to at least two consecutive glucose stimulations. In some embodiments, the cell demonstrates a GSIS response to at least three consecutive glucose stimulations. In some embodiments of the invention, the GSIS response is observed immediately after transplantation of the cell to a human or animal. In some embodiments of the invention, a GSIS response is observed within approximately 24 hours after transplantation of the cell into a human or animal body. In some embodiments of the invention, the GSIS response is observed within approximately two weeks after transplantation of the cell into the human or animal body. In some embodiments of the invention, the stimulation index, characterized by the ratio of insulin secreted in response to high glucose concentrations and low glucose concentrations, is similar to the stimulation index of an endogenous mature pancreatic β-cell. In some embodiments, the stimulation index is greater than or equal to 1, or greater than or equal to 1.1, or greater than or equal to 1.3, or greater than or equal to 2, or greater than or equal to 2.3, or greater than or equal to 2.6. In some embodiments, the cell exhibits cytokine-induced apoptosis in response to a cytokine. In some embodiments, the cytokine is selected from the group consisting of interleukin-1β (IL-β), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and combinations thereof. In some embodiments of the invention, the secretion of insulin from the cell is enhanced in response to an antidiabetic agent. In some embodiments, the antidiabetic agent comprises a secretion stimulant selected from the group consisting of an incretin mimetic, a sulfonylurea, meglitinide, and combinations thereof. In some embodiments of the invention, the cell is monohormonal. In some embodiments, the cell exhibits a morphology that resembles that of an endogenous mature pancreatic β cell. In some embodiments of the invention under an electron microscope, encapsulated crystalline insulin granules are observed in the cell, which resemble the insulin granules of an endogenous mature pancreatic β-cell. In some embodiments, the cell exhibits a low replication rate. In some embodiments, the cell exhibits a glucose-stimulated Ca ²⁺ flux (GSCF) that resembles the GSCF of an endogenous mature pancreatic β-cell. In some embodiments, the cell exhibits a GSCF response to at least one glucose stimulation. In some embodiments, the cell exhibits a GSCF response to at least two glucose stimulations. In some embodiments, the cell exhibits a GSCF response to at least three glucose stimulations. In some embodiments, the cell exhibits increased calcium flux. In some embodiments of the invention, increased calcium flux includes increased influx or an increased ratio between influx at low and high glucose concentrations. In some embodiments, the cell expresses at least one endogenous mature pancreatic β-cell marker selected from the group consisting of insulin, C-peptide, PDX1, MAFA, NKX6-1, PAX6, NEUROD1, glucokinase (GCK), SLC2A1 , PCSK1, KCNJ11, ABCC8, SLC30A8, SNAP25, RAB3A, GAD2, PTPRN, NKX2-2, Pax4. In some embodiments, the cell does not express at least one marker selected from the group consisting of a) a hormone selected from the group consisting of i) glucagon (GCG) and ii) somatostatin (SST); or b) an acinar cell marker selected from the group consisting of i) amylase and ii) carboxypeptidase A (CPA1); c) an α cell marker selected from the group consisting of i) GCG, ii) Arx, iii) Irx1 and Irx2; and d) a ductal cell marker selected from the group consisting of i) CFTR and ii) Sox9. In some embodiments, the cell is differentiated in vitro from an insulin-positive endocrine cell, or a progenitor thereof, selected from the group consisting of a Nkx6-1 positive pancreatic progenitor cell, a Pdx1-positive pancreatic progenitor cell, and a pluripotent stem cell. In some embodiments, the pluripotent stem cell is selected from the group consisting of an embryonic stem cell and an induced pluripotent stem cell. In some embodiments of the invention, the cell is human. In some embodiments of the invention, the cell is not genetically modified. In some embodiments of the invention, the cell is genetically modified. In some embodiments, the amount of insulin produced per cell is between 0.5 and 10 μIU per 1000 cells per 30 minutes of high glucose incubation. In some embodiments, the amount of insulin produced per cell is approximately 2.5 μIU per 1000 cells per 30 minutes of high glucose incubation. In some embodiments of the invention, the incubation is carried out ex vivo.

[5] In some aspects, the invention provides a cell line containing an SC-β cell. In some embodiments of the invention, the cell line stably expresses insulin. In some embodiments of the invention, cells can be frozen, thawed and amplified with a doubling time of between about 24 and 44 hours without significant morphological changes for at least 30 passages.

[6] In some aspects, the invention provides a method for generating an SC-β cell from insulin-positive endocrine cells, comprising contacting a population of cells containing insulin-positive endocrine cells under conditions that stimulate cell clustering with at least two factors β-cell maturation, comprising a) a transforming growth factor β (TGF-β) signaling pathway inhibitor and b) a thyroid hormone signaling pathway activator to induce in vitro maturation of at least one insulin-positive endocrine cell in a population into an SC-β cell .

[7] In some embodiments, the SC-β cell demonstrates a response to at least one stimulation with glucose. In some embodiments, the SC-β cell demonstrates a response to at least two consecutive glucose stimulations. In some embodiments, the SC-β cell demonstrates a response to at least three consecutive glucose stimulations. In some embodiments, the morphology of the SC-β cell resembles that of an endogenous mature β cell. In some embodiments, the SC-β cell exhibits in vitro and/or in vivo glucose-stimulated insulin secretion (GSIS) responses. In some embodiments of the invention, the GSIS response is observed immediately after transplantation of the SC-β cell into the subject's body. In some embodiments of the invention, a GSIS response is observed within approximately 24 hours after transplantation of the SC-β cell into the subject's body. In some embodiments of the invention, a GSIS response is observed within approximately two weeks after transplantation of the SC-β cell into the subject's body. In some embodiments, the cell population is contacted with a TGF-β signaling inhibitor at a concentration of 100 nM-100 μM. In some embodiments of the invention, a population of cells is brought into contact with an inhibitor of the TGF-β signaling pathway at a concentration of 10 μm. In some embodiments, the TGF-β signaling pathway includes TGF-β type I receptor kinase signaling. In some embodiments, the TGF-β signaling inhibitor comprises an Alk5 II inhibitor. In some embodiments, the TGF-β signaling inhibitor comprises an analog or derivative of an Alk5 II inhibitor. In some embodiments, the cell population is contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM-10 μM. In some embodiments, the cell population is contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the cell population is optionally contacted with a protein kinase inhibitor. In some embodiments, the cell population is not contacted with a protein kinase inhibitor. In some embodiments of the invention, a population of cells is brought into contact with a protein kinase inhibitor. In some embodiments, the cell population is contacted with a protein kinase inhibitor at a concentration of 10 nM-1 μM. In some embodiments, the cell population is contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some embodiments of the invention, this method includes bringing the population of cells into contact with at least one additional factor maturation β-cells. In some embodiments, the at least one additional β-cell maturation factor comprises a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, the cell population is contacted with a CFTR inhibitor at a concentration of 100 nM-100 μM. In some embodiments, the cell population is contacted with a CFTR inhibitor at a concentration of 10 nM-10 μM. In some embodiments, the CFTR inhibitor comprises Gly-H101. In some embodiments, the at least one additional β-cell maturation factor comprises an O-GlcNAcase inhibitor. In some embodiments, the cell population is contacted with an O-GlcNAcase inhibitor at a concentration of 100 nM-100 μM. In some embodiments, the cell population is contacted with an O-GlcNAcase inhibitor at a concentration of 10 nM-10 μM. In some embodiments, the O-GlcNAcase inhibitor comprises Thiamet-G. In some embodiments of the invention, the cell population is cultured in a suitable culture medium. In some embodiments, a suitable culture medium includes Connought Medical Research Laboratories (CMRLS) 1066 Supplemented Islet Medium or a CMRLS component. In some embodiments of the invention, CMRLS is supplemented with serum. In some embodiments of the invention, CMRLS is supplemented with 10% fetal bovine serum. In some embodiments of the invention, the conditions that stimulate cell clustering include suspension culture. In some embodiments, the cell population is maintained in suspension culture for a period of time sufficient to induce in vitro maturation of at least one of the insulin-positive endocrine cells in the cell population into at least one SC-β cell. In some embodiments of the invention, the time period is at least 7 days. In some embodiments of the invention, the time period is from 7 days to 21 days. In some embodiments of the invention, the time period is from 7 to 14 days. In some embodiments of the invention, the time period is from 10 to 14 days. In some embodiments of the invention, the time period is 14 days. In some embodiments of the invention, β-cell maturation factors are replenished every other day. In some embodiments of the invention, at least 1% of the insulin-positive endocrine cells in the cell population are induced to mature into SC-β cells. In some embodiments, at least 99% of the insulin-positive endocrine cells in a population are induced to mature into SC-β cells. In some embodiments of the invention, at least 30% of the resulting cells in the population include SC-β cells. In some embodiments, the SC-β cells express C-peptide, insulin, NKX6-1, Pdx1 and co-express NKX6-1 and C-peptide. In some embodiments of the invention, insulin-positive endocrine cells also express Pdx1 and NKX6-1. In some embodiments of the invention, insulin-positive endocrine cells are obtained from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and induced pluripotent stem cells. In some embodiments, the SC-β cells include human cells. In some embodiments of the invention, the in vitro generation of SC-β cells is scalable.

[8] In some aspects, the invention provides an isolated population of SC-β cells obtained in accordance with the methods described herein.

[9] In some aspects, the invention provides a microcapsule containing an isolated population of SC-β cells encapsulated therein.

[10] In some aspects, the invention provides a composition comprising a population of SC-β cells obtained in accordance with the method described herein.

[11] In some aspects, the invention provides an assay method comprising an isolated population of SC-β cells obtained in accordance with the method described herein.

[12] In some embodiments, the assay is designed to identify one or more candidate agents that promote or inhibit β-cell fate selected from the group consisting of β-cell proliferation, β-cell replication, β-cell death, function β-cells, the susceptibility of β-cells to immune attack, or the susceptibility of β-cells to dedifferentiation or differentiation. In some embodiments, the assay is designed to identify one or more candidate agents that stimulate the differentiation of at least one insulin-positive endocrine cell or progenitor thereof into at least one SC-β cell.

[13] In some aspects, the invention provides a method of treating a subject in need thereof, comprising administering to the subject a composition comprising an isolated population of SC-β cells obtained in accordance with the method described herein. In some embodiments, the SC-β cells are encapsulated in a microcapsule. In some embodiments, the SC-β cells are derived from a population of pluripotent stem cells derived from the subject to whom the SC-β cells are administered. In some embodiments, the SC-β cells are derived from a population of iPS (induced pluripotent stem cells) cells, wherein the iPS cells are derived from a cell derived from the subject to whom the SC-β cells are administered. In some embodiments of the invention, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, diabetes is selected from the group of type I diabetes, type II diabetes, type 1.5 diabetes, and prediabetes. In some embodiments of the invention, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[14] In some aspects, the invention relates to the use of an isolated population of SC-β cells obtained by methods according to any one of paragraphs. 41-102 for administration to a subject in need thereof.

[15] In some embodiments, the subject is administered an isolated population of SC-β cells encapsulated in microcapsules. In some embodiments of the invention, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, diabetes is selected from the group of type I diabetes, type II diabetes, type 1.5 diabetes, and prediabetes. In some embodiments of the invention, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[16] In some aspects, the invention provides a culture medium containing a) an Alk5 inhibitor, b) triiodothyronine (T3), optionally, c) staurosporine, and optionally, d) CMRLS or a component of CMRLS.

[17] In some aspects, the invention relates to the use of a culture medium to induce in vitro maturation of insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit an in vitro and/or in vivo GSIS response.

[18] In some aspects, the invention provides a method for producing a NKX6-1 positive pancreatic progenitor cell from a Pdx1 positive pancreatic progenitor cell, comprising bringing a population of cells containing Pdx1 positive pancreatic progenitor cells into contact under conditions that stimulate cell clustering. , with at least two β-cell maturation factors, including a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of retinoic acid signaling pathway activator. acid (RA), over a period of at least five days, to induce the differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into NKX6-1-positive pancreatic progenitor cells, while NKX6-1-positive pancreatic progenitor cells express NKX6-1.

[19] In some embodiments, the cell population is contacted with at least one growth factor from the FGF family at a concentration of 1 ng/mL-100 ng/mL. In some embodiments, the cell population is contacted with at least one growth factor from the FGF family at a concentration of 50 ng/mL. In some embodiments of the invention, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments of the invention, at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10 and FGF21. In some embodiments, the cell population is not brought into contact with an activator of the RA signaling pathway. In some embodiments of the invention, a population of cells is brought into contact with an RA signaling pathway activator at a concentration of 0.01 μM-1.0 μM. In some embodiments, the cell population is contacted with an RA signaling pathway activator at a concentration of 0.1 μM. In some embodiments, the RA signaling pathway activator includes RA. In some embodiments of the invention, a population of cells is brought into contact with an inhibitor of the SHH pathway ("sonic hedgehog") at a concentration of 0.1 μm-0.5 μm. In some embodiments, the cell population is contacted with an SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some embodiments of the invention, the method comprises treating the cell population with at least one additional β-cell maturation factor. In some embodiments of the invention, at least one additional β-cell maturation factor includes at least one growth factor from the EGF family. In some embodiments of the invention, the cell population is treated with at least one growth factor from the EGF family at a concentration of 2 ng/ml-200 ng/ml. In some embodiments of the invention, the cell population is treated with at least one growth factor from the EGF family at a concentration of 20 ng/ml. In some embodiments of the invention, at least one growth factor from the EGF family is selected from the group consisting of betacellulin and EGF. In some embodiments of the invention, the cell population is cultured in a suitable culture medium. In some embodiments of the invention, conditions that stimulate cell clustering include suspension culture. In some embodiments of the invention, β-cell maturation factors are replenished every other day. In some embodiments, no protein kinase C activator is added to the suspension culture for 5 days. In some embodiments, the protein kinase C activator is removed from the suspension culture before the 5 day period has elapsed. In some embodiments, the protein kinase C activator comprises PdbU. In some embodiments of the invention, no inhibitor of the BMP (bone morphogenetic protein) signaling pathway is added to the suspension culture for 5 days. In some embodiments, the BMP signaling inhibitor is removed from the suspension culture before the 5 day period has elapsed. In some embodiments, the BMP signaling inhibitor comprises LDN193189. In some embodiments, at least 10% of the Pdx1 positive pancreatic progenitors in a population are induced to differentiate into NKX6-1 positive pancreatic progenitors. In some embodiments, at least 95% of the Pdx1 positive pancreatic progenitors in a population are induced to differentiate into NKX6-1 positive pancreatic progenitors. In some embodiments, NKX6-1 positive pancreatic progenitor cells express Pdx1, NKX6-1, and FoxA2. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are obtained from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

[20] In some aspects, the invention provides an isolated population of NKX6-1-positive pancreatic progenitor cells obtained in accordance with the methods described herein.

[21] In some aspects, the invention provides a microcapsule containing an isolated population of NKX6-1-positive pancreatic progenitor cells encapsulated therein.

[22] In some aspects, the invention provides a composition comprising a population of NKX6-1 positive pancreatic progenitor cells obtained according to the method described herein.

[23] In some aspects, the invention provides an assay method comprising an isolated population of NKX6-1 positive pancreatic progenitors obtained according to the method described herein.

[24] In some embodiments, the assay is designed to identify one or more candidate agents that stimulate the differentiation of at least one Pdx1-positive pancreatic progenitor cell or its progenitor into NKX6-1-positive pancreatic progenitor cells.

[25] In some aspects, the invention provides a method of treating a subject in need thereof, comprising administering to the subject a composition comprising an isolated population of NKX6-1 positive pancreatic progenitor cells obtained in accordance with the method described herein.

[26] In some embodiments, the NKX6-1 positive pancreatic progenitors are derived from a population of pluripotent stem cells derived from the subject receiving the NKX6-1 positive pancreatic progenitors. In some embodiments, the NKX6-1 positive pancreatic progenitors are encapsulated in a microcapsule. In some embodiments of the invention, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, diabetes is selected from the group of type I diabetes, type II diabetes, type 1.5 diabetes, and prediabetes. In some embodiments of the invention, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[27] In some aspects, the invention relates to the use of an isolated population of NKX6-1-positive pancreatic progenitor cells obtained by the methods of any one of paragraphs. 113-142 for differentiation into SC-β cells.

[28] In some aspects, the invention relates to the use of an isolated population of NKX6-1-positive pancreatic progenitor cells, obtained as described herein, for administration to a subject in need thereof.

[29] In some embodiments, the subject is administered an isolated population of NKX6-1 positive pancreatic progenitor cells encapsulated in microcapsules. In some embodiments of the invention, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, diabetes is selected from the group of type I diabetes, type II diabetes, type 1.5 diabetes, and prediabetes. In some embodiments of the invention, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[30] In some aspects, the invention provides a culture medium containing a) KGF, b) SANT1), and optionally c) RA, wherein the culture medium is substantially free of PdbU and LDN 193189. In some embodiments, the invention relates to the use of culture 160 to induce in vitro differentiation of Pdx1 positive pancreatic progenitors into NKX6-1 positive pancreatic progenitors.

[31] In some aspects, the invention provides a method for producing an insulin-positive endocrine cell from an NKX6-1-positive pancreatic progenitor cell, comprising bringing a population of cells containing NKX6-1-positive pancreatic progenitor cells into contact under conditions that stimulate clustering cells with at least two β-cell maturation factors comprising a) an inhibitor of the TGF-β signaling pathway and b) an activator of the thyroid hormone signaling pathway to induce differentiation of at least one NKX6-1-positive pancreatic progenitor cell in a population of at least one insulin-positive endocrine cell, wherein at least one insulin-positive pancreatic progenitor cell expresses insulin. In some embodiments, the cell population is contacted with a TGF-β signaling inhibitor at a concentration of 100 nM-100 μM. In some embodiments of the invention, a population of cells is brought into contact with an inhibitor of the TGF-β signaling pathway at a concentration of 10 μm. In some embodiments, the TGF-β signaling pathway includes TGF-β type I receptor kinase signaling. In some embodiments, the TGF-β signaling inhibitor comprises an Alk5 II inhibitor. In some embodiments, the cell population is contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM-10 μM. In some embodiments, the cell population is contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments of the invention, this method includes bringing the population of cells into contact with at least one additional factor maturation β-cells. In some embodiments, the at least one additional β-cell maturation factor comprises a γ-secretase inhibitor. In some embodiments of the invention, a population of cells is brought into contact with an inhibitor of γ-secretase at a concentration of 0.1 μm-10 μm. In some embodiments of the invention, a population of cells is brought into contact with a γ-secretase inhibitor at a concentration of 1 μm. In some embodiments, the γ-secretase inhibitor comprises XXI. In some embodiments, the γ-secretase inhibitor comprises DAPT. In some embodiments of the invention, at least one additional β-cell maturation factor includes at least one growth factor from the EGF family. In some embodiments, the cell population is contacted with at least one growth factor from the EGF family at a concentration of 2 ng/mL-200 ng/mL. In some embodiments, the cell population is contacted with at least one growth factor from the EGF family at a concentration of 20 ng/mL. In some embodiments of the invention, at least one growth factor from the EGF family includes betacellulin. In some embodiments of the invention, at least one growth factor from the EGF family includes EGF. In some embodiments, the at least one additional β-cell maturation factor comprises a low concentration of a retinoic acid (RA) signaling pathway activator. In some embodiments, the cell population is contacted with an RA signaling pathway activator at a concentration of 0.01 μM-1.0 μM. In some embodiments, the cell population is contacted with an RA signaling pathway activator at a concentration of 0.1 μM. In some embodiments, the RA signaling pathway activator includes RA. In some embodiments, the at least one additional β-cell maturation factor comprises a sonic hedgehog (SHH) pathway inhibitor. In some embodiments, the cell population is contacted with an SHH pathway inhibitor at a concentration of 0.1 μM-0.5 μM. In some embodiments, the cell population is contacted with an SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some embodiments, the cell population is optionally contacted with a protein kinase inhibitor. In some embodiments, the cell population is not contacted with a protein kinase inhibitor. In some embodiments of the invention, a population of cells is brought into contact with a protein kinase inhibitor. In some embodiments, the cell population is contacted with a protein kinase inhibitor at a concentration of 10 nM-1 μM. In some embodiments, the cell population is contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some embodiments of the invention, this method includes the treatment of a population of cells with glucose. In some embodiments of the invention, the cell population is treated with glucose at a concentration of 1 mm-50 mm. In some embodiments of the invention, the cell population is treated with glucose at a concentration of 25 mm. In some embodiments of the invention, the conditions that stimulate cell clustering include suspension culture. In some embodiments, the cell population is maintained in suspension culture for a period of time sufficient to induce differentiation of at least one of the NKX6-1 positive pancreatic progenitor cells in the population into an insulin positive endocrine cell. In some embodiments of the invention, the specified period of time is at least 7 days. In some embodiments of the invention, β-cell maturation factors are replenished in suspension culture every other day. In some embodiments, at least 15% of the NKX6-1 positive pancreatic progenitors in the population are induced to differentiate into insulin positive endocrine cells. In some embodiments, at least 99% of the NKX6-1 positive pancreatic progenitor cells in the population are induced to differentiate into insulin positive endocrine cells. In some embodiments, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, and/or insulin. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are derived from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

[32] In some aspects, the invention provides an isolated population of insulin-positive endocrine cells obtained in accordance with the method described herein.

[33] In some aspects, the invention provides a microcapsule containing an isolated population of insulin-positive endocrine cells encapsulated therein. In some embodiments, the invention provides a composition comprising a population of insulin-positive endocrine cells obtained in accordance with the method described herein.

[34] In some aspects, the invention provides a method of treating a subject in need thereof, comprising administering to the subject a composition comprising an isolated population of insulin-positive endocrine cells obtained in accordance with the method described herein.

[35] In some embodiments, the insulin-positive endocrine cells are derived from a population of pluripotent stem cells derived from the subject to whom the insulin-positive endocrine cells are administered. In some embodiments of the invention, insulin-positive endocrine cells are encapsulated in a microcapsule. In some embodiments of the invention, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, diabetes is selected from the group of type I diabetes, type II diabetes, type 1.5 diabetes, and prediabetes. In some embodiments of the invention, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[36] In some aspects, the invention relates to the use of an isolated population of insulin-positive endocrine cells, obtained as described herein, for differentiation into SC-β cells.

[37] In some aspects, the invention relates to the use of an isolated population of insulin-positive endocrine cells, obtained as described herein, for administration to a subject in need thereof.

[38] In some embodiments, the subject is administered an isolated population of insulin-positive endocrine cells encapsulated in microcapsules. In some embodiments of the invention, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, diabetes is selected from the group of type I diabetes, type II diabetes, type 1.5 diabetes, and prediabetes. In some embodiments of the invention, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[39] In some aspects, the invention provides a culture medium comprising a) a TGF-β signaling pathway inhibitor, b) a TH (thyroid hormone) pathway activator, and at least one additional β-cell maturation factor selected from the group consisting of i ) XXI, ii) betacellulin, iii) low concentration of RA signaling pathway activator and iv) SHH pathway inhibitor.

[40] In some aspects, the invention relates to the use of the culture medium of claim 221 to induce in vitro differentiation of NKX6-1 positive pancreatic progenitor cells into insulin positive endocrine cells.

[41] In some aspects, the invention provides a method for producing SC-β cells, comprising: bringing Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into contact under conditions that stimulate cell clustering with i) an inhibitor of the signaling pathway transforming growth factor β (TGF-β), ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor to induce in vitro maturation of at least one of Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo.

[42] In some embodiments, the GSIS response is observed (i) immediately after transplantation of the SC-β cell into the subject's body; (ii) within approximately 24 hours of transplantation of the cell into the subject's body; or (iii) within about two weeks after transplantation of the cell into the subject's body. In some embodiments, the SC-β cells demonstrate a response to (i) at least one stimulation with glucose; (ii) at least two consecutive glucose stimulations; or (iii) at least three consecutive glucose stimulations. In some embodiments of the invention, the morphology of SC-β cells is similar to the morphology of endogenous β-cells. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a TGF-β signaling inhibitor at a concentration of 100 nM-100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a TGF-β signaling inhibitor at a concentration of 10 μM. In some embodiments, the TGF-β signaling pathway includes TGF-β type I receptor kinase signaling. In some embodiments, the TGF-β signaling inhibitor comprises an Alk5 II inhibitor. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM-10 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments of the invention Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are not contacted with a protein kinase inhibitor. In some embodiments, Pdx1 positive, NKX6-1 positive, insulin positive endocrine cells are contacted with a protein kinase inhibitor. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 10 nM-1 μM. In some embodiments, Pdx1 positive, NKX6-1 positive, insulin positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some embodiments, said method comprises contacting Pdx1 positive, NKX6-1 positive, insulin positive endocrine cells with a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, Pdx1 positive, NKX6-1 positive, insulin positive endocrine cells are contacted with a CFTR inhibitor at a concentration of 100 nM-100 μM. In some embodiments, Pdx1 positive, NKX6-1 positive, insulin positive endocrine cells are contacted with a CFTR inhibitor at a concentration of 10 nM-10 μM. In some embodiments, the CFTR inhibitor comprises Gly-H101. In some embodiments of the invention, this method includes bringing Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into contact with an inhibitor of O-GlcNase. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with an O-GlcNAcase inhibitor at a concentration of 100 nM-100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with an O-GlcNAcase inhibitor at a concentration of 10 nM-10 μM. In some embodiments, the O-GlcNAcase inhibitor comprises Thiamet-G. In some embodiments of the invention Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are cultured in a suitable culture medium. In some embodiments, a suitable culture medium includes 1066 supplemented islet medium from Connought Medical Research Laboratories (CMRLS) or a CMRLS component. In some embodiments of the invention, CMRLS is supplemented with serum. In some embodiments of the invention, CMRLS is supplemented with 10% fetal bovine serum. In some embodiments of the invention, conditions that stimulate cell clustering include suspension culture. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are kept in suspension culture for a period of time sufficient to induce in vitro maturation of at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells. In some embodiments of the invention, the time period is at least 7 days. In some embodiments of the invention, the time period is from 7 days to 21 days. In some embodiments of the invention, the time period is from 7 to 14 days. In some embodiments of the invention, the time period is 14 days. In some embodiments, the suspension culture is replenished every other day. In some embodiments of the invention, at least 30% of the resulting cells contain SC-β cells. In some embodiments, the SC-β cells express C-peptide, insulin, NKX6-1, Pdx1 and co-express NKX6-1 and C-peptide. In some embodiments, the SC-β cells include human cells. In some embodiments of the invention, the in vitro generation of SC-β cells is scalable.

[43] In some embodiments of the invention, insulin-positive endocrine cells are obtained by bringing Pdx1-positive, NKX6-1-positive pancreatic progenitor cells under conditions that stimulate cell clustering, in contact with i) an inhibitor of the TGF-β signaling pathway and ii ) a thyroid hormone signaling pathway activator to induce differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, while being Pdx1-positive, NKX6-1 positive, insulin positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

[44] In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with a TGF-β signaling inhibitor at a concentration of 100 nM-100 μM. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with a TGF-β signaling inhibitor at a concentration of 10 μM. In some embodiments, the TGF-β signaling pathway includes TGF-β type I receptor kinase signaling. In some embodiments, the TGF-β signaling inhibitor comprises an Alk5 II inhibitor. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM-10 μM. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, said method comprises contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with at least one of i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor , iv) at least one growth factor from the epidermal growth factor (EGF) family, and optionally v) a protein kinase inhibitor. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with an SHH pathway inhibitor at a concentration of 0.1 μM-0.5 μM. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with an SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.01 μM-1.0 μM. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.1 μM. In some embodiments, the RA signaling pathway activator includes RA. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 0.1 μM-10 μM. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 1 μM. In some embodiments, the γ-secretase inhibitor comprises XXI. In some embodiments, the γ-secretase inhibitor comprises DAPT. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 2 ng/mL-200 ng/mL. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 20 ng/mL. In some embodiments of the invention, at least one growth factor from the EGF family includes betacellulin. In some embodiments of the invention, at least one growth factor from the EGF family includes EGF. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are not contacted with a protein kinase inhibitor. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with a protein kinase inhibitor. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 10 nM-1 μM. In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some embodiments of the invention, this method includes the treatment of a population of cells with glucose. In some embodiments of the invention, the cell population is treated with glucose at a concentration of 1 mm-50 mm. In some embodiments of the invention, the cell population is treated with glucose at a concentration of 25 mm. In some embodiments of the invention, conditions that stimulate cell clustering include suspension culture. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are maintained in suspension culture for a period of time sufficient to induce differentiation of at least some of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells in Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells. In some embodiments of the invention, the specified period of time is at least 7 days. In some embodiments, the suspension culture is replenished every other day. In some embodiments, at least 15% of the Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are induced to differentiate into Pdx1 positive, NKX6-1 positive, insulin positive endocrine cells. In some embodiments, at least 99% of Pdx1 positive, NKX6-1 positive pancreatic progenitor cells are induced to differentiate into Pdx1 positive, NKX6-1 positive, insulin positive endocrine cells.

[45] In some embodiments of the invention, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are obtained by bringing Pdx1-positive pancreatic progenitor cells under conditions that stimulate cell clustering, in contact with i) at least one growth factor from the FGF family, ii) at least one inhibitor of the SHH pathway, and optionally iii) low concentrations of an RA signaling pathway activator over a period of five days to induce differentiation of at least some Pdx1-positive pancreatic progenitor cells into Pdx1 positive, NKX6-1 positive pancreatic progenitors, while Pdx1 positive, NKX6-1 positive pancreatic progenitors express Pdx1 and NKX6-1.

[46] In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one growth factor from the FGF family at a concentration of 1 ng/mL-100 ng/mL. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one growth factor from the FGF family at a concentration of 50 ng/mL. In some embodiments of the invention, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments of the invention, at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10 and FGF21. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.1 μM-0.5 μM. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments of the invention, at least one inhibitor of the SHH pathway includes Sant1. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.01 μM-1.0 μM. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.1 μM. In some embodiments, the RA signaling pathway activator includes RA. In some embodiments of the invention, this method includes contacting Pdx1-positive pancreatic progenitor cells with at least one growth factor from the EGF family. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 2 ng/mL-200 ng/mL. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 20 ng/mL. In some embodiments of the invention, at least one growth factor from the EGF family includes betacellulin. In some embodiments of the invention, at least one growth factor from the EGF family includes EGF. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are cultured in a suitable culture medium. In some embodiments of the invention, conditions that stimulate cell clustering include suspension culture. In some embodiments, the suspension culture is replenished every other day. In some embodiments, no protein kinase C activator is added to the suspension culture for 5 days. In some embodiments, the protein kinase C activator is removed from the suspension culture before the 5 day period has elapsed. In some embodiments, the protein kinase C activator comprises PdbU. In some embodiments of the invention, no inhibitor of the BMP signaling pathway is added to the suspension culture for 5 days. In some embodiments, the BMP signaling inhibitor is removed from the suspension culture before the 5 day period has elapsed. In some embodiments, the BMP signaling inhibitor comprises LDN193189. In some embodiments, at least 10% of the Pdx1 positive pancreatic progenitors in a population are induced to differentiate into Pdx1 positive, NKX6-1 positive pancreatic progenitors. In some embodiments, at least 95% of the Pdx1 positive pancreatic progenitors are induced to differentiate into Pdx1 positive, NKX6-1 positive pancreatic progenitors.

[47] In some aspects, the invention provides a method for generating SC-β cells from pluripotent cells, comprising: a) differentiating pluripotent stem cells in a population into Pdx1-positive pancreatic progenitor cells; b) differentiating at least some of the Pdx1 positive pancreatic progenitors into Pdx1 positive, NKX6-1 positive pancreatic progenitors by contacting the Pdx1 positive pancreatic progenitors under conditions that promote cell clustering with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, every other day for a period of five days to induce differentiation of at least some Pdx1-positive pancreatic progenitors in a population of NKX6-1 positive pancreatic progenitors, wherein the NKX6-1 positive pancreatic progenitors express Pdx1 and NKX6-1; c) differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by bringing Pdx1-positive, NKX6-1-positive pancreatic cells - precursors under conditions that promote cell clustering, in contact with i) an inhibitor of the TGF-β signaling pathway, b) an activator of the TH signaling pathway, and optionally c) at least one inhibitor of the SHH pathway, ii) an activator of the RA signaling pathway, iii) a γ-secretase inhibitor; and vi) at least one growth factor from the epidermal growth factor (EGF) family, every other day for a period of five to seven days, to induce differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1, insulin-positive endocrine cells express yut Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin; and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by bringing the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells under conditions that stimulate cell clustering, in contact with i) a transforming growth factor β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor, every other day for a period of seven to 14 days, to induce in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo.

[48] In some embodiments, the invention provides a method for obtaining SC-β cells from pluripotent cells, comprising: a) differentiating at least some of the pluripotent cells in the population into Pdx1-positive pancreatic progenitor cells; b) differentiating at least some of the Pdx1 positive pancreatic progenitors into Pdx1 positive, NKX6-1 positive pancreatic progenitors by contacting the Pdx1 positive pancreatic progenitors under conditions that promote cell clustering with i) KGF, ii) Sant1 and optionally iii) low concentrations of RA, every other day for a period of five days to induce differentiation of at least some of the Pdx1-positive pancreatic progenitors in the population into NKX6-1-positive pancreatic cells- progenitors, with NKX6-1-positive pancreatic progenitor cells expressing Pdx1 and NKX6-1; c) differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by bringing Pdx1-positive, NKX6-1-positive pancreatic cells - precursors in contact with i) Alk5 inhibitor II, ii) T3 and optionally iii) Sant1, iv) RA, v) XXI and vi) betacellulin every other day for a period of five to seven days to induce differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1, insulin-positive endocrine cells express Pdx1, NKX6- 1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin; and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by bringing the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells under conditions that stimulate cell clustering, in contact with i) Alk5 inhibitor II, ii) T3, and optionally iii) staurosporine, every other day for a period of seven to 14 days to induce in vitro maturation of at least some Pdx1-positive, NKX6 -1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo.

[49] In some aspects, the invention provides an artificial islet containing SC-β cells differentiated in vitro from pluripotent stem cells.

[50] In some aspects, the invention provides an artificial pancreas comprising SC-β cells differentiated in vitro from pluripotent stem cells.

[51] In the practice of the present invention, as a rule, unless otherwise indicated, conventional methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid technology (for example, DNA), immunology and RNA interference are used. (RNAi), which correspond to the art. Non-limiting descriptions of specific methods can be found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all published by John Wiley & Sons, N.Y., December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R.I., "Culture of Animal Cells, A Manual of Basic Technique", 5th ed., John Wiley & Sons, Hoboken, NJ, 2005. Non-limiting information regarding therapeutic agents and human diseases can be found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton &Lange; 10th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders can be found in McKusick, V.A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or more recent online databases: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), accessed May 1, 2010, World Wide Web URL: http:/ /www.ncbi.nlm.nih.gov/omim/ and in the Online Mendelian Inheritance in Animals (OMIA), a database of genes, hereditary disorders and characteristics of different animal species (other than humans and mice), at http://omia .angis.org.au/contact.shtml. All patents, patent publications, and other publications (eg, scientific articles, books, web pages, and databases) referenced in this document are incorporated by reference in their entirety. In the event of any conflict between the specification and the incorporated references, the specification (including any modifications thereof that may be based on the incorporated reference) shall prevail. Unless otherwise indicated, this document uses standard, accepted in the art meanings of terms. This document uses standard abbreviations for various terms.

BRIEF DESCRIPTION OF GRAPHICS

[52] The file of the patent or application contains at least one graphic image, made in color. Color graphic copies of this patent or patent application will be made available by the Patent Office upon request and upon payment of the required fee.

[53] In FIG. 1A and 1B show a comparison of a previously published control differentiation method and a new guided differentiation method. FIG. 1A shows a schematic comparison of an exemplary guided differentiation method according to the invention for generating INS+ cells from hPSCs and a previously published control differentiation method. FIG. 1B illustrates histological sections of undifferentiated HUES8 (top), DE-differentiated (middle) and PP1-differentiated (bottom), and stained with OCT4, SOX17, and PDX1, respectively, using a previously published control differentiation method. Scale mark = 100 µm.

[54] FIG. 2A, 2B and 2C demonstrate that in vitro derived stem cell-derived β-cells (SC-β) secrete insulin in response to repeated sequential stimulation with high concentrations of glucose, like primary human β-cells. FIG. 2A, 2B, and 2C are graphs illustrating ELISA-derived measurements of human insulin secreted from SC-β (FIG. 2A), primary β-cells (FIG. 2B), and RN cells (FIG. 2C) that were sequentially stimulated with 2 , 20, 2, 20, 2 and 20 mM glucose. Following sequential low/high glucose stimulation, cells were depolarized with 30 mM KCl.

[55] FIG. 3A, 3B and 3C show additional biological replicas of in vitro derived SC-β cells that secrete insulin in response to repeated sequential stimulation with high concentrations of glucose as primary β cells. The left panels are the same as in FIG. 2. SC-β cells (SC-β; FIG. 3A), primary β-cells (1° β; FIG. 3B) and RN cells (FIG. 3C) were sequentially stimulated 2, 20, 2, 20, 2 and 20 mm glucose and 30 mm KCl, and the level of human insulin was measured using ELISA.

[56] FIG. 4A, 4B, 4C, 4D and 4E demonstrate cytosolic Ca ²⁺ flux in SC-β cells in response to repeated sequential stimulation with high concentrations of glucose, as in the case of primary β-cells. FIG. 4A is a diagram showing cytosolic Ca ²⁺ detection at population and single cell levels by Fluo-4 AM staining. Population-level measurements were performed on individual whole clusters (indicated by the large red circle in the diagram), and individual cells within intact clusters (indicated by small red circles) were analyzed by single cell analysis. FIG. 4B is a graph illustrating population measurements of dynamic normalized Fluo-4 fluorescence intensity for SC-β cells, primary β cells, and PH cells stimulated sequentially with 2, 20, 2, 20, 2, and 20 mM glucose and 30 mM KCl. FIG. 4C shows fluorescent images obtained with Fluo-4 AM staining used for single cell analysis. FIG. 4D shows exemplary images indicating the location of single cells that responded to 3 (yellow), 2 (orange), 1 (blue), and 0 (red) glucose stimulations. FIG. 4E shows a graphical quantification of the frequency of SC-β cells (n=156), primary β cells (n=114), and PH cells (n=138) that showed a response to 20 mM glucose. Scale mark = 100 µm.

[57] FIG. 5A, 5B, 5C, 5D, 5E and 5F demonstrate that SC-β express markers of human β-cells at the level of protein and gene expression. FIG. 5A shows immunohistochemical images of cells stained for C-peptide (green), NKX6-1 (red), and somatostatin (grey). FIG. 5B shows immunohistochemical images of cells stained for C-peptide (green) and PDX1 (red). FIG. 5C shows immunohistochemical images of cells stained for C-peptide (green) and glucagon (red) with the respective DAPI dye (blue). FIG. 5D shows exemplary flow cytometry dot plots and the percentage of cells in the population stained for C-peptide and NKX6-1. FIG. 5E shows a hierarchical cluster analysis based on all genes measured by microarray analysis of undifferentiated HUES8, PH cells, fetal β-cells and primary adult β-cells sorted based on INS (data from Hrvatin et al. (Hrvatin et al. ., 2014)), and SC-β (SC-β) cells sorted based on INS and NKX6-1. FIG. 5F shows a heat map of the 100 genes with the highest variability among all samples. CP = C-peptide, SST = somatostatin, GCG = glucagon. Scale mark = 100 µm.

[58] FIG. 6 illustrates the histology of a cluster of SC-β cells stained for DAPI (blue), insulin (green), C-peptide (red). Scale mark = 100 µm.

[59] FIG. 7A, 7B and 7C show additional histological staining of SC-β cells. FIG. 7A illustrates staining for C-peptide (green) and ISL1 (red). FIG. 7B illustrates staining for C-peptide (green) and MAFA (red). FIG. 7C illustrates staining for C-peptide (green) and MAFB (red). Scale mark = 100 µm.

[60] FIG. 8A, 8B and 8C show exemplary flow cytometric dot plots and the percentage of SC-β cells and PH cells in the population stained for C-peptide and SST (FIG. 8A), C-peptide and GCG (FIG. 8B) and SST and GCG (FIG. 8C).

[61] FIG. 9A, 9B and 9C demonstrate that SC-β cell granules are structurally similar to primary human β cell granules. FIG. 9A shows electron microscope images of granules highlighting typical crystallized insulin granules (red), early insulin granules (yellow), and mixed endocrine granules (blue). Scale mark = 500 nm. FIG. 9B shows enlarged images of the granules highlighted in (FIG. 9A). Scale mark = 500 nm. FIG. 9C shows electron microscope images of cells labeled with immunological gold staining illustrating beads that contain insulin (smaller 5 nm black dots) and/or glucagon (larger 15 nm black dots). Typical gold particles are marked with red arrows (insulin) and blue arrows (glucagon). Scale mark = 100 nm.

[62] FIG. 10A and 10B demonstrate that stem cell-derived β-cells (SC-β) derived from hiPSC in vitro secrete insulin in response to repeated sequential stimulation with high concentrations of glucose, like primary human β-cells. FIG. 10A and 10B are graphs illustrating method ELISA measurements of human insulin secreted from SC-β derived from non-diabetic cells (FIG. 10A) and type 1 diabetic cells (FIG. 10B) sequentially stimulated with 2, 20, 2, 20 , 2 and 20 mM glucose.

[63] In FIG. 11A, 11B, 11C, 11D, 11E and 11F show exemplary flow cytometry scatter plots and percentage of cells stained for C-peptide and NKX6-1 from multiple hiPSC lines. FIG. 11A, 11B and 11C show exemplary flow cytometric dot plots and percentage of cells stained for C-peptide and NKX6-1 from non-diabetic hiPSC lines. FIG. 11D, 11E and 11F show exemplary flow cytometric dot plots and percentage of cells stained for C-peptide and NKX6-1 from type 1 diabetic hiPSC lines.

[64] FIG. 12A, 12B, 12C, and 12D demonstrate that transplanted SC-β cells rapidly begin to function in vivo. FIG. 12A is a graph illustrating ELISA measurements of human insulin from the serum of individual mice transplanted with SC-β cells (cultured for 1 week at the final in vitro step), primary human β cells (1° β), or PH cells. . Measurements were made before (white bars) and 30 minutes after (black bars) injection of glucose into mice two weeks after transplantation. FIG. 12B shows immunohistochemical images of transplanted cells with (FIG. 12A) stained for C-peptide (green) and PDX1 (red) to confirm the presence of a graft. FIG. 12C is a graph illustrating ELISA measurements of human insulin from the serum of individual mice transplanted with pancreatic progenitor cells. Measurements were made before (white bars) and 30 minutes after (black bars) injection of glucose into mice two weeks after transplantation. FIG. 12D is a graph illustrating ELISA measurements of human insulin from the serum of individual mice transplanted with SC-β cells cultured for 2 weeks at the final in vitro stage. Measurements were taken 30 minutes after (black bars) glucose injection in mice two weeks after transplantation, n/a = not determined Scale mark = 100 µm.

[65] FIG. 13A and 13B illustrate additional histological sections of SC-β cells and PH cells transplanted into mice for 2 weeks. FIG. 13A shows low magnification images of grafts stained for DAPI (blue), C-peptide (green), and GCG (red). Scale mark = 200 µM. FIG. 13B shows higher magnification images of grafts stained for C-peptide (green) and GCG (red). Scale mark = 100 μM.

[66] FIG. 14A, 14B and 14C demonstrate the use of media at the last stage of differentiation to enable SC-β cells to secrete more insulin in vivo. FIG. 14A is a diagram illustrating the use of various media at various stages of the differentiation process. FIG. 14B illustrates that addition of additional factors such as Sant1, XXI, and SSP to CMRL medium at the last stage of differentiation results in a better response of SC-β cells consisting of glucose-stimulated insulin secretion (GSIS), as determined by the stimulation index when stimulated with high and low concentrations of glucose. FIG. 14C illustrates that the addition of additional factors such as Sant1, XXI and SSP to CMRL medium at the last stage of differentiation results in a better response of SC-β cells consisting of glucose-stimulated insulin secretion (GSIS) as measured by the amount of insulin released.

[67] FIG. 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I demonstrate protocol modifications that can improve survival and quality of resulting SC-β cells. FIG. 15A is a schematic illustration of the protocol. FIG. 15B shows how much more pure NKX6.1+ endocrine cell clusters can be obtained (FIG. 15B) using the modified protocol. FIG. 15C demonstrates how the use of a Rock inhibitor in steps 3-5 can improve cell survival. FIG. 15D demonstrates how the use of activin A together with nicotinamide can lead to downregulation of SOX2 and improve cell survival. FIG. 15E shows that SOX2 and NKX6-1 are mutually exclusive. FIG. 15F demonstrates how the use of staurosporine in step 6 generates a substantially pure population of endocrine cells, and FIG. 15G demonstrates how the use of staurosporine in step 6 results in a higher percentage of NKX6-1/C-peptide+ cells. FIG. 15I demonstrates how using XXI in combination with Alk5i and T3 in steps 5-6 increases the NeuroD+ population compared to using Alk5i and T3 alone (FIG. 15H).

[68] FIG. 16A, 16B, 16C, 16D, 16E, 16F, 16G, 16H, and 16I demonstrate the clinical use of SC-β cells as a diabetes therapy or drug discovery platform. FIG. 16A is a schematic illustration of the use of SC-β cells for the treatment of diabetes or screening for drugs to improve function or replication. FIG. 16B is a table listing the investigational diabetes drugs and their general therapeutic category. FIG. 16C is a graph illustrating human insulin measurements obtained by the ELISA method from seeded SC-β cells treated with the indicated drugs in 2 and 20 mM glucose. These p-values allow comparison of the insulin level in 20 mM glucose between drug and control. FIG. 16D is an untreated immunofluorescence image of dispersed and seeded SC-β cells stained with DAPI (blue), C-peptide (green) and Ki67 (red). FIG. 16E is an immunofluorescence image of dispersed and seeded SC-β cells stained with DAPI (blue), C-peptide (green), and Ki67 (red) treated with prolactin for 48 hours. FIG. 16F shows a graphical quantification of the proportion of cells that co-express C-peptide and Ki67. *p<0.05. FIG. 16G is a graph illustrating fasting blood glucose measurements for Akita mice transplanted with SC-β cells (n=6) or PH cells (n=6). *p<0.05 when comparing two groups of cells on the same day. FIG. 16H is a graph illustrating blood glucose measurements in advanced diabetic Akita mice transplanted with SC-β cells or PH cells. Measurements were taken before (white bars) and 20 minutes after (black bars) injection of glucose into mice 2 weeks after transplantation. Saturation in glucose measurements was observed at 550 mg/dL. *p<0.05 comparing two groups of cells at the same time after glucose injection. FIG. 16I is a graph illustrating ELISA measurements of human insulin from the serum of Akita mice 20 minutes after glucose injection. Mice were stimulated with glucose 2 weeks after transplantation. *p<0.05 when comparing two groups of cells. Scale mark = 50 µm.

[69] FIG. 17 is a graph illustrating the body weight of Akita mice transplanted with SC-β cells (n=6) or PH cells (n=6). *p<0.05 when comparing two groups of cells at the time point corresponding to 18 and 28 days.

DETAILED DESCRIPTION OF THE INVENTION

[70] Aspects of the invention relate to compositions, methods, kits, and agents for generating stem cell-derived β-cells (SC-β) (e.g., mature pancreatic β-cells) from at least one insulin-positive endocrine cell or progenitor thereof. (e.g., iPS cells, hESCs, definitive endoderm cells, subembryonic cavity cells, Pdx1-positive pancreatic progenitors, Pdx1-positive, NKX6-1-positive pancreatic progenitors, Ngn3-positive endocrine progenitors, etc. ), as well as SC-β cells produced by these compositions, methods, kits, and agents for use in cell therapy, assay methods (eg, drug screening), and various treatments.

[71] In addition, aspects of the invention relate to methods for identifying SC-β cells that are detected based on morphological criteria without the need for a selectable marker, as well as functional characteristics, such as the ability to express insulin, secrete insulin in response to one or more stimulations. glucose, exhibit a mature GSIS response, and form islets in pancreatic tissue in vivo, and typically contain small, spindle-shaped cells about 9-15 µm in diameter.

[72] In addition, aspects of the invention relate to methods for detecting β-cell maturation factors. It is obvious or not difficult for a person skilled in the art to check whether a particular β-cell maturation factor is functional using methods known in the art. For example, the ability of the β-cell maturation factor to convert at least one insulin-positive endocrine cell or its progenitor into an SC-β cell can be assessed using the assay methods described herein. Other convenient assays include determining the ability to activate transcription of a reporter construct containing a β-cell marker binding site operably linked to a nucleotide sequence encoding a detectable marker, such as luciferase. One assay involves determining whether a candidate β-cell maturation factor induces at least one insulin-positive endocrine cell to become an SC-β cell, or expression of β-cell markers, or functional characteristics of a mature β-cell, as described. in this document. Determination of the expression of β-cell markers can be carried out using any suitable method, for example, immunoblotting. Such assays can be readily adapted to detect or confirm the activity of agents that directly convert at least one insulin-positive endocrine cell or progenitor thereof to an SC-β cell.

[73] In vitro-mature SC-β cells (i.e., pancreatic β-cells) obtained in accordance with the methods of the invention described herein demonstrate a large number of advantages, for example, they perform glucose-stimulated insulin secretion in vitro, are similar by gene expression and ultrastructure with human islet β-cells, secrete human insulin and alleviate hyperglycemia when transplanted into mice, represent a new platform for cell therapy (for example, transplantation into the body of a subject in need of additional and/or functional β-cells), drug screening drugs (eg, insulin production/secretion, survival, differentiation, etc.), research (eg, determining the difference between normal and diabetic β-cell function), and tissue engineering (eg, using SC-β cells as a first type cells during islet reconstruction).

[74] DEFINITIONS

[75] For convenience, individual terms used in this document - in the description of the invention, examples and the attached claims - are collected below. Unless otherwise indicated, all technical and scientific terms used herein have the meanings that are generally understood by a person skilled in the art to which this invention pertains.

[76] The term "differentiated cell", as defined herein, means any primary cell that is not pluripotent in its native form. According to another definition, the term "differentiated cell" refers to a cell of a more specialized type, derived from a cell of a less specialized type (eg, a stem cell, such as an induced pluripotent stem cell) during the process of cellular differentiation. Without being limited by theory, it can be said that during normal ontogeny, a pluripotent stem cell can differentiate first into an endodermal cell, which is capable of forming pancreatic cells and other types of endoderm cells. Further differentiation of endodermal cells triggers the pancreatic pathway, with ^~ 98% of cells becoming exocrine, ductular, or matrix cells and ^~ 2% becoming endocrine cells. Early endocrine cells are the precursors of islets, which can then differentiate into insulin-producing cells (eg, functional endocrine cells) that secrete insulin, glucagon, somatostatin, or a pancreatic polypeptide. Endodermal cells can also differentiate into other cells of endodermal origin, such as cells of the lung, liver, intestines, eyes, and glands, etc.

[77] As used herein, the term "somatic cell" refers to any cells that form the body of an organism, as opposed to germline cells. In the case of mammals, germline cells (also known as "gametes") are sperm and eggs that fuse during fertilization to form a cell called a zygote from which the entire mammalian embryo develops. Every other type of cell in the mammalian body - with the exception of sperm and eggs, the cells from which they are formed (gametocytes) and undifferentiated stem cells - are somatic cells: internal organs, skin, bones, blood and connective tissue are made up of somatic cells. In some embodiments of the invention, the somatic cell is a "non-embryonic somatic cell", which means a somatic cell that is not present in the embryo or is not derived from the embryo, and is not the result of the proliferation of such a cell in vitro. In some embodiments, the somatic cell is an "adult somatic cell", which refers to a somatic cell that is present in the body or derived from an organism other than an embryo or fetus, or is the result of the proliferation of such a cell in vitro. Unless otherwise indicated, methods for converting at least one insulin-positive endocrine cell or progenitor thereof into an insulin-producing, glucose-sensitive cell can be performed both in vivo and in vitro (in vivo methods are practiced when at least one the insulin-positive endocrine cell or precursor thereof is present in the subject, and the in vitro methods are practiced using at least one isolated insulin-positive endocrine cell or precursor contained in culture).

[78] As used herein, the term "adult cell" refers to a cell that can be found in the body after embryonic development.

[79] As used herein, the term "endodermal cell" refers to a cell from one of the three primary germ cell layers at a very early embryonic stage (the other two germ cell layers are mesoderm and ectoderm). The endoderm is the deepest layer of the three. The endoderm may differentiate first into the primary intestinal tube of the embryo, and then into the lining of the respiratory and digestive tracts (eg, intestines), liver, and pancreas.

[80] As used herein, the term "cell of endodermal origin" refers to any cell that has developed or differentiated from an endodermal cell. For example, a cell of endodermal origin includes cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without being limited by theory, liver and pancreatic progenitors (also referred to as pancreatic progenitors) develop from endoderm cells in the fetal foregut. Shortly after specification, progenitor cells of the liver and pancreas quickly acquire significantly different cellular functions and regenerative abilities. These changes are driven by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in organ development and regeneration has been fueled by the urgent need for hepatocytes and pancreatic β-cells in the therapeutic treatment of liver failure and type I diabetes. Studies conducted in various model organisms and in humans have revealed evolutionarily conserved inductive signals and transcription factor systems that lead to the differentiation of liver and pancreatic cells and serve as a guide for stimulating the differentiation of hepatocytes and β-cells from various types of stem cells and cells- predecessors.

[81] As used herein, the term "definitive endoderm" refers to a cell differentiated from an endodermal cell that can differentiate into an SC-β cell (eg, a pancreatic β cell). The definitive endoderm cell expresses the Sox17 marker. Other markers specific to cells of the definitive endoderm include, but are not limited to, MIXL2, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, cerberus, OTX2, goosecoid, C-Kit, CD99, CMKOR1, and CRIP1. In particular, the definitive endoderm cells described herein express Sox17 and, in some embodiments, Sox17 and HNF3B, and do not express GATA4, SPARC, APF, or DAB at a significant level. Definitive endoderm cells are not positive for the Pdx1 marker (eg, they are Pdx1 negative). The cells of the definitive endoderm have the ability to differentiate into cells, including cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Expression of Sox17 and other markers of the definitive endoderm can be assessed by any method known to one of skill in the art, such as immunochemistry, for example with an anti-Sox17 antibody, or quantitative RT-PCR.

[82] The term "pancreatic endoderm" refers to a cell of endodermal origin that is capable of differentiating into multiple pancreatic lineages, including pancreatic β-cells, but can no longer differentiate into non-pancreatic lineages.

[83] As used herein, the term "primary intestinal tube cell" or "intestinal tube cell" refers to a cell differentiated from an endodermal cell that can differentiate into an SC-β cell (eg, a pancreatic β cell). The primary intestinal tube cell expresses at least one of the following markers: HNF1-β, HNF3-β, or HNF4-α. The cells of the primary intestinal tube have the ability to differentiate into cells, including those of the lungs, liver, pancreas, stomach, and intestines. The expression of HNF1-β and other markers of the primary intestinal tube can be assessed by any method known to the person skilled in the art, such as immunochemistry, for example using an anti-HNF1-β antibody.

[84] The terms "pancreatic progenitor cell", "pancreatic endocrine progenitor cell", "pancreatic progenitor", or "pancreatic endocrine progenitor" are used interchangeably herein and refer to a stem cell that is capable of becoming a pancreatic hormone-expressing cell capable of forming pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic ductal cells. These cells are designed to differentiate into at least one type of pancreatic cell, such as beta cells that produce insulin; alpha cells that produce glucagon; delta cells (or D cells), which produce somatostatin; and/or F cells that produce a pancreatic polypeptide. Such cells express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6 or ARX.

[85] As used herein, the term "pdx1-positive pancreatic progenitor cell" refers to a cell that is a pancreatic endoderm (PE) cell that can differentiate into SC-β cells such as pancreatic β cells. The Pdx1-positive pancreatic progenitor cell expresses the Pdx1 marker. Other markers include, but are not limited to, Cdcp1 or Ptfla or HNF6 or NRx2.2. Pdx1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry using an anti-Pdx1 antibody or quantitative RT-PCR.

[86] As used herein, the term "pdx1-positive, NKX6-1-positive pancreatic progenitor cell" refers to a cell that is a pancreatic endoderm (PE) cell that can differentiate into insulin-producing cells such as pancreatic β- cells. The pdx1-positive, NKX6-1-positive pancreatic progenitor cell expresses the Pdx1 and NKX6-1 markers. Other markers include, but are not limited to, Cdcp1 or Ptfla or HNF6 or NRx2.2. Expression of NKX6-1 can be assessed by any method known to one of skill in the art, such as immunochemistry using an anti-NKX6-1 antibody or quantitative RT-PCR.

[87] As used herein, the term "Ngn3-positive endocrine progenitor cell" refers to progenitors of pancreatic endocrine cells expressing the neurogenin-3 (Ngn3) transcription factor. Progenitor cells are more differentiated than multipotent stem cells and can only differentiate into a small number of cell types. In particular, Ngn3-positive endocrine progenitor cells have the ability to differentiate into five types of pancreatic endocrine cells (α, β, δ, ε and PP). Expression of Ngn3 can be assessed by any method known to those skilled in the art, such as immunochemistry using an anti-Ngn3 antibody or quantitative RT-PCR.

[88] The terms "NeuroD" and "NeuroD1" are used interchangeably herein and refer to a protein expressed in pancreatic endocrine progenitor cells and the gene encoding it.

[89] The terms “insulin-positive β-like cell” and “insulin-positive endocrine cell” refer to a cell (e.g., pancreatic endocrine cell) that exhibits at least one marker characteristic of a pancreatic β-cell and also expresses insulin but does not exhibit the characteristic endogenous β-cell GSIS response.

[90] The term "its progenitor" as used in relation to an insulin-positive endocrine cell refers to any cell that is capable of differentiating into an insulin-positive endocrine cell, including, for example, a pluripotent stem cell, a definitive endoderm cell, a primary intestinal tube cell, a pancreatic progenitor cell or an endocrine progenitor cell, when cultured under conditions suitable for differentiation of the progenitor cell into an insulin-positive endocrine cell.

[91] The terms "stem cell-derived β-cell", "SC-β cell", "functional β-cell", "functional pancreatic β-cell" and "mature SC-β cell" refer to cells (e.g., pancreatic β-cells) that exhibit at least one marker characteristic of the pancreatic β-cell (eg, PDX-1 or NKX6-1), express insulin, and exhibit a GSIS response characteristic of the endogenous mature β-cell. In some embodiments, "SC-β cell" includes mature pancreatic β cells. It should be understood that SC-β cells need not be derived (eg, directly) from stem cells, as the methods of the invention make it possible to obtain SC-β cells from any insulin-positive endocrine cell or progenitor thereof, using as starting material any cell (for example, embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells can be used (for example, a somatic cell that has been reprogrammed to an intermediate state between the induced pluripotent stem cell and the somatic cell from which it was derived ), multipotent cells, totipotent cells, transdifferentiated versions of any of the above cells, etc., as the invention is not limited in this sense). In some embodiments, the SC-β cells exhibit a response to multiple glucose stimulations (eg, at least one, at least two, or at least three or more consecutive glucose stimulations). In some embodiments, the response resembles that of endogenous islets (eg, human islets) to repeated glucose stimulation. In some embodiments of the invention, the morphology of the SC-β cell resembles that of an endogenous β-cell. In some embodiments, the SC-β cell exhibits an in vitro GSIS response that resembles the endogenous β-cell GSIS response. In some embodiments, the SC-β cell exhibits an in vivo GSIS response that resembles the endogenous β-cell GSIS response. In some embodiments, the SC-β cell exhibits both in vitro and in vivo a GSIS response that resembles the endogenous β-cell GSIS response. The GSIS response of an SC-β cell can be observed up to two weeks after transplantation of the SC-β cell into a host (eg, human or animal). In some embodiments, the insulin in SC-β cells is packaged into secretory granules. In some embodiments of the invention, encapsulated insulin crystalline granules are observed in SC-β cells. In some embodiments, the SC-β cells have a stimulation index greater than 1. In some embodiments, the SC-β cells have a stimulation index greater than 1.1. In some embodiments, the SC-β cells have a stimulation index greater than 2. In some embodiments, the SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from SC-β cells is increased in response to known anti-diabetic drugs (eg, secretagogues). In some embodiments, the SC-β cells are monohormonal. In some embodiments, the SC-β cells are not characterized by abnormal co-expression of other hormones such as glucagon, somatostatin, or pancreatic polypeptide. In some embodiments of the invention, SC-β cells show a low replication rate. In some embodiments of the invention, intracellular Ca ²⁺ is increased in SC-β cells in response to glucose. As used herein, the term "exocrine cell" refers to a cell of an exocrine gland, i. gland, the secretion of which is carried out through the duct. In specific embodiments, exocrine cells refer to a pancreatic exocrine cell, which is a pancreatic cell that produces enzymes that are secreted into the small intestine. These enzymes help break down food as it passes through the gastrointestinal tract. Pancreatic exocrine cells are also known as the islets of Langerhans, which secrete two hormones, insulin and glucagon. A pancreatic exocrine cell can be one of several types: alpha-2 cells (which produce the hormone glucagon); or β-cells (which produce the hormone insulin); and alpha-1 cells (which produce the regulatory agent somatostatin). In the context of this document non-insulin producing exocrine cells refer to alpha-2 cells or alpha-1 cells. It should be noted that the term "pancreatic exocrine cells" includes "pancreatic endocrine cells", which refers to a pancreatic cell that produces hormones (e.g., insulin (produced by β-cells), glucagon (produced by alpha-2 cells), somatostatin ( produced by delta cells) and a pancreatic polypeptide (produced by F cells), which are secreted into the bloodstream.

[92] As used herein, the term "insulin-producing cell" refers to a cell differentiated from a pancreatic progenitor cell, or a progenitor thereof, that secretes insulin. An insulin-producing cell, as used herein, includes pancreatic β-cells as well as pancreatic β-like cells (e.g., insulin-positive endocrine cells) that synthesize (i.e., transcribe the insulin gene, translate proinsulin mRNA and modify proinsulin mRNA into insulin protein), express (ie, exhibit the phenotypic trait that the insulin gene carries), or secrete (release insulin into the extracellular space) insulin in a constant or inducible manner. The population of insulin-producing cells, for example, obtained by differentiation of insulin-positive endocrine cells or their progenitors into SC-β cells in accordance with the methods of the present invention, may be pancreatic β-cells or β-like cells (for example, cells that have at least one or at least two characteristics of an endogenous β-cell and exhibit a GSIS response similar to that of an endogenous adult β-cell). The novelty of the presented compositions and methods is not offset by the presence in the population of cells that produce insulin naturally (for example, β-cells). It is also contemplated that a population of insulin-producing cells, such as those obtained by the methods disclosed herein, may contain mature pancreatic β-cells or SC-β cells, and may also contain non-insulin-producing cells (i.e., cells with a phenotype similar to β-cells except that they do not produce or secrete insulin).

[93] As used herein, the terms "endogenous β-cell", "endogenous mature pancreatic β-cell" or "endogenous pancreatic β-cell" refer to an insulin-producing pancreatic cell or a cell having the phenotype of a pancreatic β-cell (β -cells). The phenotype of the pancreatic β cell is well known to those skilled in the art and includes, for example, insulin secretion in response to elevated glucose levels, expression of markers such as c-peptide, Pdx1 polypeptide and Glut 2, and distinct morphological features such as the formation islets in the pancreas in vivo, and typically includes small, spindle-shaped cells about 9-15 µm in diameter.

[94] As used herein, the terms "SC-β cell", "pancreatic β-like cell", and "mature pancreatic β-like cell" refer to cells produced by the methods disclosed herein that express at least 15% of the amount insulin expressed by an endogenous pancreatic β-cell, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or more than 100%, such as at least about 1.5 times, or at least about 2 times, or at least about 2.5 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or more than 5 times multiple of the amount of insulin secreted by the endogenous pancreatic β-cell, or alternatively, demonstrate have at least one or at least two endogenous pancreatic β-cell characteristics, such as, without limitation, insulin secretion in response to glucose, and expression of β-cell markers such as, for example, c-peptide, Pdx1 and glut-2. In one embodiment, the SC-β cell is not an immortalized cell (eg, proliferates in culture indefinitely). In one embodiment, the SC-β cell is not a transformed cell, eg, a cell that exhibits transformational properties such as growth in soft agar or lack of contact inhibition.

[95] The term "β-cell marker" refers, without limitation, to proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic β-cells. Exemplary β-cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (Pdx1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI/3, B2, Nkx2.2, NKX6-1, GLUT2, PC2, ZnT-8, Isll, Pax6, Pax4, NeuroD, Hnflb, Hnf-6, Hnf-3beta and MafA as well as those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the β-cell marker is a nuclear β-cell marker. In some embodiments, the β-cell marker is Pdx1 or PH3.

[96] The term "pancreatic endocrine marker" refers, without limitation, to proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

[97] As used herein, the term "non-insulin producing cell" refers to any cell of endodermal origin that does not naturally synthesize, express or secrete insulin continuously or after induction. Thus, the term "non-insulin producing cells" as used herein does not include pancreatic β-cells. Examples of non-insulin producing cells that can be used in the methods of the present invention include pancreatic cells other than β-cells such as amylase-producing cells, acinar cells, cells from ductal adenocarcinoma cell lines (e.g., CD18, CD11, and Capan- I (see Busik et al., 1997; Schaffert et al. 1997) Non-pancreatic cells of endodermal origin may also be used, for example, non-pancreatic stem cells of other endocrine or exocrine organs, including, for example, liver cells, thymus cells, thyroid cells. , intestinal cells, lung cells, and pituitary cells.In some embodiments, the non-insulin-producing endodermal cells can be mammalian cells or, more specifically, human cells.This document provides examples of the proposed method using mammalian pancreatic non-islet cells, pancreatically x mammalian amylase-producing cells, mammalian pancreatic acinar cells.

[98] The term "phenotype" refers to one or more general biological characteristics that are characteristic of a cell or organism under the influence of certain external conditions and factors, regardless of the actual genotype.

[99] As used herein, the term "pluripotent" refers to a cell that has the ability under different conditions to differentiate into more than one differentiated cell type and preferentially differentiate into cell types characteristic of all three germ layers of cells. Pluripotent cells are characterized primarily by their ability to differentiate into more than one cell type, preferably into cells from all three germ layers, for example, using the athymic mouse teratoma formation assay. Pluripotency is also indicated by the expression of embryonic stem (ES) cell markers, although the preferred sign of pluripotency is the ability to differentiate into cells from each of the three germ layers. It should be noted that culturing such cells by itself is not enough to determine their pluripotency. Reprogrammed pluripotent cells (e.g., as used herein, iPS cells) also have the characteristic ability to pass for extended periods without losing the ability to grow, compared to the original parental cells, which generally have a limited number of divisions in culture.

[100] As used herein, the terms "iPS cell" and "induced pluripotent stem cell" are used interchangeably and refer to a pluripotent stem cell artificially derived (e.g., by induction or complete reversal) from a non-pluripotent cell, typically an adult somatic cell. for example, by inducing increased expression of one or more genes.

[101] The terms "precursor cell" or "progenitor" are used interchangeably herein and refer to cells having a cellular phenotype that is more primitive (i.e., refers to an earlier stage of development or progress than a fully differentiated cell) compared to the cells that can be obtained from them in the process of differentiation. Often progenitor cells have significant or very high proliferative potential. Progenitor cells can develop into a large number of different types of differentiated cells or a single type of differentiated cell, depending on the developmental pathway and the environment in which the cells develop and differentiate.

[102] As used herein, the term "stem cell" refers to an undifferentiated cell capable of proliferating and developing into new progenitor cells with the ability to generate a large number of mother cells, which in turn can develop into differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and give rise to progeny that subsequently differentiate into one or more mature cell types while retaining one or more cells with the ability to develop like the parent cells. The term "stem cell" refers to a subgroup of progenitor cells that have the ability or capacity under certain circumstances to differentiate into a more specialized or differentiated phenotype and which, under certain circumstances, retain the ability to proliferate without any significant differentiation. In one embodiment of the invention, the term stem cell generally refers to a mother cell of natural origin, whose progeny (progeny) specialize, often in different directions, by differentiation, for example, acquiring completely individual characteristics, as occurs during the progressive diversification of embryonic cells and fabrics. Cell differentiation is a complex process, usually involving a large number of cell division steps. A differentiated cell may be derived from a multipotent cell, which in turn is derived from a multipotent cell, and so on. While any of these multipotent cells can be considered stem cells, the range of cell types that can develop from each of them can vary greatly. Some differentiated cells also have the ability to develop into cells with a greater ability to develop. This ability may be natural or may be artificially induced by treatment with various factors. In many biological systems, stem cells are also "multipotent" in that they can produce offspring belonging to more than one cell type, although this is not a necessary condition for a "stem cell". Self-renewal represents another classic part of the definition of stem cells and is important in the context of this document. According to the theory, self-renewal can occur through either of two mechanisms. Stem cells can divide asymmetrically, with one daughter cell remaining a stem cell and the other daughter cell expressing some distinct specific function and phenotype. Alternatively, some of the stem cells in the population may divide symmetrically in two steps, such that the population as a whole retains some stem cells while other cells in the population produce highly differentiated progeny. Formally, it is possible for cells that were originally stem cells to develop to a differentiated phenotype, but then "reverse" and re-express the stem cell phenotype; this phenomenon is often referred to by those skilled in the art as "dedifferentiation" or "reprogramming" or "retrodifferentiation". As used herein, the term "pluripotent stem cell" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[103] In the context of cellular ontogeny, the adjective "differentiated" or "differentiating" is a relative term denoting a "differentiated cell" - a cell that has made more developmental progress than the cell to which it is being compared. Thus, stem cells can differentiate into lineage-specific progenitor cells (such as mesodermal stem cells), which in turn can differentiate during development into other progenitor cell types (such as cardiomyocyte progenitors) and then into the final differentiated cell. , which plays a characteristic role in a certain type of tissue and may or may not retain the ability to further proliferate.

[104] The term "embryonic stem cell" is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see US Pat. Nos. 5,843,780, 6,200,806). Such cells can be obtained from the inner cell mass of blastocysts obtained by somatic cell nuclear transfer (see, for example, US patent No. 5945577, 5994619, 6235970). The distinctive characteristics of the embryonic stem cell determine the phenotype of the embryonic stem cell. Accordingly, a cell has an embryonic stem cell phenotype if it has one or more of the unique characteristics of an embryonic stem cell to the extent that the cell can be distinguished from other cells. Exemplary distinguishing characteristics of an embryonic stem cell include, without limitation, gene expression profile, proliferation ability, differentiation ability, karyotype, susceptibility to specific culture conditions, and the like.

[105] The term "adult stem cell" or "ASC" ("adult stem cell") is used to refer to any multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile and adult tissue. Stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, thyroid, skeletal muscle, and cardiac muscle. Any of these stem cells can be characterized based on gene expression, susceptibility to factors, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. As stated above, stem cells have been found in virtually all tissues. Accordingly, the present invention contemplates that stem cell populations can be isolated from virtually any animal tissue.

[106] The term "pancreas" refers to a glandular organ that secretes digestive enzymes and hormones. In humans, the pancreas is a yellowish organ about 7 inches (17.8 cm) long and 1.5 inches (3.8 cm) wide. It is located below the stomach and is connected to the small intestine, the muscular, tube-like part of the gastrointestinal tract that extends from the lower end of the stomach (pylorus) to the anus. Most of the pancreatic tissue is made up of clusters of cells that produce a clear fluid (pancreatic juice) that enters the duodenum through the common duct along with bile from the liver. Pancreatic juice contains three digestive enzymes: tryptase, amylase, and lipase, which, together with intestinal enzymes, complete the digestion of proteins, carbohydrates, and fats, respectively. Scattered among the enzyme-producing cells of the pancreas are small groups of endocrine cells called the islets of Langerhans, which secrete two hormones, insulin and glucagon. The pancreatic islets contain several types of cells: alpha-2 cells, which produce the hormone glucagon; β-cells (also referred to herein as "pancreatic β-cells"), which produce the hormone insulin, and alpha-1 cells, which produce the regulatory agent somatostatin. These hormones are secreted directly into the bloodstream and together they regulate blood glucose levels. Insulin lowers blood sugar and increases the amount of glycogen (a storage form of carbohydrates) in the liver, glucagon has the opposite effect.The inability of insulin-secreting cells to function properly leads to diabetes or diabetes mellitus.

[107] As used herein, the term "reprogramming" refers to a process that alters or reverses the differentiation state of a somatic cell. Before reprogramming, the cell may be partially or completely differentiated. Reprogramming involves the complete reversal of the state of differentiation of a somatic cell into a pluripotent cell. This complete reversal of differentiation results in an induced pluripotent (iPS) cell. In the context of this document, reprogramming also includes the partial reversal of a cell differentiation state, for example, to a multipotent state or to a somatic cell that is neither pluripotent nor multipotent, but is a cell that has lost one or more specific characteristics of the differentiated cell from which it was obtained, for example, in the case of direct reprogramming of a differentiated cell into a different type of somatic cell. In general, reprogramming involves changing, for example, reversing, at least some of the inherited features of nucleotide modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cell differentiation when the zygote develops into adult organism.

[108] As used herein, the term "agent" refers to any compound or substance, such as, without limitation, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An "agent" can be any chemical, compound, or component, including, without limitation, synthetic and naturally occurring proteinaceous and non-proteinaceous compounds. In some embodiments, the agent is a nucleic acid, nucleic acid analogs, proteins, antibodies, peptides, aptamers, nucleic acid oligomers, amino acids, or carbohydrates, including, but not limited to, proteins, oligonucleotides, ribozymes, DNA enzymes, glycoproteins, shRNA, lipoproteins, aptamers, as well as their modifications and combinations, etc. In certain embodiments of the invention, the agents are small molecules containing a chemical moiety. For example, chemical moieties include unsubstituted or substituted alkyl, aromatic, or heterocyclic compounds, including macrolides, leptomycins, and related natural products and analogs. The compounds may be known to have the desired activity and/or properties, or they may be selected from a library of various compounds.

[109] As used herein, the term "contacting" (i.e., bringing at least one insulin-positive endocrine cell or progenitor thereof into contact with a β-cell maturation factor or combination of β-cell maturation factors) includes co-incubation of β-cell maturation factor and the cell in vitro (eg, adding β-cell maturation factors to cells in culture). In some embodiments, the term "contacting" does not include in vivo cell exposure to compounds as described herein that may occur naturally in the subject's body (i.e., exposure that may occur as a result of natural physiological process). The step of bringing at least one insulin-positive endocrine cell or progenitor thereof into contact with the β-cell maturation factor, as described in the embodiments of the invention related to the production of SC-β cells, can be carried out by any suitable method. For example, the cells can be treated in an adherent culture or in a suspension culture. In some embodiments of the invention, cells are treated under conditions that stimulate cell clustering. The invention contemplates any conditions that stimulate cell clustering. Examples of conditions that promote cell clustering include, without limitation, suspension culture in low adhesion tissue culture plates, roller bottles, AggreWell plates. In some embodiments of the invention, the inventors observed that the clusters remained stable in media containing 10% serum. In some embodiments of the invention, conditions that stimulate cell clustering include a low serum environment.

[110] It should be understood that cells that are brought into contact with a β-cell maturation factor can also be simultaneously or sequentially brought into contact with another agent, such as a growth factor or other differentiation agent, or media for cell stabilization or for further cell differentiation. .

[111] Similarly, at least one insulin-positive endocrine cell or progenitor thereof can be contacted with at least one β-cell maturation factor and then contacted with at least another β-cell maturation factor. In some embodiments, the cell is brought into contact with at least one β-cell maturation factor, wherein the contact is separated in time, and in some embodiments, the cell is brought into contact with at least one β-cell maturation factor substantially simultaneously. In some embodiments of the invention, the cell is brought into contact with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight with at least nine or at least 10 β-cell maturation factors.

[112] As used herein, the term "cell culture medium" (also referred to herein as "culture medium" or "medium") is a cell culture medium containing nutrients that maintain cell viability and promote proliferation. The cell culture medium may contain any of the following in suitable combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum substitute, and other components such as peptide growth factors, etc. Cell culture media traditionally used for specific cell types are known to those skilled in the art.

[113] the Term "cell line" refers to a population of mostly or almost identical cells, which, as a rule, were obtained from the same parent cell or from a specific and/or almost identical population of parent cells. The cell line could be or can be kept in culture for an extended period of time (eg, months, years, or an unlimited period of time). It could undergo a spontaneous or induced transformation process, which resulted in an unlimited lifespan of cells in culture. Cell lines include all cell lines known in the art. It should be understood that over time, cells accumulate mutations and possible epigenetic changes, whereby at least some of the properties of individual cells of a cell line may differ from each other. In some embodiments, the cell line contains the SC-β cell described herein.

[114] The term "exogenous" refers to a substance present in a cell or organism other than its native source. For example, the terms "exogenous nucleic acid" or "exogenous protein" refer to a nucleic acid or protein that has been introduced by a human process into a biological system, such as a cell or organism, in which it is not normally present or in which it is present in lesser amounts. quantities. A substance is considered exogenous if it is introduced into a cell or progenitor cell that inherits the substance. The term "endogenous", in contrast, refers to a substance that is native to a biological system.

[115] The term "expression" refers to cellular processes associated with the production of RNA and proteins and, as appropriate, the secretion of proteins, including, if necessary, but not limited to, for example, transcription, translation, folding, modification and processing. Expression products"include RNA transcribed from the gene and polypeptides obtained by translation of the mRNA transcribed from the gene.

[116] As used herein, the terms "genetically modified" or "engineered" cell refer to a cell into which, by a human process, an exogenous nucleic acid has been introduced (or the progeny of such a cell that has inherited at least a portion of that nucleic acid). A nucleic acid may, for example, contain a sequence that is exogenous to a cell, it may contain native sequences (i.e., sequences that can be found in cells in their natural state), but in an order that is not natural (for example, when the coding region is linked to the promoter of another gene), or altered versions of native sequences, etc. The process of transferring a nucleic acid into a cell can be carried out in any suitable manner. Suitable methods include calcium phosphate or lipid mediated transfection, electroporation, and transduction or infection with a viral vector. In some embodiments of the invention, a polynucleotide or a portion thereof is integrated into the cell's genome. The nucleic acid may subsequently be removed or omitted from the genome, provided that such removal or omission results in a detectable change in the cell compared to an unmodified but otherwise equivalent cell. It should be understood that the term genetically modified includes the introduction of modified RNA directly into the cell (eg, synthetically modified RNA). Such synthetic modified RNAs include modifications that prevent rapid degradation by endo- and exonucleases and avoid or reduce the cellular natural immune or interferon response to the RNA. Modifications include, but are not limited to, for example (a) terminal modifications, e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted bonds, etc.), 3' end modifications (conjugation, DNA nucleotides, bonds, etc.), (b) base modifications, for example, substitution with modified bases, stabilizing bases, destabilizing bases or bases that are capable of pairing with an extended set of partners, or conjugated bases, (c) sugar modifications (for example, in position 2' or position 4') or sugar substitution, and (d) modification of internucleoside bonds, including modification or replacement of phosphodiester bonds. In the event that such modifications interfere with translation (i.e., result in a reduction in translation of 50% or more in the absence of modification, for example, as determined by in vitro analysis of rabbit reticulocyte translation), such modification is not suitable for use in the applications described in this document methods and compositions. In some embodiments, the SC-β cell is genetically modified to express neurogenin 3. In some embodiments, the genetic modification of the SC-β cell includes the introduction of a synthetically modified mRNA encoding neurogenin 3. It is believed that the genetic modification of SC-β cells with synthetic modified mRNA encoding neurogenin 3 increases insulin production from cells. It is expected that such a genetic modification of any insulin-producing cell will increase insulin production in that cell.

[117] In some aspects, the invention provides an SC-β cell genetically modified to contain a detectable marker at the insulin locus. In some embodiments, the SC-β cell is modified so that both alleles at the insulin locus are replaced with a detectable marker. In some embodiments, the SC-β cell is genetically modified such that a detectable marker is inserted into the insulin locus and co-expressed with insulin in the SC-β cell in response to glucose stimulation. In some embodiments, the SC-β cell is genetically modified such that a detectable marker is inserted at the insulin locus in place of insulin and expressed instead of insulin in the SC-β cell in response to glucose stimulation. It is contemplated that any detectable marker can be inserted into the insulin locus, including, for example, a nucleic acid encoding a fluorescent protein (eg, GFP). Those skilled in the art will appreciate that such genetically modified SC-β cells can be used in various screening methods, for example, to detect agents that stimulate the expression and/or secretion of insulin from β cells by analyzing a detectable marker in response to the agent. For example, an SC-β cell genetically modified so that the insulin gene is substituted (for example, GFP) in both alleles can be brought into contact with the agent of interest, and those agents that cause SC-β cells to fluoresce due to GFP expression can be considered candidate agents that are capable of activating insulin gene expression in β-cells. In other words, the detectable marker can be used as a surrogate marker for insulin expression in such genetically modified SC-β cells.

[118] As used herein, the term "identity" refers to the degree of sequence similarity between two or more nucleic acids or polypeptides. The percentage identity between a sequence of interest and a second sequence within an evaluation window, e.g., over the sequence of interest, can be assessed by aligning the sequences, determining the number of residues (nucleotides or amino acids) within the comparison window that are opposite the identical residue, while allowing the insertion gaps to maximize identity, dividing by the total number of residues in the sequence of interest or in the second sequence (whichever is longer) that falls within the comparison window, and multiplying by 100. When calculating the number of identical residues required to achieve a specific percentage of identity, fractional numbers should be rounded to the nearest whole number. Percent identity can be calculated using a wide variety of computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc. create alignments and calculate percent identity between sequences of interest. Algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990), modified according to Karlin and Altschul, Proc. Natl. Acad. ScL USA 90:5873-5877, 1993, included in the NBLAST and XBLAST programs developed by Altschul et al. (Altschul, et al., J. MoI. Biol. 215:403-410, 1990). To obtain alignments containing gaps for comparison purposes, Gapped BLAST was used as described by Altschul et al. (Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, you can use the default settings of the respective programs. You can use the PAM250 or BLOSUM62 matrix. BLAST analysis software is publicly available from the National Center for Biotechnology Information (NCBI). For these programs, see the website with the URL: "ncbi.nlm nih.gov". In a specific embodiment of the invention, percent identity is calculated using BLAST2 with default parameters provided by NCBI.

[119] As used herein, the term "isolated" or "partially purified" in the case of a nucleic acid or polypeptide refers to a nucleic acid or polypeptide separated from at least one of the other components (e.g., nucleic acid or polypeptide) that are present together. with the nucleic acid or polypeptide in a natural source and/or which would be present with the nucleic acid or polypeptide if expressed by the cell or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide or synthesized by in vitro transcription/translation are considered "isolated".

[120] As used herein, the term "isolated cell" refers to a cell that has been removed from the organism in which it was originally located, or to the progeny of this cell. Optionally, the cell was cultured in vitro, eg in the presence of other cells. Optionally, the cell is later introduced into a second organism or re-introduced into the organism from which it (or the cell of which it is a progeny) was isolated.

[121] The term "isolated population" as used herein in relation to an isolated population of cells refers to a population of cells that has been removed from and separated from a mixed heterogeneous population of cells. In some embodiments, the isolated population is a substantially purified population of cells compared to the heterogeneous population from which the cells were isolated or enriched.

[122] The term "substantially pure" as used in relation to a particular cell population refers to a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90% % and most preferably at least about 95% pure compared to the cells constituting the total cell population. Then the terms "substantially pure" or "substantially purified" as used in relation to a population of SC-β cells refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not SC-β cells, as used herein. In some embodiments, the present invention includes methods for expanding a population of SC-β cells, wherein the expanded population of SC-β cells is a substantially pure population of SC-β cells.

[123] Similarly, the terms "substantially pure" or "substantially purified" as used in relation to a population of insulin-positive endocrine cells refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10 %, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not insulin-positive endocrine cells, as used herein. In some embodiments, the present invention includes methods for expanding the population of insulin-positive endocrine cells, wherein the expanded population of insulin-positive endocrine cells is a substantially pure population of insulin-positive endocrine cells.

[124] Similarly, the terms "substantially pure" or "substantially purified" as used in relation to a population of Ngn3-positive endocrine progenitor cells refer to a population of cells that contains less than about 20%, more preferably less than about 15% , 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not Ngn3-positive endocrine progenitor cells or progeny thereof, according to interpretation of these terms in this document. In some embodiments, the present invention includes methods for expanding a population of Ngn3-positive endocrine progenitors, wherein the expanded population of Ngn3-positive endocrine progenitors is a substantially pure population of Ngn3-positive endocrine progenitors.

[125] Similarly, the terms "substantially pure" or "substantially purified" as used in relation to a population of Pdx1-positive, NKX6-1-positive pancreatic progenitor cells refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not Pdx1 positive, NKX6- 1-positive pancreatic progenitor cells or their progeny, as used herein. In some embodiments, the present invention includes methods for expanding the population of Pdx1-positive, NKX6-1-positive pancreatic progenitors, wherein the expanded population of Pdx1-positive, NKX6-1-positive pancreatic progenitors is a substantially pure population of Pdx1- positive, NKX6-1-positive pancreatic progenitor cells.

[126] Similarly, the terms "substantially pure" or "substantially purified" as used in relation to a population of Pdx1-positive pancreatic progenitors refer to a population of cells that contains less than about 20%, more preferably less than about 15% , 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not Pdx1-positive pancreatic progenitors or progeny thereof, according to interpretation of these terms in this document. In some embodiments, the invention includes methods for expanding a population of Pdx1 positive pancreatic progenitors, wherein the expanded population of Pdx1 positive pancreatic progenitors is a substantially pure population of Pdx1 positive pancreatic progenitors.

[127] Similarly, the terms "substantially pure" or "substantially purified" as used in relation to a population of cells of the primary intestinal tube, refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10% , 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not primary intestinal tube cells or their progeny, as used herein . In some embodiments, the present invention includes methods for expanding a population of primary intestinal tube cells, wherein the expanded population of primary intestinal tube cells is a substantially pure population of primary intestinal tube cells.

[128] Similarly, the terms "substantially pure" or "substantially purified" as used in relation to a definitive endoderm cell population refer to a cell population that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not definitive endoderm cells or progeny thereof, as used herein. In some embodiments, the present invention includes methods for expanding the definitive endoderm cell population, wherein the expanded definitive endoderm cell population is a substantially pure definitive endoderm cell population.

[129] Similarly, the terms "substantially pure" or "substantially purified" as used in relation to a population of pluripotent cells refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10%, 8 %, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not pluripotent cells or their progeny, as used herein. In some embodiments, the present invention includes methods for expanding a population of pluripotent cells, wherein the expanded population of pluripotent cells is a substantially pure population of pluripotent cells.

[130] The terms "enriching" or "enriched" are used interchangeably herein and mean that the yield (part) of cells of one type is increased by at least 10% compared to the part of cells of that type in the starter culture or preparation.

[131] The terms "renewal" or "self-renewal" or "proliferation" are used interchangeably herein and refer to the ability of stem cells to renew themselves by dividing into the same non-specialized cell types over an extended period of time and/or many months to years. In some cases, proliferation refers to an increase in the number of cells by repeated divisions of single cells into two identical daughter cells.

[132] As used herein, the term "lines of differentiation" describes cells of a common origin or cells with a common developmental path. For example, in the context of a cell that is of endodermal origin or belongs to an "endodermal lineage", this means that the cell has been derived from an endoderm cell and can differentiate along a pathway limited by the endodermal lineage, e.g., along one or more lineage developmental pathways, which give rise to cells of the definitive endoderm, which, in turn, can differentiate into cells of the liver, thymus, pancreas, lungs and intestines.

[133] As used herein, the term "xenogeneic" refers to cells derived from different species.

[134] As used herein, the term "marker" is used to describe the characteristics and/or phenotype of a cell. The markers can be used to select cells having characteristics of interest. The markers are cell specific. Markers are characteristics, both morphological, functional, or biochemical (enzymatic) characteristics of a particular cell type, and the molecules that that cell type expresses. Preferably, such markers are proteins and more preferably contain antibody epitopes or other binding molecules known in the art. In this case, the marker may contain any molecule that can be found in the cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids. Examples of morphological characteristics or features include, but are not limited to, shape, size, and the ratio between nucleus and cytoplasm. Examples of functional characteristics or traits include, but are not limited to, the ability to bind to specific substrates, the ability to include or exclude specific dyes, the ability to migrate under specific conditions, and the ability to differentiate according to specific lineages. The markers can be detected by any means known to the person skilled in the art. Also, markers can be the absence of any morphological characteristic or the absence of proteins, lipids, etc. The markers can be a panel of unique characteristics corresponding to the presence or absence of polypeptides and other morphological characteristics.

[135] The term "modulate" is used in accordance with its meaning adopted in the art, ie. means to cause or contribute to a qualitative or quantitative change, transformation or modification in a process, pathway or phenomenon of interest. Without limitation, such a change may be an increase, decrease, or change in the relative strength or activity of different components or directions of a process, pathway, or phenomenon. A "modulator" is an agent that causes or promotes a qualitative or quantitative change, transformation, or modification in a process, pathway, or phenomenon of interest.

[136] As used herein, the term "DNA" means deoxyribonucleic acid.

[137] The term "polynucleotide" is used interchangeably herein with the term "nucleic acid" and refers to a polymer of nucleosides. Typically, a polynucleotide of the invention consists of nucleosides that can be found in DNA or RNA in their natural state (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester linkages. However, this term also includes molecules containing nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified skeletons, etc., whether or not they can be found in nucleic acids of natural origin, and such molecules can be preferred in certain applications. Whenever a polynucleotide is mentioned in this application, it is to be understood that both DNA and RNA are meant, and in each case both single and double stranded forms (as well as complementary molecules of single stranded molecules). As used herein, "polynucleotide sequence" may refer to the polynucleotide material itself and/or sequence information (ie, the order of letters used as base abbreviations) that biochemically characterizes a particular nucleic acid. Presented in this document, the polynucleotide sequence is presented in the direction from 5' to 3', unless otherwise indicated.

[138] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically 2 to 60 amino acids in length. Polypeptides in the context of this document, as a rule, contain amino acids, such as 20 L-amino acids, which are most often found in proteins. Other amino acids and/or analogs of amino acids known in the art may be used. One or more amino acids in a polypeptide may be modified, for example, by adding a chemical moiety such as a carbohydrate group, a phosphate group, a fatty acid group, or a linker for conjugation, functionalization, etc. A polypeptide that contains a non-polypeptide component covalently or non-covalently associated with it is considered a "polypeptide". Typical modifications include glycosylation and palmitoylation. Polypeptides can be isolated from natural sources, produced by recombinant DNA technology, synthesized by chemical means such as conventional solid phase peptide synthesis, and so on. As used herein, the term "polypeptide sequence" or "amino acid sequence" may refer to the polypeptide material itself and/or sequence information (i.e., the order of letters or three-letter codes used as abbreviations for amino acid names) that characterizes the polypeptide biochemically. . Presented in this document, the polypeptide sequence is presented in the direction from the N-terminus to the C-terminus, unless otherwise indicated.

[139] The term "variant" as used in relation to a polypeptide can be, for example, a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the full length polypeptide. The variant may be a fragment of a full length polypeptide. The variant may be a naturally occurring splice variant. A variant may be a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a polypeptide fragment, whereby the length of the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of the full length of the wild-type polypeptide, or a domain thereof, having an activity of interest, such as the ability to detect the presence of an SC-β cell or an insulin-positive endocrine cell or progenitor thereof, from which an SC-β cell was obtained. In some embodiments, the domain is at least 100, 200, 300, or 400 amino acids long, starting at any amino acid position in the sequence and extending towards the C-terminus. It is preferred to avoid variations known in the art to eliminate or significantly reduce protein activity. In some embodiments, the variant is missing the N- and/or C-terminal portion of the full-length polypeptide, eg, missing up to 10, 20, or 50 amino acids from either end. In some embodiments, the polypeptide has the sequence of a mature (full-length) polypeptide, which refers to a polypeptide in which one or more parts, such as a signal peptide, have been removed during normal intracellular proteolytic processing (for example, during co-translational or post-translational processing). In some embodiments of the invention, in which the protein is obtained by other than isolation from cells that express it naturally, the protein is a chimeric polypeptide, which means that it contains parts obtained from two or more different species. In some embodiments of the invention, in which the protein is obtained by other than isolation from cells that express it naturally, the protein is a derivative, which means that the protein contains additional sequences that are unrelated to this protein, provided that these sequences are significantly do not reduce the biological activity of the protein.

[140] As used herein, the term "functional fragment" means a polypeptide having an amino acid sequence that is smaller but substantially homologous to the polypeptide of which it is a fragment, and wherein the polypeptide sequence of the functional fragment has at least about 50% or 60% or 70% or 80% or 90% or 100% or more than 100% e.g. 1.5x, 2x, 3x, 4x or more than 4x effective biological effect compared to the polypeptide of which it is a fragment. Functional polypeptide fragments may have additional functions, which may include reduced antigenicity, increased DNA binding capacity (as in the case of transcription factors), or altered RNA binding capacity (as in the case of regulation of RNA stability or degradation).

[141] The term "vector" refers to a DNA molecule carrying, in which the DNA sequence can be inserted for introduction into the host cell. Preferred vectors are those capable of autonomous replication and/or expression of the nucleic acids to which they are associated. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions needed for the expression of a gene of interest in a host cell. In some embodiments, the gene of interest is operably linked to another sequence in the vector. The vectors may be viral vectors and non-viral vectors. In the case of viral vectors, it is preferred that the viral vectors be replication defective, which can be achieved, for example, by removing all viral nucleic acids that encode replication. The replication-deficient viral vector retains its infecting properties and invades the cell in the same way as the replicating adenoviral vector, while after entering the cell, the replication-defective viral vector is unable to reproduce or multiply. Also, vectors include liposomes and nanoparticles, as well as other means of delivering a DNA molecule into a cell.

[142] The term "operably linked" means that the regulatory sequences necessary for the expression of the coding sequence are located in the DNA molecule at appropriate positions relative to the coding sequence so as to influence the expression of the coding sequence. The same definition is sometimes applied to the location of coding sequences and transcriptional regulatory elements (eg, promoters, enhancers, and termination elements) in an expression vector. The term "operably linked" includes the presence of an appropriate start signal (e.g., ATG) at the beginning of the polynucleotide sequence to be expressed and maintaining the correct reading frame to allow expression of the polynucleotide sequence under the control of an expression control sequence and production of the desired polypeptide encoded by the polynucleotide. sequence.

[143] The term "viral vectors" refers to the use of viruses or virus-associated vectors as carriers of nucleotide constructs for introduction into a cell. The constructs can be integrated and packaged into non-replicating defective viral genomes such as adenovirus, adeno-associated virus (AAV) or herpes simplex virus (HSV) or others, including retroviral and lentiviral vectors, for infection or cell transduction. The vector may or may not integrate into the cellular genome. If desired, the constructs may contain viral sequences for transfection. Alternatively, the construct may be incorporated into vectors capable of episomal replication, such as the EPV and EBV vectors.

[144] The terms "regulatory sequence" and "promoter" are used interchangeably herein and refer to nucleotide sequences, such as initiation signals, enhancers, and promoters, that induce or regulate transcription of the protein coding sequences to which they are operably linked. In some examples, the transcription of the recombinant gene is under the control of a promoter sequence (or other transcriptional control sequence) that controls the expression of the recombinant gene in the cell type in which expression is to occur. It should also be understood that the recombinant gene may be under the control of transcriptional regulatory sequences that are the same as or that are different from those sequences that control the transcription of the natural form of the protein. In some cases, the promoter sequence is recognized by the cellular machinery of synthesis or introduced by the synthesis machinery necessary to initiate transcription of a particular gene.

[145] As used herein, the term "transcription factor" refers to a protein that binds to specific portions of DNA via DNA-binding domains and is part of a system that directs the transfer (or transcription) of genetic information from DNA to RNA. As used herein, "proliferating" or "proliferation" refers to an increase in the number of cells in a population (growth) through cell division. It is generally understood that cell proliferation is the result of coordinated activation of multiple signal transduction pathways in response to environmental factors, including growth factors and other mitogens. Cell proliferation can also be stimulated by the release of intra- and extracellular signals and mechanisms that block or adversely affect cell proliferation.

[146] The term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers on a cell a selectable phenotype, such as resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), nutrient prototrophy, or expression of a particular protein, which can be used as a basis for distinguishing between cells that express a protein and cells that do not. Proteins whose expression can be easily detected, such as a fluorescent or luminescent protein, or an enzyme whose action on a substrate leads to the production of a colored, fluorescent or luminescent substance (“detectable markers”) constitute a subgroup of selectable markers. The presence of a selectable marker associated with expression control elements native to a gene that is normally selectively or exclusively expressed in pluripotent cells makes it possible to identify and select somatic cells that have been reprogrammed to a pluripotent state. A large number of selectable marker genes can be used, such as neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyl transferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC) gene , hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene, and hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange and blue fluorescent proteins, as well as variants of any of them. Luminescent proteins such as luciferase (eg firefly luciferase or Renilla luciferase) can also be used. One of ordinary skill in the art will appreciate that the term "selectable marker" as used herein may refer to a gene or a product of gene expression, such as an encoded protein.

[147] In some embodiments, the selectable marker provides cells that express it with a proliferation and/or viability advantage over cells that do not express it or that express it at a substantially lower level. Such a proliferation and/or viability advantage generally occurs when the cells are kept under certain conditions, ie. "selective conditions". To ensure selection is effective, a population of cells can be maintained under conditions and for a sufficient period of time so that cells that do not express the marker do not proliferate and/or survive and are excluded from the population or constitute only a very small part of the population. The process of selecting cells that express a marker that provides a proliferation and/or viability advantage by maintaining a population of cells under selective conditions so as to largely or completely eliminate cells that do not express that marker is referred to herein as "positive selection". ", and the marker is considered "usable for positive selection". Negative selection and markers applicable for negative selection are also of interest in the context of some of the methods described herein. The expression of such markers provides cells that express the marker with a proliferation and/or viability disadvantage compared to cells that do not express the marker or that express it at a significantly lower level (or, in other words, cells that do not express the marker, have a proliferation and/or viability advantage over cells that express the marker). Therefore, cells that express the marker can be largely or completely excluded from the cell population when maintained under selective conditions for a sufficient period of time.

[148] As used herein, the term "reporter gene" includes any gene genetically introduced into a cell that complements the phenotype of a stem cell. The reporter genes disclosed herein include fluorescent, luminescent, enzymatic, and resistance-conferring genes, as well as other genes that can be readily identified by those skilled in the art. In some embodiments, reporter genes are used as markers to identify specific stem cells, cardiovascular stem cells, and their differentiated progeny. In general, a reporter gene is operably linked to sequences that control its expression in a manner that depends on one or more conditions that are monitored by determining the expression of the reporter gene. In some cases, reporter gene expression can be determined in living cells. In cases where live cell reporter gene analysis is used, reporter gene expression can be monitored for a number of time points, such as 2, 3, 4, 5, 6, 8, or 10 or more time points. In some instances where live cell reporter gene assays are used, reporter gene expression is monitored at a frequency of at least about 10 to about 24 hours, such as 20 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours. hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, or any other integer value from about 10 minutes to about 24 hours.

[149] The terms “subject” and “individual” are used interchangeably herein and refer to an animal, such as a human, from which cells can be obtained and/or treated, including prophylactic treatment with cells, as described herein. document. In the case of the treatment of those infections, pathological or disease states that are specific to a particular animal, such as a human, the term subject refers to that particular animal. The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and monkeys. The term "subject" also includes any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Wherein, the subject is predominantly a mammal, such as a human, or other mammals, such as domestic mammals, such as dogs, cats, horses, and the like, or agricultural mammals, such as cows, pigs, and the like.

[150] The terms "diabetes" and "diabetes mellitus" are used interchangeably herein. According to the World Health Organization definition, in the case of diabetes mellitus, the diagnostic value of fasting plasma glucose is 7.0 mmol/L (126 mg/dL) and higher (in whole blood - 6.1 mmol/L or 110 mg/dL) or the level glucose 2 hours after a meal is 11.1 mmol/L or higher (200 mg/dL or higher). Other indicators that suggest or indicate a high risk of diabetes include high blood pressure of 140/90 mm Hg. Art. or higher; elevated plasma triglycerides (1.7 mmol/L; 150 mg/dL) and low HDL cholesterol (less than 0.9 mmol/L, 35 mg/dL for men; less than 1.0 mmol/L, 39 mg /dl for women); central obesity (men: waist-to-hip ratio greater than 0.90; women: waist-to-hip ratio greater than 0.85) and/or a body mass index greater than 30 kg/m ² ; microalbuminuria, in which the urinary albumin excretion rate is 20 µg/min or more, or the albumin:creatinine ratio is 30 mg/g or more). The term diabetes includes all forms of diabetes, such as type I, type II and type 1.5.

[151] The terms "treat" ("treat"), "processing" ("treatment"), etc., used in relation to an isolated cell, include exposing the cell to a process or condition of any type, or performing any type of manipulation or procedure with cell. When used in relation to a subject, these terms refer to the provision of medical or surgical care, observation or treatment of an individual. Typically, an individual has a disease or injury or is at an increased risk of developing a disease compared to the average member of the population and is in need of such care, observation or treatment.

[152] As used herein, the term "treatment" refers to administering to a subject an effective amount of a composition such that the subject experiences an amelioration of at least one symptom of a disease or an improvement in the disease, eg, favorable or desired clinical results. In the context of this invention, beneficial or desirable clinical results include, but are not limited to, alleviation of one or more symptoms, reduction in the degree of the disease, stabilization (i.e., no worsening) of the state of the disease, arrest or slowing of the progression of the disease, improvement or alleviation of the state of the disease, and remission (both partial and complete), both detected and not detected. Treatment may refer to prolongation of survival compared to life expectancy without treatment. Thus, one of ordinary skill in the art will recognize that treatment may lead to an improvement in the disease state, but may not lead to a complete cure of the disease. As used herein, the term "treatment" includes prophylaxis. Alternatively, the treatment is "effective" if the progression of the disease is slowed or stopped. "Treatment" can also mean prolonging the lifespan compared to the expected lifespan without treatment. Those in need of treatment include those who have already been diagnosed with a cardiac condition as well as those who are at high risk of developing a cardiac condition due to genetic susceptibility or other factors such as weight, diet and health status.

[153] As used herein, the terms "introduction", "introduction" and "transplantation" are used interchangeably in the context of introducing cells, for example, SC-β cells according to the invention, into the body of a subject by a method or route that results in at least partial placement introduced cells in the required area. Cells, such as SC-β cells (eg, pancreatic β-cells or pancreatic β-like cells), can be implanted directly into the pancreas or, alternatively, they can be administered by any suitable route that results in delivery to the desired site in the body of the subject, while at least part of the implanted cells or cell components remain viable. The period of viability of cells after introduction into the body of a subject can be from several hours, for example, twenty-four hours, to several days and up to several years. In some cases, the cells can also be administered not to the pancreas, but, for example, to the liver or subcutaneously, for example, in a capsule (eg, microcapsule), in order to keep the implanted cells at the implantation site and avoid the migration of the implanted cells.

[154] As used herein, the expressions "parenteral administration" and "parenterally administered" mean modes of administration other than enteral and topical administration, usually by injection and including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal and intrasternal injection and infusion. As used herein, the expressions "systemic administration", "systemically administered", "peripherally administered" and "peripherally administered" mean the administration of cardiovascular stem cells and/or their progeny and/or a compound and/or other material other than direct administration into the central nervous system in such a way that it enters the body of the animal and undergoes metabolism and other such processes, for example, as in the case of subcutaneous administration.

[155] The term "tissue" refers to a group or layer of specialized cells that together perform certain functions. The term "tissue-specific" refers to the source of cells from a particular tissue.

[156] The terms "reduced", "reduced", "decrease", "decrease" or "inhibition" are generally used to mean a decrease by a statically significant amount. However, for the avoidance of doubt, "reduced", "reduction" or "reduction" or "inhibition" means a reduction of at least 10% compared to a control level, for example, a reduction of at least about 20% or at least by about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% reduction (i.e., no levels compared to the control), or any reduction corresponding to a value in the range of 10-100% compared to the control.

[157] The terms "increased", "increase" or "increase" or "activate" are generally used to mean an increase by a statically significant amount; for the avoidance of doubt, the terms "increased", "increase" or "increase", or "activate" means an increase of at least 10% over a control level, e.g., an increase of at least about 20% or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% increase, or any increase corresponding to a value in the range of 10-100% compared to the control level, or at least about 2-fold, or at least about 3- fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, or any increase corresponding to the range from 2-fold to 10-fold higher than the control level.

[158] The term "statistically significant" or "significantly" refers to statistical significance and generally means twice the standard deviation (2SD) of the marker concentration from or below normal. This term refers to the statistical evidence of a difference. It is defined as the probability of deciding to reject the null hypothesis when the null hypothesis is in fact true. This decision is often made using a p-value.

[159] As used herein, the terms "comprising" or "comprises" are used in relation to compositions, methods and their respective component(s) that are relevant to this invention, but also refer to the inclusion of arbitrary elements, both essential and and no.

[160] As used herein, the term "consists predominantly of" refers to those elements that are necessary for this embodiment of the invention. The term allows for the presence of additional elements that do not significantly affect the main(s) or functional(s) characteristic(s) of this embodiment of the invention.

[161] The term "consisting of" refers to the compositions, methods, and their respective components described herein, which do not include elements not listed in this description of an embodiment of the invention.

[162] In the description of the invention and the accompanying claims, the use of the singular form includes reference to the plural, unless otherwise clearly provided by the context. Thus, for example, reference to "method" refers to one or more of the methods and/or steps of the method described herein, as will become apparent to those skilled in the art upon reading the specification, etc.

[163] Stem Cells

[164] Stem cells are cells that retain the ability to self-renew through mitotic cell division and can differentiate into a wide range of specialized cell types. The two main types of mammalian stem cells are embryonic stem (ES) cells, which are found in blastocysts, and adult stem cells, which are found in adult tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem cells and progenitor cells serve as the body's repair system, replenishing a population of specialized cells, and also support the normal renewal of regenerative organs such as blood, skin, or intestinal tissue. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[165] Although certain embodiments of the invention are described below in connection with the use of stem cells to produce SC-β cells (e.g., pancreatic β-cells or β-like cells) or their progenitors, germ cells can be used instead of or together with stem cells to obtaining at least one SC-β cell using protocols similar to the exemplary protocols described herein. Suitable germ cells can be obtained, for example, from primordial cells that are present in human embryonic material taken approximately 8-11 weeks after the last menstrual cycle. Illustrative methods for obtaining germ cells are described, for example, in Shamblott et al., Proc. Natl. Acad. sci. USA 95:13726, 1998 and US Pat. No. 6,090,622.

[166] ES cells, such as human embryonic stem cells (hESC) or mouse embryonic stem cells (mESC), with the ability to replicate almost endlessly and differentiate into most cell types, represent in principle an unlimited starting material for generating differentiated cells for clinical therapy. (http://stemcells.nih.gov/info/scireport/2006report.htm, 2006). One possible application of ES cells is to generate new pancreatic β-cells for cell replacement therapy of type I diabetes by obtaining endoderm, e.g. definitive endoderm, from e.g. hESC, and further differentiation of definitive endoderm into at least one insulin-positive endocrine cell. or its precursor, and then differentiating at least one insulin-positive endocrine cell or its precursor into an SC-β cell.

[167] hESCs are described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and Thomson et al. (Science 282:1145, 1998); embryonic stem cells from other primates, rhesus monkey stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol. Reprod. 55:254, 1996) and human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998) can also be used in the methods described herein. mESCs are described, for example, in Tremml et al. (Curr Protoc Stem Cell Biol. Chapter 1: Unit 1C.4, 2008). Stem cells can be, for example, unipotent, totipotent, multipotent, or pluripotent. In some examples, the methods disclosed herein can use any primate-derived cells that are capable of producing offspring that are derived from at least one germ layer or all three germ layers.

[168] In some examples, ES cells can be isolated, for example, as described in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and US Pat. No. 5,843,780, and Thomson et al., Proc. Natl. Acad. sci. USA 92:7844, 1995. For example, hESC can be obtained from human blastocyst cells using the methods described in Thomson et al. (U.S. Patent No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. HESC-equivalent cell types include their pluripotent derivatives, such as cells like primary ectoderm (EPL) cells, as set forth, for example, in WO 01/51610 (Bresagen). hESC can also be obtained from human pre-implantation embryos. Alternatively, in vitro fertilized (IVF) embryos may be used, or single-celled human embryos may be grown to the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from the formed blastocysts by short exposure to pronase (Sigma). The internal cell mass can be isolated by immunosurgery, during which the blastocysts are exposed for 30 minutes to a 1:50 solution of rabbit anti-human antiserum against spleen cells, then washed three times for 5 minutes in DMEM and exposed to a 1:5 solution for 3 minutes. guinea pig complement (Gibco) (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ECM) by gentle pipetting, and the ECM is seeded onto mEF feed sheets. After 9-15 days, the outgrowth of the inner cell mass can be grouped by treatment with calcium- and magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by treatment with dispase or trypsin, or by mechanical separation using a micropipette; and then subcult on mEF to fresh medium. Growing colonies with undifferentiated morphology can be collected individually with a micropipette, mechanically grouped and subcultured. The ES-like morphology has the characteristics of compact colonies with an apparently high nucleus-to-cytoplasm ratio and prominent nucleoli. The resulting hESCs can then be routinely separated every 1-2 weeks, e.g., by short trypsin treatment, Dulbecco's PBS treatment (containing 2 mM EDTA), type IV collagenase treatment (about 200 U/mL; Gibco), or by picking single colonies. using a micropipette. In some examples, optimal group sizes are about 50-100 cells. mESC can be obtained using the methods described, for example, in Conner et al. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).

[169] Embryonic stem cells can be isolated from primate blastocysts (US Pat. No. 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) can be obtained from human blastocyst cells using the methods described in Thomson et al. (US patent No. 6200806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and in Reubinoff et al, Nature Biotech. 18:399, 2000. HES cell-equivalent cell types include their pluripotent derivatives, such as cells like primary ectoderm cells (EPL), as set forth, for example, in WO 01/51610 (Bresagen).

[170] In some alternative embodiments of the invention, hES cells can be obtained from human pre-implantation embryos. Alternatively, in vitro fertilized (IVF) embryos may be used, or single-celled human embryos may be grown to the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from the formed blastocysts by short exposure to pronase (Sigma). The inner cell mass is isolated by immunosurgery, during which the blastocysts are exposed for 30 min to a 1:50 solution of rabbit anti-human antiserum against spleen cells, then washed three times for 5 min in DMEM and exposed to a 1:5 solution of complement for 3 min. guinea pig (Gibco) (Sorter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ECM) by gentle pipetting, and the ECM is seeded onto mEF feed sheets.

[171] After 9-15 days, the outgrowth of the inner cell mass is divided into groups by treatment with calcium and magnesium-free phosphate-buffered saline (PBS) with 1 mm EDTA, by treatment with dispase or trypsin, or by mechanical separation using a micropipette; and then subcultured to mEF in fresh medium. Growing colonies with undifferentiated morphology are collected individually with a micropipette, mechanically divided into groups and subcultured. The ES-like morphology has the characteristics of compact colonies with an apparently high nucleus-to-cytoplasm ratio and prominent nucleoli. The resulting ES cells are then routinely separated every 1-2 weeks by brief trypsin treatment, Dulbecco's PBS treatment (containing 2 mM EDTA), type IV collagenase ( ^~ 200 U/mL; Gibco), or by collecting single colonies using a micropipette. . The optimal group sizes are about 50-100 cells.

[172] In some embodiments, human embryonic germ cells (hEG) are pluripotent stem cells that can be used in the methods described herein to differentiate into primary endoderm cells. hEG cells can be obtained from primordial cells that are present in human fetal material taken approximately 8-11 weeks after the last menstrual cycle. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. sci. USA 95:13726, 1998 and US Pat. No. 6,090,622, which are incorporated herein by reference in their entirety.

[173] Briefly, sex cords are processed to form disaggregated cells. EG growth medium consists of DMEM, 4500 mg/l D-glucose, 2200 mg/l mM NaHCO ₃ ; 15% suitable ES fetal bovine serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U/ml human recombinant leukemia inhibitory factor (LIF, Genzyme); 1-2 ng/ml human recombinant bFGF (Genzyme); and 10 μM forskolin (in 10% DMSO). Ninety-six well tissue culture plates are coated with a subconfluent layer of feeder cells (e.g., STO cells, ATCC # CRL 1503), cultured for 3 days in modified EG growth medium containing no LIF, bFGF, or forskolin, ^~ 0. 2 ml of inactivated γ-irradiation in 5000 rad suspension of primary germ cells (PGC). The first passage is completed after 7-10 days in EG growth medium by transferring the contents of each well to one of the wells of a 24-well culture plate pretreated with irradiated STO mouse fibroblasts. Cells are cultured with daily medium replacement until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages.

[174] In certain examples, prior to treatment with at least one β-cell maturation factor in accordance with the methods disclosed herein, the stem cells may be undifferentiated (e.g., a cell not related to a particular lineage), while in other examples it may be desirable to differentiate stem cells into one or more intermediate cell types prior to treatment with at least one β-cell maturation factor described herein. For example, stem cells may display morphological, biological, or physical characteristics of undifferentiated cells by which they can be distinguished from differentiated cells of an embryo or mature organism. In some instances, undifferentiated cells may appear in two dimensions when viewed microscopically in colonies of cells with a high nucleus/cytoplasm ratio and prominent nucleoli. Stem cells can be on their own (eg, in the virtual absence of any undifferentiated cells) or they can be used in the presence of differentiated cells. In certain instances, stem cells can be cultured in the presence of suitable nutrients and optionally other cells so that the stem cells can grow and optionally differentiate. For example, embryonic fibroblasts or fibroblast-like cells may be present in culture to promote stem cell growth. Fibroblasts may be present at one of the stages of stem cell growth, but not necessarily at all stages. For example, fibroblasts can be added to stem cell cultures in the first culture step and not added to stem cell cultures in one or more of the subsequent culture steps.

[175] Stem cells used in all aspects of the present invention can be any cell derived from any type of tissue (for example, embryonic tissue such as fetal or prefetal tissue, or adult tissues), and the stem cells are characterized by the ability to appropriate conditions to give offspring belonging to different types of cells, for example, derivatives of at least one of 3 germ layers (endoderm, mesoderm and ectoderm). These cell types may be in the form of a stable cell line, or they may be directly obtained from primary embryonic tissue and used directly for differentiation. Included are cells listed in the NIH human embryonic stem cell registry, eg hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments of the invention, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into mature insulin-positive cells is not associated with the destruction of the human embryo.

[176] In another embodiment, stem cells can be isolated from tissue, including solid tissue. In some embodiments of the invention, the tissue is skin, adipose tissue (eg, adipose tissue), muscle tissue, heart, or cardiac tissue. In other embodiments of the invention, the tissue is, without limitation, for example, cord blood, placenta, bone marrow, or cartilage.

[177] Stem cells of interest also include different types of embryonic cells, exemplified by the human embryonic stem (hES) cells described in Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also of interest are lineage-specific stem cells such as mesodermal stem cells and other early cardiogenic cells (see Reyes et al. (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2) :205-16; etc.). These stem cells can be obtained from any mammalian species, such as humans, horses, ruminants, pigs, dogs, cats, rodents, such as mice, rats, hamsters, primates, etc. In some embodiments of the invention, the human embryo is not subjected to destruction to obtain a source of pluripotent cells used in the methods and compositions disclosed herein.

[178] ES cells are considered undifferentiated if they do not belong to a particular lineage. Such cells exhibit morphological characteristics that distinguish them from differentiated embryonic or adult cells. Those skilled in the art will readily recognize undifferentiated ES cells, which typically appear in two dimensions when viewed microscopically in colonies of cells with a high nucleus/cytoplasm ratio and prominent nucleoli. Undifferentiated ES cells express genes that can be used as markers to detect the presence of undifferentiated cells and whose polypeptide products can be used as markers for negative selection. For example, see US Patent Application No. 2003/0224411 A1; Bhattacharya (2004) Blood 103(8):2956-64; and Thomson (1998), supra, each of which is incorporated herein by reference. Human ES cell lines express cell surface markers that are characteristic of undifferentiated monkey ES cells and human ES cells, including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The GL7 globo-series glycolipid, which carries the SSEA-4 epitope, is formed by adding sialic acid to the GbS globo-series glycolipid, which carries the SSEA-3 epitope. Thus, GL7 reacts with both SSEA-3 and SSEA-4 antibodies. Undifferentiated human ES cell lines do not stain with SSEA-1, but differentiated cells stain strongly with SSEA-I. Methods for the proliferation of hES cells in undifferentiated form are described in WO 99/20741, WO 01/51616 and WO 03/020920.

[179] A mixture of cells from a suitable source of endothelial, muscle and/or neural stem cells can be obtained from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (ie, stimulated), can be collected from a patient. Alternatively, bone marrow may be obtained from a mammal, such as a human patient undergoing autotransplantation. In some embodiments, stem cells can be obtained from the adipose tissue of subjects, for example, using the Cytori CELUTION™ SYSTEM as disclosed in US Pat.

[180] In some embodiments, the methods disclosed herein use human umbilical cord blood cells (HUCBC). Human UBC cells are considered a rich source of hematopoietic and mesenchymal progenitor cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113). Previously, cord and placental blood were considered by-products that usually accompany the birth of a child. Cord blood cells are used as a source of transplantable stem and progenitor cells and as a source of repopulating bone marrow cells for the treatment of malignant diseases (i.e., acute lymphoid leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and neuroblastoma) and non-malignant diseases, such as Fanconi anemia and aplastic anemia (Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79; 1874-1881; Lu et al., 1996 Crit. Rev Oncol Hematol 22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503). An important advantage of HUCBC is the immature immunity of these cells, very similar to fetal cells, which greatly reduces the risk of host rejection (Taylor & Bryson, 1985J. Immunol. 134:1493-1497). Human cord blood contains mesenchymal and hematopoietic progenitors, as well as endothelial progenitors, which can be expanded in tissue culture (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113; Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79; 1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol 22:61-78; Lu et al. al., 1995 Cell Transplantation 4:493-503; Taylor & Bryson, 1985 J. Immunol. 134:1493-1497 Broxmeyer, 1995 Transfusion 35:694-702; Chen et al., 2001 Stroke 32:2682-2688; Nieda et al. al., 1997 Br. J. Haematology 98:775-777; Erices et al., 2000 Br. J. Haematology 109:235-242). The total content of hematopoietic progenitors in cord blood is equal to or greater than that in bone marrow and, in addition, the number of highly proliferative hematopoietic cells is eight times higher in HUCBC than in bone marrow and they express hematopoietic markers such as CD14, CD34 and CD45 (Sanchez - Ramos et al., 2001 Exp Neur 171:109-115; Bicknese et al., 2002 Cell Transplantation 11:261-264; Lu et al., 1993 J. Exp Med 178:2089-2096).

[181] In another embodiment, the pluripotent cells are cells in the hematopoietic microenvironment, such as circulating peripheral blood, preferably from peripheral blood mononuclear fraction, cord blood, bone marrow, fetal liver, or mammalian yolk sac. Stem cells, especially neural stem cells, can also be obtained from the central nervous system, including the lining of the brain.

[182] In another embodiment, the pluripotent cells present in the embryoid bodies are obtained by harvesting ES cells with a short protease treatment and allowing small groups of undifferentiated human ESCs to grow in suspension culture. Differentiation is induced by removing the conditioned medium. The resulting embryoid bodies are plated on semi-solid substrates. The formation of differentiated cells can be observed after a time ranging from about 7 days to about 4 weeks. The selection of viable differentiating cells from in vitro cultures of stem cells is carried out by partial dissociation of embryoid bodies or similar structures to obtain cell aggregates. The selection of aggregates containing cells of interest is carried out on the basis of phenotypic traits, using methods that allow to a large extent to preserve the contacts between cells in the aggregate.

[183] In an alternative embodiment, the stem cells may be reprogrammed stem cells, such as stem cells derived from somatic or differentiated cells. In such an embodiment, the dedifferentiated stem cells may, for example, be, without limitation, neoplastic cells, tumor cells, and cancer cells, or alternatively, induced reprogrammed cells such as induced pluripotent stem cells or iPS cells.

[184] Cloning and cell culture

[185] Illustrative methods of molecular genetics and genetic engineering that can be used in the methods described herein can be found, for example, in modern editions of Molecular Cloning: A Laboratory Manual., (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.); and Current Protocols in Molecular Biology (F.M. Ausubel et al. eds., Wiley & Sons). Materials relating to cell biology, protein chemistry, and antibody-related techniques can be found, for example, in Current Protocols in Protein Science (J.E. Colligan et al. eds., Wiley &Sons); Current Protocols in Cell Biology (J.S. Bonifacino et al., Wiley & Sons) and Current protocols in Immunology (J.E. Colligan et al. eds., Wiley & Sons.). Exemplary reagents, cloning vectors, and genetic manipulation kits are commercially available from, for example, BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[186] A general description of suitable cell culture techniques can be found, for example, in the current Culture of Animal Cells: A Manual of Basic Technique (R.I. Freshney ed., Wiley &Sons); General Techniques of Cell Culture (M.A. Harrison & I.F. Rae, Cambridge Univ. Press) and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press). Suitable tissue culture nutrients and reagents are available from, for example, Gibco/BRL, Nalgene-Nunc International., Sigma Chemical Co. and ICN Biomedicals.

[187] A person skilled in the art can grow pluripotent stem cells in culture for a long time using culture conditions that stimulate proliferation but do not stimulate differentiation. A typical serum-containing medium for ES cells consists of 80% DMEM (e.g. Knock-Out DMEM, Gibco), 20% chemically defined fetal bovine serum (FBS, Hyclone) replacement serum (WO 98/30679), 1% non-essential amino acids, 1 mM L-glutamine and 0.1 mM β-mercaptoethanol. Just prior to use, human bFGF is added to 4 ng/ml (WO 99/20741, Geron Corp.). Traditionally, ES cells are cultured on a layer of nurse cells, usually fibroblasts, derived from embryonic or fetal tissue.

[188] Scientists at Geron found that pluripotent SCs can be maintained in an undifferentiated state even in the absence of nurse cells. The nutrient-free culture medium includes a suitable culture substrate, namely an extracellular matrix such as Matrigel® or laminin. Typically, enzymatic digestion is stopped until the cells are completely dispersed (eg, this corresponds to about 5 minutes of collagenase IV treatment). Groups containing ^~ 10 to 2000 cells are then seeded directly onto the substrate without additional scatter.

[189] Nutrient-free cultures are maintained with a nutrient medium containing factors that stimulate cell proliferation without differentiation. Such factors can be introduced into the medium by culturing the medium with cells secreting such factors, such as irradiated ( ^~ 4000 rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells. The medium can be conditioned by seeding feedstock at a density of ^~ 5-6×10 ⁴ cm ^-2 in serum-free medium such as KO DMEM supplemented with 20% serum replacement and 4 ng/ml bFGF. Medium conditioned for 1-2 days is supplemented with additional bFGF and used to maintain a culture of pluripotent SCs for 1-2 days. Features of the nutrient-free culture method are further discussed in International Patent Publication WO 01/51616; and in Xu et al., Nat. Biotechnol. 19:971, 2001.

[190] When observed under a microscope, ES cells are characterized by a high ratio between the nucleus and cytoplasm and pronounced nucleoli, as well as the formation of compact colonies with weak intercellular contacts. Primate ES cells express stage-specific embryonic antigens (SSEA) 3 and 4, as well as markers detected by antibodies called Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Mouse ES cells can be used as a positive control for SSEA-1 and as a negative control for SSEA-4, Tra-1-60 and Tra-1-81. SSEA-4 is compulsorily present in human embryonic carcinoma (hEC) cells. Differentiation of pluripotent SCs in vitro leads to the cessation of expression of SSEA-4, Tra-1-60 and Tra-1-81 and an increase in the expression of SSEA-1, which can also be found in undifferentiated hEG cells.

[191] Stem cell-derived β-cell (SC-β)

[192] In some aspects, the invention provides a stem cell-derived β-cell (SC-β). The SC-β cells disclosed herein share many of the hallmarks of β cells but differ from them in some aspects (eg, gene expression profiles). In some embodiments, the SC-β cell is not native. In the context of this document, "non-native" means that the SC-β cells are significantly different in some respects from naturally occurring β-cells, ie. native β-cells. It should be understood, however, that these significant differences typically relate to structural features that can result in SC-β cells exhibiting certain functional differences, for example, when gene expression levels of SC-β cells differ from native β- cells, SC-β cells behave in a manner similar to native β-cells, but some functions can be changed (eg improved) compared to native β-cells. For example, as shown in FIG. 2E, the response of SC-β cells to 20 mM glucose occurs at a higher frequency than that exhibited by native β cells. Other differences between SC-β cells and native β cells will become apparent to those skilled in the art based on the data disclosed herein.

[193] The SC-β cells of the invention have many characteristics of β-cells that are important for the normal functioning of β-cells. In some embodiments, the SC-β cell exhibits a glucose-stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the SC-β cell exhibits a GSIS response in vivo. In some embodiments, the SC-β cell demonstrates in vitro and in vivo GSIS responses. In some embodiments, the GSIS response resembles the GSIS response of an endogenous mature pancreatic β cell. In some embodiments, the SC-β cell demonstrates a GSIS response to at least one glucose stimulation. In some embodiments, the SC-β cell demonstrates a GSIS response to at least two consecutive glucose stimulations. In some embodiments, the SC-β cell demonstrates a GSIS response to at least three consecutive glucose stimulations. In some embodiments, the GSIS response resembles the GSIS response of endogenous human islets to repeated glucose stimulation. In some embodiments of the invention, the GSIS response is observed immediately after transplantation of the cell into the human or animal body. In some embodiments of the invention, a GSIS response is observed within approximately 24 hours after transplantation of the cell into a human or animal body. In some embodiments of the invention, a GSIS response is observed within approximately one week after transplantation of the cell into a human or animal body. In some embodiments of the invention, the GSIS response is observed within approximately two weeks after transplantation of the cell into the human or animal body. In some embodiments of the invention, the stimulation index, characterized by the ratio of insulin secreted in response to high glucose concentrations and low glucose concentrations, is similar to the stimulation index of an endogenous mature pancreatic β-cell. In some embodiments, the SC-β cell exhibits a stimulation index greater than 1. In some embodiments, the SC-β cell exhibits a stimulation index greater than or equal to 1. In some embodiments, the SC-β cell exhibits a stimulation index greater than 1, one. In some embodiments, the SC-β cell exhibits a stimulation index greater than or equal to 1.1. In some embodiments, the SC-β cell exhibits a stimulation index greater than 2. In some embodiments, the SC-β cell exhibits a stimulation index greater than or equal to 2. In some embodiments, the SC-β cell exhibits a stimulation index of at least least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3 .3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 , 4.6, 4.7, 4.8, 4.9 or 5.0 or more.

[194] In some embodiments, the SC-β cell exhibits cytokine-induced apoptosis in response to cytokines. In some embodiments, the SC-β cell exhibits cytokine-induced apoptosis in response to a cytokine selected from the group consisting of interleukin-1β (IL-β), interferon-γ (IFN-γ), tumor necrosis factor α (TNF- α) and their combinations.

[195] In some embodiments, insulin secretion from the SC-β cell is increased in response to known antidiabetic drugs (e.g., antidiabetic drugs that act on β cells ex vivo or in vitro, and/or antidiabetic drugs in general). case in vivo). The present invention provides for any anti-diabetic drug. In some embodiments of the invention, insulin secretion from the SC-β cell is enhanced in response to a secretion stimulator. In some embodiments, the secretion stimulant is selected from the group consisting of an incretin mimetic, a sulfonylurea, meglitinide, and combinations thereof.

[196] In some embodiments, the SC-β cell is monohormonal. In some embodiments, the SC-β cell exhibits a morphology that resembles that of an endogenous mature pancreatic β cell. In some embodiments, the SC-β cell encapsulates crystalline insulin beads. In some embodiments of the invention, encapsulated crystalline insulin granules are observed in the SC-β cell under an electron microscope, which resemble the insulin granules of an endogenous mature pancreatic β-cell. In some embodiments, the SC-β cell exhibits a low replication rate. In some embodiments, the SC-β cell exhibits a low replication rate. In some embodiments, the SC-β cell exhibits a low but increased replication rate as measured by C-peptide and Ki67 staining in response to prolactin treatment.

[197] In some embodiments of the invention in the cell SC-β increases intracellular Ca ²⁺ in response to glucose. In some embodiments, the SC-β cell exhibits glucose-stimulated Ca ²⁺ flux (GSCF) that resembles the GSCF of an endogenous mature pancreatic β cell. In some embodiments, the SC-β cell exhibits a GSCF response to at least three consecutive glucose stimulations that resembles the GSCF response of an endogenous mature pancreatic β cell to multiple glucose stimulations.

[198] In some embodiments, the SC-β cell expresses at least one endogenous mature pancreatic β-cell marker selected from the group consisting of insulin, C-peptide, PDX1, MAFA, NKX6-1, PAX6, NEUROD1, glucokinases (GCK), SLC2A1, PCSK1, KCNJ11, ABCC8, SLC30A8, SNAP25, RAB3A, GAD2 and PTPRN.

[199] In some embodiments, the SC-β cell does not express at least one marker (e.g., a marker not expressed by endogenous mature pancreatic β-cells) selected from the group consisting of a) a hormone selected from the group consisting of i) glucagon (GCG); and ii) somatostatin (SST); b) acinar cell marker selected from the group consisting of i) amylase and ii) carboxypeptidase A (CPA1), c) α-cell marker selected from the group consisting of i) GCG, Arx, Irx1 and Irx2, d) marker ductal cells selected from the group consisting of i) CFTR and ii) Sox9.

[200] SC-β cells are differentiated in vitro from any starter cell, as the invention is not limited to the type of starter cell from which the SC-β cells are derived. Exemplary starter cells include, without limitation, insulin positive endocrine cells or any progenitors thereof such as Nkx6-1 positive pancreatic progenitor cell, Pdx1 positive pancreatic progenitor cell and pluripotent stem cell, embryonic stem cell and induced pluripotent stem cell. . In some embodiments, SC-β cells are differentiated in vitro from a reprogrammed cell, a partially reprogrammed cell (i.e., a somatic cell, e.g., a fibroblast, that has been partially reprogrammed to exist in an intermediate state between an induced pluripotent cell and a somatic cell, from which it was derived) of the transdifferentiated cell. In some embodiments, the SC-β cells disclosed herein can be differentiated in vitro from an insulin positive endocrine cell or a precursor thereof. In some embodiments, the SC-β cell is differentiated in vitro from a progenitor cell selected from the group consisting of an Nkx6-1 positive pancreatic progenitor cell, a Pdx1 positive pancreatic progenitor cell, and a pluripotent stem cell. In some embodiments, the pluripotent stem cell is selected from the group consisting of an embryonic stem cell and an induced pluripotent stem cell. In some embodiments, the SC-β cell or the pluripotent stem cell from which the SC-β cell is derived is human. In some embodiments, the SC-β cell is human.

[201] In some embodiments, the SC-β cell is not genetically modified. In some embodiments of the invention, in the absence of genetic modification of cells, the SC-β cell shares features with native β-cells. In some embodiments of the invention, the SC-β cell is genetically modified.

[202] In some embodiments, the amount of insulin produced per SC-β cell is at least 0.5 μIU per 1000 cells per 30 minutes of incubation (eg, ex vivo) at a high glucose concentration.

[203] In some embodiments, the amount of insulin produced per SC-β cell is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 μIU per 1000 cells per 30 minutes of high glucose incubation. In some embodiments, the amount of insulin produced per SC-β cell is 0.5-10 μIU per 1000 cells per 30 minutes of high glucose incubation. In some embodiments, the amount of insulin produced per SC-β cell is approximately 2.5 μIU per 1000 cells per 30 minutes of high glucose incubation.

[204] In some aspects, the invention provides a cell line containing the SC-β cell described herein. In some embodiments, the SC-β cells stably express insulin. In some embodiments of the invention, the SC-β cell can be frozen, thawed and amplified with a doubling time of about 24-44 hours without significant morphological changes for at least 30 passages.

[205] Generation of SC-β cells

[206] Aspects of the invention relate to the generation of SC-β cells (eg, pancreatic β-cells). In general, at least one SC-β cell or progenitor thereof, e.g., pancreatic progenitor cells, obtained in accordance with the methods disclosed herein, may contain a mixture or combination of different cells, for example, a mixture of cells, such as Pdx1- positive pancreatic progenitor cells, pancreatic progenitor cells co-expressing Pdx1 and NKX6-1, Ngn3-positive endocrine progenitor cell, insulin-positive endocrine cell (e.g., β-like cell), and insulin-positive endocrine cell, and/or other pluripotent or stem cells.

[207] At least one SC-β cell or its precursor can be obtained in accordance with any suitable culture protocol for differentiating a stem cell or pluripotent cell to the desired stage of differentiation. In some embodiments of the invention, at least one SC-β cell or its precursor is obtained by culturing at least one pluripotent cell for a period of time and under conditions suitable for at least one pluripotent cell to differentiate into at least one an SC-β cell or its precursor.

[208] In some embodiments, the at least one SC-β cell or progenitor thereof belongs to a substantially purified population of SC-β cells or progenitors thereof. In some embodiments of the invention, the population of SC-β cells or their progenitors contains a mixture of pluripotent cells or differentiated cells. In some embodiments of the invention, the population of SC-β cells or their progenitors contains little or no embryonic stem cells or pluripotent cells or iPS cells.

[209] In some embodiments, a somatic cell, such as a fibroblast, can be isolated from a subject's body, such as in the form of a tissue biopsy, such as, for example, a skin biopsy, and reprogrammed into an induced pluripotent stem cell for further differentiation to obtain at least one SC-β cell or progenitor thereof for use in the compositions and methods described herein. In some embodiments, the somatic cell, such as a fibroblast, is maintained in culture by methods known to one of skill in the art, and in some embodiments, is propagated prior to transformation into SC-β cells by the methods disclosed herein.

[210] In some embodiments, at least one SC-β cell or its progenitor is maintained in culture by methods known to one of skill in the art, and in some embodiments, propagated prior to conversion to SC-β cells by the methods disclosed herein.

[211] In addition, at least one SC-β cell or its precursor, for example, a pancreatic progenitor cell, may be from any mammalian species, and non-limiting examples of such cells include murine, bovine, simian, porcine, equine, ovine, or human. cell. For convenience and simplification, the description of the methods provided herein refers to at least one mammalian SC-β cell or progenitor thereof, but it should be understood that all methods described herein can readily be applied to other cell types to which at least one SC-β cell or its precursor belongs to. In some embodiments of the invention, at least one SC-β cell or its precursor is derived from a human.

[212] Induction of differentiation of pluripotent stem cells into definitive endoderm cells

[213] Included in aspects of the invention are cells of the definitive endoderm. The definitive endoderm cells used herein can be obtained from any source or generated according to any suitable protocol. In some aspects, pluripotent stem cells, such as iPSCs or hESCs, are differentiated into endoderm cells. In some aspects, endoderm cells are further differentiated, for example, into subembryonic cavity cells, Pdx1-positive pancreatic progenitors, NKX6-1-positive pancreatic progenitors, Ngn3-positive endocrine progenitors, or insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[214] In some embodiments, the stem cells can be seeded on a new substrate, or the medium can be changed to remove extracellular matrix or soluble factors that inhibit differentiation. This is sometimes referred to as the "direct differentiation method", which is broadly described in International Patent Publication WO 01/51616 and US Patent Publication 2002/0019046, which are incorporated herein by reference in their entirety. In a direct differentiation method, it is generally preferable to start with a nutrient-free stem cell culture to avoid potential complications in the differentiation process that can be caused by residual nurse cells. Another approach is to place undifferentiated stem cells in suspension culture, which will result in continuous aggregate formation of differentiated and undifferentiated cells. For example, stem cells can be harvested by short exposure to collagenase, clustered, and passaged in culture plates coated with non-adherent cells. Aggregates can be fed every few days and then harvested after an appropriate period of time, typically 4-8 days. Depending on the conditions, the aggregates generally begin to form a heterogeneous population of different types of cells, including fairly common endoderm cells. The aggregates can then be dispersed and plated on substrates such as laminin or fibronectin for the next step in the differentiation process; or passage in suspension culture using, for example, non-adherent plates and a suitable medium.

[215] During direct differentiation or differentiation in aggregates, the presence of endoderm cells can be monitored using suitable markers such as those described in US Pat. No. 7,326,572. In some preferred embodiments, the presence of endoderm cells can be monitored during differentiation using markers such as Sox17. Once a sufficient proportion of endoderm cells has been obtained, the cells can be subcultured or otherwise manipulated to initiate another stage of differentiation. Under certain conditions, cell differentiation or maintenance can be enhanced by having cells in microclusters (eg, 50 to 5000 cells). Additional differentiation steps contemplated by the invention are shown in FIG. one.

[216] In some embodiments, definitive endoderm cells are obtained by contacting (e.g., culturing) a pluripotent stem cell with a compound of formula (I) as described in US Pat. this document by reference. The compound having formula (I) as described in the 274th patent is a small molecule capable of entering the cell and regulating cellular processes by modulating signal transduction, gene expression or metabolism pathways, and is effectively used in stem cell differentiation protocols. Small molecules can be synthesized in large quantities and with a high degree of purity, and they are also conveniently delivered or removed, which provides them with great potential in therapeutic applications. A huge amount of research has been done to identify new small molecules that can support self-renewal of ES cells (Chen et al., 2006; Desbordes et al., 2008), mouse ES cells with a cardiogenic specification (Wu et al., 2004), or neural cells α-progenitors (Diamandis et al., 2007), as well as inducing certain cell types, in particular neuronal and muscle cells (reviewed by Ding and Schultz, 2004). It is expected that the compounds of formula (I) of the 274th patent can be used to differentiate a pluripotent stem cell into a definitive endoderm cell.

[217] In some embodiments of the invention, the compound of formula (I) of the 274th patent contains:

[218]

[219]

[220] where:

[221] R ¹ and R ² are independently H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, or cyclyl, each of which may be optionally substituted and/or one or more of O, N, S, S(O) and C(O);

[222] R ³ and R ⁴ are independently H, halogen, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cyclyl, or cyclyl, each of which may be optionally substituted, or R ³ and R ⁴ together with a carbon atom, to which they are attached form an optionally substituted cyclyl or heterocyclyl; and

[223] L is C ₁ -C ₁₀ alkylenyl, C ₂ -C ₁₀ alkenylene, or C ₂ -C ₁₀ alkynylene, each of which may be optionally substituted and/or one or more of O, N, S, S(O) and C(O).

[224] In some embodiments of the invention, the compound of formula (I) of the 274th patent contains the following IDE1:

[225]

[226] In some embodiments of the invention, the compound of formula (I) of the 274th patent contains the following IDE2:

[227]

[228] The 274th patent describes methods for verifying the identity of the definitive endoderm cell thus obtained, as well as methods for isolating, storing, propagating and further differentiating the definitive endoderm, which, as it is clear to a person skilled in the art, can be used in the described in this document compositions and methods.

[229] In some embodiments, definitive endoderm cells can be obtained by differentiating at least some of the pluripotent cells in a population into definitive endoderm cells, for example, by contacting a population of pluripotent cells with i) at least one growth factor from the TGF- β and ii) a WNT signaling pathway activator to induce differentiation of at least some pluripotent cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of the definitive endoderm.

[230] The invention contemplates the use of any growth factor from the TGF-β superfamily that induces differentiation of pluripotent stem cells into definitive endoderm cells (eg, alone or in combination with a WNT signaling pathway activator). In some embodiments, at least one growth factor from the TGF-β superfamily comprises activin A. In some embodiments, at least one growth factor from the TGF-β superfamily comprises growth and differentiation factor 8 (GDF8).

[231] The invention contemplates the use of any WNT signaling activator that induces differentiation of pluripotent stem cells into definitive endoderm cells (eg, alone or in combination with a growth factor from the TGF-β superfamily). In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator comprises a recombinant Wnt3a protein.

[232] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) employed may vary. In some embodiments, the pluripotent cells are contacted with at least one growth factor from the TGF-β superfamily at a concentration of 10 ng/mL - 1000 ng/mL. In some embodiments, the pluripotent cells are contacted with at least one growth factor from the TGF-β superfamily at a concentration of 100 ng/mL. In some embodiments, the pluripotent cells are contacted with at least one growth factor from the TGF-β superfamily at a concentration of 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/ml, 80 ng/ml or 90 ng/ml. In some embodiments, the pluripotent cells are contacted with at least one growth factor from the TGF-β superfamily at a concentration of 91 ng/mL, 92 ng/mL, 93 ng/mL, 94 ng/mL, 95 ng/mL, 96 ng/ml, 97 ng/ml, 98 ng/ml or 99 ng/ml. In some embodiments, the pluripotent cells are contacted with at least one growth factor from the TGF-β superfamily at a concentration of 110 ng/mL, 120 ng/mL, 130 ng/mL, 140 ng/mL, 150 ng/mL, 160 ng/ml, 170 ng/ml, 180 ng/ml or 190 ng/ml. In some embodiments, the pluripotent cells are contacted with at least one growth factor from the TGF-β superfamily at a concentration of 101 ng/mL, 102 ng/mL, 103 ng/mL, 104 ng/mL, 105 ng/mL, 106 ng/ml, 107 ng/ml, 108 ng/ml or 109 ng/ml.

[233] In some embodiments, the pluripotent cells are contacted with a WNT signaling pathway activator at a concentration of 1.4 μg/mL - 140 μg/mL. In some embodiments, the pluripotent cells are contacted with a WNT signaling pathway activator at a concentration of 14 μg/ml. In some embodiments of the invention, pluripotent cells are brought into contact with a WNT signaling pathway activator at a concentration of 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml. ml, 9 µg/ml, 10 µg/ml, 11 µg/ml, 12 µg/ml or 13 µg/ml. In some embodiments of the invention, pluripotent cells are brought into contact with a WNT signaling pathway activator at a concentration of 15 μg/ml, 16 μg/ml, 17 μg/ml, 18 μg/ml, 19 μg/ml, 20 μg/ml, 21 μg/ml. ml, 22 µg/ml, 23 µg/ml, 24 µg/ml, 25 µg/ml, 26 µg/ml, 27 µg/ml, 28 µg/ml, 29 µg/ml or 30 µg/ml. In some embodiments of the invention, pluripotent cells are brought into contact with a WNT signaling pathway activator at a concentration of 13.1 μg/ml, 13.2 μg/ml, 13.3 μg/ml, 13.4 μg/ml, 13.5 μg/ ml, 13.6 µg/ml, 13.7 µg/ml, 13.8 µg/ml or 13.9 µg/ml. In some embodiments of the invention, pluripotent cells are brought into contact with a WNT signaling pathway activator at a concentration of 14.1 μg/ml, 14.2 μg/ml, 14.3 μg/ml, 14.4 μg/ml, 14.5 μg/ml. ml, 14.6 µg/ml, 14.7 µg/ml, 14.8 µg/ml or 14.9 µg/ml.

[234] In general, the pluripotent cells are maintained in a suitable culture medium (eg, in suspension culture) for a period of time sufficient to induce differentiation of at least some of the pluripotent cells into definitive endoderm cells. A typical suitable culture medium is shown in Table 1 below.

[235]

[236] In some embodiments, a suitable culture medium for differentiating pluripotent cells into definitive endoderm cells includes S1 medium.

[237] In some embodiments of the invention, bringing into contact pluripotent cells is carried out in suspension culture. In some embodiments of the invention, the suspension culture is maintained in a roller bottle. In some embodiments of the invention, the time period is 3 days. In some embodiments, at least one growth factor from the TGF-β superfamily and a WNT signaling pathway activator are added to the suspension culture on the first day. In some embodiments of the invention, at least one growth factor from the TGF-β superfamily is replenished in the suspension culture on the second day. In some embodiments of the invention, the WNT signaling pathway activator is not replenished in the suspension culture on the second day. In some embodiments, the WNT signaling pathway activator is removed from the suspension culture on the second day. In some embodiments, at least one growth factor from the TGF-β superfamily is replenished in the suspension culture on the second day and the WNT signaling pathway activator is removed or not replenished in the suspension culture on the second day. In some embodiments of the invention, neither at least one growth factor from the TGF-β superfamily nor the WNT signaling pathway activator is replenished in the suspension culture on the third day. In some embodiments of the invention, on the third day, both at least one growth factor from the TGF-β superfamily and the WNT signaling pathway activator are removed from the suspension culture.

[238] Using these methods, at least one pluripotent cell in a population of cells can be induced to differentiate into a definitive endoderm cell. In general, any pluripotent cell can be differentiated into a definitive endoderm cell using the method described herein. In some embodiments, the pluripotent cells include induced pluripotent cells. In some embodiments, the pluripotent cells include embryonic stem cells. In some embodiments, the pluripotent cells include human cells.

[239] In some embodiments, differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells is achieved by contacting the population of pluripotent cells with i) Activin A and ii) CHIR99021 to induce differentiation of at least some of the pluripotent cells in the population. into cells of the definitive endoderm, wherein the cells of the definitive endoderm express at least one marker characteristic of the definitive endoderm.

, et al., US 2005/0158853 авторства K.

, et al. и US 2005/0260749 авторства Jon Odorico, et al., соответствующие разделы которых включены в данный документ посредством ссылки.[240] Other methods for producing definitive endoderm cells are known in the art, including, for example, those described in United States patent application US 2006/0003446 by G. Keller, et al.; US 2006/0003313 by K.

, et al., US 2005/0158853 by K.

et al. and US 2005/0260749 by Jon Odorico, et al., the relevant sections of which are incorporated herein by reference.

[241] In some embodiments, the definitive endoderm cell obtained by the methods described herein expresses at least one marker selected from the group consisting of: Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3gl2, Ripk4, Rab15, Npnt , Clic6, Cldn8, Cacna1b, Bnipl, Anxa4, Emb, FoxA1, Sox17 and Rbm35a, while in the cell of the definitive endoderm there is an increased expression of at least one marker by a statistically significant amount compared to the pluripotent stem cell from which it was obtained. In some embodiments of the invention, the definitive endoderm cell obtained by the methods described herein does not express in a statistically significant amount at least one marker selected from the group consisting of: Gata4, SPARC, AFP and Dab2, compared to a pluripotent stem cell, from which it was derived. In some embodiments of the invention, the definitive endoderm cell obtained by the methods described herein does not express in a statistically significant amount at least one marker selected from the group consisting of: Zicl, Pax6, Flk1 and CD31, compared with a pluripotent stem cell, from which it was derived.

[242] In some embodiments, the definitive endoderm cell obtained by the methods described herein has a statistically significant higher level of Smad2 phosphorylation compared to the pluripotent stem cell from which it was obtained. In some embodiments of the invention, the definitive endoderm cell obtained by the methods described herein has the ability to form an intestinal tube in vivo. In some embodiments, a definitive endoderm cell produced by the methods described herein can differentiate into a cell with a gut cell morphology, wherein the cell with a gut cell morphology expresses FoxA2 and/or Claudin6. In some embodiments, a definitive endoderm cell produced by the methods described herein can further differentiate into a cell of endoderm origin.

[243] In some embodiments, a population of pluripotent stem cells is cultured in the presence of at least one β-cell maturation factor prior to any differentiation or in the first stage of differentiation. Any pluripotent stem cell, such as a human pluripotent stem cell or a human iPS cell, or any pluripotent stem cell or other suitable pluripotent stem cells discussed herein may be used. In some embodiments, the β-cell maturation factor described herein may be present in the culture medium of the pluripotent stem cell population, or it may be added repeatedly or intermittently during growth (eg, replication or expansion) of the pluripotent stem cell population. In certain instances, a population of pluripotent stem cells may be exposed to at least one β-cell maturation factor prior to any differentiation. In other examples, a population of pluripotent stem cells can be exposed to at least one β-cell maturation factor in the first stage of differentiation.

[244] Induction of differentiation of definitive endoderm cells into cells of the primary intestinal tube

[245] Aspects of the invention include cells of the primary intestinal tube. Used in this document, the cells of the primary intestinal tube can be obtained from any source or generated in accordance with any suitable protocol. In some aspects, the cells of the definitive endoderm differentiate into cells of the primary intestinal tube. In some aspects, primary intestinal tube cells further differentiate, for example, into Pdx1-positive pancreatic progenitors, NKX6-1-positive pancreatic progenitors, Ngn3-positive endocrine progenitors, insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[246] In some embodiments, primary intestinal tube cells can be obtained by differentiating at least some of the definitive endoderm cells in a population into primary intestinal tube cells, for example, by contacting the definitive endoderm cells with at least one growth factor from the family of factors fibroblast growth (FGF) to induce differentiation of at least some cells of the definitive endoderm into cells of the primary intestinal tube, wherein the cells of the primary intestinal tube express at least one marker characteristic of cells of the primary intestinal tube.

[247] The invention contemplates the use of any growth factor from the FGF family that induces differentiation of definitive endoderm cells into primary intestinal tube cells (eg, alone or in combination with other factors). In some embodiments of the invention, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments of the invention, at least one growth factor from the FGF family includes FGF2. In some embodiments of the invention, at least one growth factor from the FGF family includes FGF8B. In some embodiments of the invention, at least one growth factor from the FGF family includes FGF10. In some embodiments of the invention, at least one growth factor from the FGF family includes FGF21.

[248] One skilled in the art will appreciate that the concentrations of growth factors used may vary. In some embodiments of the invention, cells of the definitive endoderm are contacted with at least one growth factor from the FGF family at a concentration of 5 ng/ml -500 ng/ml. In some embodiments of the invention, cells of the definitive endoderm are contacted with at least one growth factor from the FGF family at a concentration of 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng /ml or 40 ng/ml. In some embodiments of the invention, cells of the definitive endoderm are contacted with at least one growth factor from the FGF family at a concentration of 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng /ml, 90ng/ml, 95ng/ml or 100ng/ml. In some embodiments of the invention, cells of the definitive endoderm are contacted with at least one growth factor from the FGF family at a concentration of 41 ng/ml, 42 ng/ml, 43 ng/ml, 44 ng/ml, 45 ng/ml, 46 ng /ml, 47ng/ml, 48ng/ml or 49ng/ml. In some embodiments of the invention, cells of the definitive endoderm are contacted with at least one growth factor from the FGF family at a concentration of 51 ng/ml, 52 ng/ml, 53 ng/ml, 54 ng/ml, 55 ng/ml, 56 ng /ml, 57ng/ml, 58ng/ml or 59ng/ml. In some embodiments, cells of the definitive endoderm are contacted with at least one growth factor from the FGF family at a concentration of 50 ng/mL.

[249] In some embodiments, the definitive endoderm cells are cultured in a suitable culture medium.

[250] In general, definitive endoderm cells are maintained in a suitable culture medium (eg, suspension culture) for a period of time sufficient to induce differentiation of at least some of the definitive endoderm cells into primary intestinal tube cells. A typical suitable culture medium is shown in Table 2 below.

[251]

[252] In some embodiments, a suitable culture medium for differentiating definitive endoderm cells into primary intestinal tube cells includes S2 medium.

[253] In some embodiments of the invention, bringing into contact cells of the definitive endoderm is carried out in suspension culture. In some embodiments of the invention, the suspension culture is maintained in a roller bottle. In some embodiments of the invention, the time period is from 2 days to 5 days. In some embodiments of the invention, the time period is 3 days. In some embodiments, the suspension culture is replenished every other day.

[254] In some embodiments, definitive endoderm cells can be obtained by differentiating at least some of the definitive endoderm cells in a population into primary intestinal tube cells, for example, by contacting definitive endoderm cells with KGF to induce differentiation of at least some of the cells. of the definitive endoderm into cells of the primary intestinal tube, wherein the cells of the primary intestinal tube express at least one marker characteristic of the definitive endoderm.

[255] Inducing differentiation of primary intestinal tube cells into Pdx1-positive pancreatic progenitor cells

[256] Aspects of the invention include Pdx1 positive pancreatic progenitor cells. Used in this document Pdx1-positive pancreatic progenitor cells can be obtained from any source or generated in accordance with any suitable protocol. In some aspects, the cells of the primary intestinal tube differentiate into Pdx1-positive pancreatic progenitor cells. In some aspects, Pdx1 positive pancreatic progenitors further differentiate, for example, into NKX6-1 positive pancreatic progenitors, Ngn3 positive endocrine progenitors, insulin positive endocrine cells, followed by induction or maturation into SC-β cells.

[257] In some aspects, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primary intestinal tube cells in a population into Pdx1-positive pancreatic progenitor cells, for example, by bringing primary intestinal tube cells into contact with i) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator; and v) at least one protein kinase C activator to induce differentiation of at least some cells of the primary intestinal tube into Pdx1-positive pancreatic progenitors, wherein the Pdx1-positive pancreatic progenitors express Pdx1.

[258] The invention contemplates the use of any inhibitor of the BMP signaling pathway that induces differentiation of primary intestinal tube cells into Pdx1-positive pancreatic progenitors (e.g., alone or in combination with at least one growth factor from the FGF family, at least one an SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator). In some embodiments, the BMP signaling inhibitor comprises LDN193189.

[259] The invention contemplates the use of any growth factor from the FGF family that induces differentiation of primary intestinal tube cells into Pdx1-positive pancreatic progenitors (e.g., alone or in combination with at least one inhibitor of the BMP signaling pathway, at least one an SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator). In some embodiments of the invention, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments of the invention, at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10 and FGF21.

[260] The invention contemplates the use of any SHH pathway inhibitor that induces differentiation of primary intestinal tube cells into Pdx1-positive pancreatic progenitors (e.g., alone or in combination with at least one inhibitor of the BMP signaling pathway, at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator). In some embodiments, the SHH pathway inhibitor comprises Sant1.

[261] The invention contemplates the use of any RA signaling pathway activator that induces differentiation of primary intestinal tube cells into Pdx1-positive pancreatic progenitors (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth from the FGF family, at least one SHH pathway inhibitor, and at least one protein kinase C activator). In some embodiments, the RA signaling pathway activator comprises retinoic acid.

[262] The invention provides for the use of any protein kinase C (PKC) activator that induces differentiation of primary intestinal tube cells into Pdx1-positive pancreatic progenitors (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, and at least one retinoic acid signaling pathway activator). In some embodiments, the PKC activator includes PdbU. In some embodiments of the invention, the PKC activator includes TRV.

[263] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) employed may vary. In some embodiments of the invention, cells of the primary intestinal tube are brought into contact with an inhibitor of the BMP signaling pathway at a concentration of 20 nM - 2000 nM. In some embodiments, primary intestinal tube cells are contacted with a BMP signaling pathway inhibitor at a concentration of 30-40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM or 190 nM. In some embodiments, primary intestinal tube cells are contacted with a BMP signaling pathway inhibitor at a concentration of 191 nM, 192 nM, 193 nM, 194 nM, 195 nM, 196 nM, 197 nM, 198 nM, or 199 nM. In some embodiments, primary intestinal tube cells are contacted with a BMP signaling pathway inhibitor at a concentration of 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1100 nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM or 1900 nM. In some embodiments, primary intestinal tube cells are contacted with a BMP signaling pathway inhibitor at a concentration of 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280 nM, or 290 nM. In some embodiments, primary intestinal tube cells are contacted with a BMP signaling pathway inhibitor at a concentration of 200 nM.

[264] In some embodiments, primary intestinal tube cells are contacted with at least one growth factor from the FGF family at a concentration of 5 ng/mL - 500 ng/mL. In some embodiments, primary intestinal tube cells are contacted with at least one growth factor from the FGF family at a concentration of 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/ml or 40 ng/ml. In some embodiments, primary intestinal tube cells are contacted with at least one growth factor from the FGF family at a concentration of 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/ml, 90 ng/ml, 95 ng/ml or 100 ng/ml. In some embodiments, primary intestinal tube cells are contacted with at least one growth factor from the FGF family at a concentration of 41 ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46 ng/ml, 47 ng/ml, 48 ng/ml or 49 ng/ml. In some embodiments, primary intestinal tube cells are contacted with at least one growth factor from the FGF family at a concentration of 51 ng/mL, 52 ng/mL, 53 ng/mL, 54 ng/mL, 55 ng/mL, 56 ng/ml, 57 ng/ml, 58 ng/ml or 59 ng/ml. In some embodiments, primary intestinal tube cells are contacted with at least one growth factor from the FGF family at a concentration of 50 ng/mL.

[265] In some embodiments of the invention, cells of the primary intestinal tube are brought into contact with at least one inhibitor of the SHH signaling pathway at a concentration of 0.1 μm - 0.5 μm. In some embodiments, primary intestinal tube cells are contacted with at least one SHH signaling pathway inhibitor at a concentration of 0.11 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, 0. 16 µM, 0.17 µM, 0.18 µM, 0.19 µM, 0.2 µM, 0.21 µM, 0.22 µM, 0.23 µM or 0.24 µM. In some embodiments, primary intestinal tube cells are contacted with at least one SHH signaling pathway inhibitor at a concentration of 0.26 μM, 0.27 μM, 0.28 μM, 0.29 μM, 0.30 μM, 0. 31 µM, 0.32 µM, 0.33 µM, 0.34 µM, 0.35 µM, 0.36 µM, 0.37 µM, 0.38 µM, 0.39 µM, 0.40 µM, 0, 41 µM, 0.42 µM, 0.43 µM, 0.44 µM, 0.45 µM, 0.46 µM, 0.47 µM, 0.48 µM, 0.49 µM. In some embodiments, primary intestinal tube cells are contacted with at least one inhibitor of the SHH signaling pathway at a concentration of 0.25 μM.

[266] In some embodiments, primary intestinal tube cells are contacted with an RA signaling pathway activator at a concentration of 0.01 μM - 1.0 μM. In some embodiments, primary intestinal tube cells are contacted with RA signaling pathway activator at a concentration of 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0 .08 µM or 0.09 µM. In some embodiments, primary intestinal tube cells are contacted with an RA signaling pathway activator at a concentration of 0.20 μM, 0.30 μM, 0.40 μM, 0.05 μM, 0.60 μM, 0.70 μM, 0 .80 µM or 0.90 µM. In some embodiments, primary intestinal tube cells are contacted with an RA signaling pathway activator at a concentration of 0.1 μM.

[267] In some embodiments, primary intestinal tube cells are contacted with a PKC activator at a concentration of 50 nM - 5000 nM. In some embodiments, primary intestinal tube cells are contacted with a PKC activator at a concentration of 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 460 nM, 470 nM, 480 nM, or 490 nM. In some embodiments, primary intestinal tube cells are contacted with a PKC activator at a concentration of 491 nM, 492 nM, 493 nM, 494 nM, 495 nM, 496 nM, 497 nM, 498 nM, or 499 nM. In some embodiments, primary intestinal tube cells are contacted with a PKC activator at a concentration of 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1100 nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM or 2000 nM. In some embodiments, primary intestinal tube cells are contacted with a PKC activator at a concentration of 501 nM, 502 nM, 503 nM, 504 nM, 505 nM, 506 nM, 507 nM, 508 nM, or 509 nM, 510 nM, 520 nM , 530 nM, 540 nM, 550 nM, 560 nM, 570 nM, 580 nM or 590 nM. In some embodiments, primary intestinal tube cells are contacted with a PKC activator at a concentration of 500 nM.

[268] In general, primary intestinal tube cells are maintained in a suitable culture medium (eg, suspension culture) for a period of time sufficient to induce differentiation of at least some primary intestinal tube cells into Pdx1-positive pancreatic progenitor cells. A typical suitable culture medium is shown in Table 3 below.

[269]

[270] In some embodiments of the invention, S3 medium can be used as a suitable culture medium for differentiating primary intestinal tube cells into pancreatic progenitor cells.

[271] In some embodiments of the invention, bringing into contact the cells of the primary intestinal tube is carried out in suspension culture. In some embodiments of the invention, the suspension culture is maintained in a roller bottle. In some embodiments of the invention, the time period is at least 2 days. In some embodiments of the invention, the suspension culture is replenished every day.

[272] In some embodiments, primary intestinal tube cells can be obtained by differentiating at least some of the primary intestinal tube cells in a population into Pdx1-positive pancreatic progenitor cells, for example, by contacting primary intestinal tube cells with i) LDN193189, ii) KGF, iii) Sant1; iv) RA; and iv) PdbU to induce differentiation of at least some cells of the primary intestinal tube into Pdx1 positive pancreatic progenitors, wherein the Pdx1 positive pancreatic progenitors express Pdx1.

[273] Induction of differentiation of Pdx1-positive pancreatic progenitors into NKX6-1 + pancreatic progenitors

[274] Aspects of the invention include NKX6-1 positive pancreatic progenitor cells. The NKX6-1 positive pancreatic progenitors used herein can be obtained from any source or generated according to any suitable protocol. In some aspects, Pdx1 positive pancreatic progenitors differentiate into NKX6-1 positive pancreatic progenitors. In some aspects, NKX6-1 positive pancreatic progenitor cells further differentiate, for example, into Ngn3 positive endocrine progenitor cells or insulin positive endocrine cells, followed by induction or maturation into SC-β cells.

[275] In some aspects, a method for producing a NKX6-1 positive pancreatic progenitor cell from a Pdx1 positive pancreatic progenitor cell comprises bringing a cell population (e.g., under conditions that promote cell clustering) containing Pdx1 positive pancreatic progenitor cells into contact with at least two β-cell maturation factors, including a) at least one growth factor from the fibroblast growth factor (FGF) family, b) an inhibitor of the sonic hedgehog pathway, and optionally c) a low concentration of retinoic acid signaling pathway activator acid (RA) over a period of at least five days to induce differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into an NKX6-1-positive pancreatic progenitor cell, wherein the NKX6-1- positive pancreatic progenitor cells express NKX6-1.

[276] In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitors are produced by contacting Pdx1-positive pancreatic progenitors under conditions that promote cell clustering with i) at least one growth factor from the FGF family, ii) at least one inhibitor of the SHH pathway, and optionally iii) low concentrations of RA signaling pathway activator over a period of five days to induce differentiation of at least some Pdx1-positive pancreatic progenitor cells into Pdx1 positive, NKX6-1 positive pancreatic progenitors, while Pdx1 positive, NKX6-1 positive pancreatic progenitors express Pdx1 and NKX6-1.

[277] In some embodiments, Pdx1 positive, NKX6-1 positive pancreatic progenitors are obtained by contacting Pdx1 positive pancreatic progenitors with i) at least one growth factor from the FGF family, ii) at least at least one SHH pathway inhibitor; and iii) an RA signaling pathway activator to induce differentiation of at least some Pdx1-positive pancreatic progenitors into Pdx1-positive, NKX6-1-positive pancreatic progenitors, wherein Pdx1-positive, NKX6- 1-positive pancreatic progenitors express Pdx1 HNKX6-1.

[278] In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are obtained from a population of pluripotent cells. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are obtained from a population of iPS cells. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are obtained from a population of ESC cells. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are obtained from a population of definitive endoderm cells. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are obtained from a population of cells of the primary intestinal tube.

[279] The invention provides for the use of any growth factor from the FGF family that induces the differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitors (e.g., alone or in combination with at least one SHH pathway inhibitor or optionally at least one retinoic acid signaling pathway activator). In some embodiments of the invention, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments of the invention, at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10 and FGF21.

[280] The invention contemplates the use of any inhibitor of the SHH pathway that induces differentiation of Pdx1-positive pancreatic progenitors into NKX6-1-positive pancreatic progenitors (e.g., alone or in combination with at least one growth factor from the FGF family or at least one retinoic acid signaling pathway activator). In some embodiments, the SHH pathway inhibitor comprises Sant1.

[281] The invention contemplates the use of any RA signaling pathway activator that induces differentiation of Pdx1-positive pancreatic progenitors into NKX6-1-positive pancreatic progenitors (e.g., alone or in combination with at least one growth factor from the FGF family or at least one SHH pathway inhibitor). In some embodiments, the RA signaling pathway activator comprises retinoic acid.

[282] In some embodiments of the invention, this method includes bringing a population of cells (eg, Pdx1-positive pancreatic progenitor cells) into contact with at least one additional maturation factor β-cells. In some embodiments of the invention, at least one additional β-cell maturation factor includes at least one growth factor from the EGF family. In some embodiments of the invention, this method includes bringing Pdx1-positive pancreatic progenitor cells into contact with at least one growth factor from the EGF family. The invention provides for the use of any growth factor from the EGF family that facilitates the differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitors (for example, together with any combination of at least one growth factor from the FGF family, according to at least one SHH pathway inhibitor and optionally at least one RA signaling pathway activator). In some embodiments of the invention, at least one growth factor from the EGF family includes betacellulin. In some embodiments of the invention, at least one growth factor from the EGF family includes EGF.

[283] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) employed may vary. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one growth factor from the FGF family at a concentration of 1 ng/mL - 100 ng/mL. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are brought into contact with at least one growth factor from the FGF family at a concentration of 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml. ml, 30 ng/ml, 35 ng/ml or 40 ng/ml. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are brought into contact with at least one growth factor from the FGF family at a concentration of 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml. ml, 85 ng/ml, 90 ng/ml, 95 ng/ml or 100 ng/ml. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are brought into contact with at least one growth factor from the FGF family at a concentration of 41 ng/ml, 42 ng/ml, 43 ng/ml, 44 ng/ml, 45 ng/ml. ml, 46 ng/ml, 47 ng/ml, 48 ng/ml or 49 ng/ml. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are brought into contact with at least one growth factor from the FGF family at a concentration of 51 ng/ml, 52 ng/ml, 53 ng/ml, 54 ng/ml, 55 ng/ml. ml, 56 ng/ml, 57 ng/ml, 58 ng/ml or 59 ng/ml. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one growth factor from the FGF family at a concentration of 50 ng/mL.

[284] In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one inhibitor of the SHH pathway at a concentration of 0.1 μM - 0.5 μM. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are contacted with at least one inhibitor of the SHH pathway at a concentration of 0.11 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, 0.16 µM, 0.17 µM, 0.18 µM, 0.19 µM, 0.2 µM, 0.21 µM, 0.22 µM, 0.23 µM or 0.24 µM. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.26 μM, 0.27 μM, 0.28 μM, 0.29 μM, 0.30 μM, 0.31 µM, 0.32 µM, 0.33 µM, 0.34 µM, 0.35 µM, 0.36 µM, 0.37 µM, 0.38 µM, 0.39 µM, 0.40 µM, 0.41 µM, 0.42 µM, 0.43 µM, 0.44 µM, 0.45 µM, 0.46 µM, 0.47 µM, 0.48 µM, 0.49 µM. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.25 μM.

[285] In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are contacted with an activator of the HA signaling pathway at a concentration of 0.01 μM - 1.0 μM. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 µM, 0.08 µM or 0.09 µM. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.20 μM, 0.30 μM, 0.40 μM, 0.05 μM, 0.60 μM, 0.70 µM, 0.80 µM or 0.90 µM. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.1 μM.

[286] In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 2 ng/mL - 200 ng/mL. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are brought into contact with at least one growth factor from the EGF family at a concentration of 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml. ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 16 ng/ml, 17 ng/ml, 18 ng/ml ml or 19 ng/ml. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are brought into contact with at least one growth factor from the EGF family at a concentration of 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml. ml, 55 ng/ml, 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml or 100 ng/ml ml. In some embodiments of the invention, Pdx1-positive pancreatic progenitor cells are brought into contact with at least one growth factor from the EGF family at a concentration of 21 ng/ml, 22 ng/ml, 23 ng/ml, 24 ng/ml, 25 ng/ml. ml, 26 ng/ml, 27 ng/ml, 28 ng/ml or 29 ng/ml. In some embodiments, Pdx1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 20 ng/mL.

[287] In general, Pdx1-positive pancreatic progenitors are maintained in a suitable culture medium for a period of time sufficient to induce differentiation of at least some of the Pdx1-positive pancreatic progenitors in the population into Pdx1-positive, NKX6-1- positive pancreatic progenitor cells. An exemplary suitable culture medium is shown in Table 3 above. In some embodiments, conditions that promote cell clustering include suspension culture. In some embodiments of the invention, the suspension culture is maintained in a roller bottle. In some embodiments of the invention, the time period is at least 5 days. In some embodiments, the suspension culture is replenished every other day. In some embodiments of the invention, β-cell maturation factors are replenished every other day.

[288] In some embodiments, no protein kinase C activator is added to the suspension culture for 5 days. In some embodiments, the protein kinase C activator is removed from the suspension culture before the 5 day period has elapsed. In some embodiments, the protein kinase C activator comprises PdbU. In some embodiments of the invention, no inhibitor of the BMP signaling pathway is added to the suspension culture for 5 days. In some embodiments, the BMP signaling inhibitor is removed from the suspension culture before the 5 day period has elapsed. In some embodiments, the BMP signaling inhibitor comprises LDN193189.

[289] In some embodiments, at least 10% of the Pdx1-positive pancreatic progenitors in a population are induced to differentiate into Pdx1-positive, NKX6-1-positive pancreatic progenitors. In some embodiments, at least 95% of the Pdx1 positive pancreatic progenitors are induced to differentiate into Pdx1 positive, NKX6-1 positive pancreatic progenitors.

[290] In general, any Pdx1 positive pancreatic progenitor cell can differentiate into a Pdx1 positive, NKX6-1 positive pancreatic progenitor cell. In some embodiments, the NKX6-1 positive pancreatic progenitors express Pdx1, NKX6-1 and/or FoxA2.

[291] In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitors are produced by contacting Pdx1-positive pancreatic progenitors under conditions that promote cell clustering with i) at least one growth factor from the FGF family, ii) at least one inhibitor of the SHH pathway, and optionally iii) low concentrations of an RA signaling pathway activator over a period of five days to induce differentiation of at least some Pdx1-positive pancreatic progenitor cells into Pdx1 positive, NKX6-1 positive pancreatic progenitors, while Pdx1 positive, NKX6-1 positive pancreatic progenitors express Pdx1 and NKX6-1.

[292] In some embodiments, NKX6-1 positive pancreatic progenitors can be obtained by differentiating at least some of the Pdx1-positive pancreatic progenitors into Pdx1-positive, NKX6-1-positive pancreatic progenitors by bringing Pdx1- positive pancreatic progenitor cells under conditions that promote cell clustering, in contact with i) at least one growth factor from the FGF family, ii) at least one inhibitor of the SHH pathway, and optionally iii) an RA signaling pathway activator every other day over a period of five days to induce differentiation of at least some of the Pdx1-positive pancreatic progenitors in the population into NKX6-1-positive pancreatic progenitors, wherein the NKX6-1-positive pancreatic progenitors express Pdx1 and NKX6 -one.

[293] In some embodiments, NKX6-1 positive pancreatic progenitors can be obtained by differentiating at least some of the Pdx1-positive pancreatic progenitors in a population into Pdx1-positive, NKX6-1 positive pancreatic progenitors, e.g. , by bringing Pdx1-positive pancreatic progenitor cells into contact with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator to induce differentiation of at least least some Pdx1-positive pancreatic progenitors in the population into NKX6-1-positive pancreatic progenitors, while NKX6-1-positive pancreatic progenitors express Pdx1 and NKX6-1.

[294] Induction of differentiation of NKX6-1+ pancreatic insulin progenitor cells + endocrine cells

[295] Aspects of the invention include insulin-positive endocrine cells. As used herein, the insulin-positive endocrine cells may be obtained from any source or generated according to any suitable protocol. In some aspects, NKX6-1 positive pancreatic progenitor cells differentiate into insulin positive endocrine cells. In some aspects, insulin-positive endocrine cells further differentiate, for example, by induction or maturation into SC-β cells.

[296] In some aspects, a method of obtaining insulin-positive endocrine cells from NKX6-1-positive pancreatic progenitor cells includes bringing a population of cells (for example, under conditions that stimulate cell clustering) containing NKX6-1-positive pancreatic progenitor cells, in contact with at least two β-cell maturation factors, including a) an inhibitor of the TGF-β signaling pathway and b) an activator of the thyroid hormone signaling pathway, to induce the differentiation of at least one NKX6-1-positive pancreatic progenitor cell in a population of an insulin-positive endocrine cell, wherein the insulin-positive pancreatic progenitor cell expresses insulin.

[297] The invention contemplates the use of any TGF-β signaling inhibitor that induces differentiation of NKX6-1 positive pancreatic progenitor cells into insulin positive endocrine cells (e.g., alone or in combination with other β cell maturation factors, e.g., activator of the thyroid hormone signaling pathway). In some embodiments, the TGF-β signaling pathway includes TGF-β type I receptor kinase signaling. In some embodiments, the TGF-β signaling inhibitor comprises an Alk5 II inhibitor.

[298] The invention contemplates the use of any thyroid hormone signaling pathway activator that induces differentiation of NKX6-1 positive pancreatic progenitor cells into insulin positive endocrine cells (e.g., alone or in combination with other β-cell maturation factors, e.g. signaling pathway TGF-β). In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3).

[299] In some embodiments, the method comprises contacting a population of cells (eg, NKX6-1 positive pancreatic progenitor cells) with at least one additional β-cell maturation factor. In some embodiments, said method comprises contacting Pdx1 positive, NKX6-1 positive pancreatic progenitor cells with at least one of i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor. , iv) at least one growth factor from the epidermal growth factor (EGF) family, and optionally v) a protein kinase inhibitor.

[300] In some embodiments, the at least one additional β-cell maturation factor comprises a γ-secretase inhibitor. The invention contemplates the use of any γ-secretase inhibitor that is capable of inducing differentiation of NKX6-1 positive pancreatic progenitor cells in a population into insulin positive endocrine cells (e.g., alone or in combination with any component of a TGF-β signaling pathway inhibitor and/or or an activator of the thyroid hormone signaling pathway). In some embodiments, the γ-secretase inhibitor comprises XXI. In some embodiments, the γ-secretase inhibitor comprises DAPT.

[301] In some embodiments, the at least one additional β-cell maturation factor includes at least one growth factor from the EGF family. The invention contemplates the use of any growth factor from the EGF family that is capable of inducing differentiation of NKX6-1 positive pancreatic progenitor cells in a population into insulin positive endocrine cells (e.g., alone or in combination with any component of a TGF-β signaling inhibitor and /or an activator of the thyroid hormone signaling pathway). In some embodiments of the invention, at least one growth factor from the EGF family includes betacellulin. In some embodiments of the invention, at least one growth factor from the EGF family includes EGF.

[302] In some embodiments, the at least one additional β-cell maturation factor comprises a low concentration of retinoic acid (RA) signaling pathway activator. The invention contemplates the use of any RA signaling pathway activator that induces differentiation of NKX6-1 positive pancreatic progenitor cells into insulin positive endocrine cells (e.g., alone or in combination with any component of a TGF-β signaling inhibitor and/or a signaling activator). thyroid hormone pathway). In some embodiments, the RA signaling pathway activator includes RA.

[303] In some embodiments, the at least one additional β-cell maturation factor comprises a sonic hedgehog (SHH) pathway inhibitor. The invention contemplates the use of any SHH pathway inhibitor that induces differentiation of NKX6-1 positive pancreatic progenitors into insulin positive endocrine cells (e.g., alone or in combination with any component of a TGF-β signaling inhibitor and/or a signaling pathway activator). thyroid hormone). In some embodiments, the SHH pathway inhibitor comprises Sant1.

[304] In some embodiments, a population of cells (eg, NKX6-1 positive pancreatic progenitor cells) is treated with glucose.

[305] In some embodiments, the cell population is optionally contacted with a protein kinase inhibitor. In some embodiments, the cell population is not contacted with a protein kinase inhibitor. In some embodiments of the invention, a population of cells is brought into contact with a protein kinase inhibitor. The invention contemplates the use of any protein kinase inhibitor that is capable of inducing differentiation of NKX6-1-positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone or in combination with any component of a TGF-β signaling inhibitor and/or an activator thyroid hormone signaling pathway). In some embodiments, the protein kinase inhibitor comprises staurosporine.

[306] In some embodiments, insulin-positive endocrine cells are obtained by bringing Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into contact with i) at least one SHH pathway inhibitor, ii) an RA signaling pathway activator, iii ) a γ-secretase inhibitor, iv) an inhibitor of the TGF-β signaling pathway, v) an activator of the TH signaling pathway, and vi) at least one growth factor from the epidermal growth factor (EGF) family to induce the differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

[307] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) used may vary.

[308] In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one TGF-β signaling inhibitor at a concentration of 100 nM - 100 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one TGF-β signaling inhibitor at a concentration of 10 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one TGF-β signaling inhibitor at a concentration of 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM , 800 nM or 900 nM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one TGF-β signaling inhibitor at a concentration of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM or 9 μM. In some embodiments of the invention, NKX6-1-positive pancreatic progenitor cells are brought into contact with at least one inhibitor of the TGF-β signaling pathway at a concentration of 9.1 μM, 9.2 μM, 9.3 μM, 9.4 μM, 9.5 µM, 9.6 µM, 9.7 µM, 9.8 µM or 9.9 µM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one TGF-β signaling inhibitor at a concentration of 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM , 18 µM or 19 µM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one inhibitor of the TGF-J3 signaling pathway at a concentration of 10.1 μM, 10.2 μM, 10.3 μM, 10.4 μM, 10.5 µM, 10.6 µM, 10.7 µM, 10.8 µM or 10.9 µM.

[309] In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM - 10 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments of the invention, NKX6-1-positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 µM, 0.8 µM or 0.9 µM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 µM, 1.7 µM, 1.8 µM or 1.9 µM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM.

[310] In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 0.1 μM - 10 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 1 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0 .7 µM, 0.8 µM or 0.9 µM. In some embodiments of the invention, NKX6-1-positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1 .6 µM, 1.7 µM, 1.8 µM or 1.9 µM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM.

[311] In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 2 ng/ml - 200 ng/ml. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/ml or 19 ng/ml. In some embodiments of the invention, NKX6-1-positive pancreatic progenitor cells are brought into contact with at least one growth factor from the EGF family at a concentration of 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL or 100 ng/ml. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, 25 ng/ml, 26 ng/ml, 27 ng/ml, 28 ng/ml or 29 ng/ml. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 20 ng/mL.

[312] In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.01 μM - 1.0 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0 .07 µM, 0.08 µM or 0.09 µM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.20 μM, 0.30 μM, 0.40 μM, 0.05 μM, 0.60 μM, 0 .70 µM, 0.80 µM or 0.90 µM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.1 μM.

[313] In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a low concentration of RA signaling pathway activator at a concentration of 0.01 μM - 1.0 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a low concentration of RA signaling pathway activator at a concentration of 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0. 06 µM, 0.07 µM, 0.08 µM or 0.09 µM. In some embodiments of the invention, NKX6-1-positive pancreatic progenitor cells are contacted with a low concentration of RA signaling pathway activator at a concentration of 0.20 μM, 0.30 μM, 0.40 μM, 0.05 μM, 0 .60 µM, 0.70 µM, 0.80 µM, or 0.90 µM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a low concentration of 0.1 μM RA signaling pathway activator.

[314] In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.1 μM - 0.5 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.11 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 µM, 0.16 µM, 0.17 µM, 0.18 µM, 0.19 µM, 0.2 µM, 0.21 µM, 0.22 µM, 0.23 µM or 0.24 µM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.26 μM, 0.27 μM, 0.28 μM, 0.29 μM, 0.30 µM, 0.31 µM, 0.32 µM, 0.33 µM, 0.34 µM, 0.35 µM, 0.36 µM, 0.37 µM, 0.38 µM, 0.39 µM, 0.40 μM, 0.41 μM, 0.42 μM, 0.43 μM, 0.44 μM, 0.45 μM, 0.46 μM, 0.47 μM, 0.48 μM, 0.49 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.25 μM.

[315] In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 10 nM - 1 μM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, or 190 nM. In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM.

[316] In some embodiments, NKX6-1 positive pancreatic progenitor cells are contacted with glucose at a concentration of 1 mM - 50 mM. In some embodiments, NKX6-1 positive pancreatic progenitors are contacted with 25 mM glucose.

[317] In some embodiments, insulin-positive endocrine cells can be obtained by differentiating at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells. by contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells under conditions that promote cell clustering with i) an inhibitor of the TGF-β signaling pathway, b) an activator of the TH signaling pathway, and optionally c) at least one SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, and vi) at least one growth factor from the epidermal growth factor (EGF) family, every other day for a period of five to seven days, to induce differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX 6-1, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

[318] In some embodiments, insulin-positive endocrine cells can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive pancreatic progenitors in a population into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, for example, by bringing Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into contact with i) at least one SHH pathway inhibitor, ii) RA signaling pathway activator, iii) γ-secretase inhibitor, iv) inhibitor signaling pathway TGF-β, v) an activator of the TH signaling pathway, and vi) at least one growth factor from the family of epidermal growth factors (EGF) to induce the differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells c Pdx1-positive, NKX6-1, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

[319] In general, a population of cells is maintained in a suitable culture medium for a period of time sufficient to induce differentiation of at least one NKX6-1 positive pancreatic progenitor cell in the population into an insulin positive endocrine cell. A typical suitable culture medium is shown in Table 4.

[320]

[321] In some embodiments, BE5 medium can be used as a suitable culture medium for differentiating NKX6-1 positive pancreatic progenitor cells into insulin positive endocrine cells. In some embodiments of the invention, a suitable culture medium is shown in Table 5.

[322] In some embodiments, conditions that promote cell clustering include suspension culture. In some embodiments of the invention, the time period corresponds to a period of time sufficient to maximize the number of cells co-expressing C-peptide and Nkx6-1. In some embodiments of the invention, the time period is at least 5 days. In some embodiments of the invention, the time period is from 5 days to 7 days. In some embodiments of the invention, the specified period of time is at least 7 days. In some embodiments of the invention, the suspension culture is replenished every day (eg, β-cell maturation factors). In some embodiments of the invention, a period of time ranging from 5 days to 7 days, allows you to maximize the number of cells co-expressing C-peptide and Nkx6-1.

[323] In some embodiments, at least 15% of the NKX6-1-positive pancreatic progenitor cells in the population are induced to differentiate into insulin-positive endocrine cells.

[324] In some embodiments, at least 99% of the NKX6-1 positive pancreatic progenitors in a population are induced to differentiate into insulin positive endocrine cells.

[325] Induction of maturation of insulin + endocrine cells into SC-β cells

[326] Aspects of the invention include SC-β cells. Used in this document, the SC-β cells can be obtained from any source or generated in accordance with any suitable protocol. In some aspects, insulin-positive endocrine cells are induced to mature into SC-β cells.

[327] In some aspects, the invention provides a method for generating mature glucose-dependent β-cells from insulin-positive endocrine cells, comprising bringing a population of cells (for example, under conditions that stimulate cell clustering) containing insulin-positive endocrine cells into contact with at least with at least two β-cell maturation factors, including a) a transforming growth factor-β (TGF-β) signaling inhibitor, b) a thyroid hormone (TH) signaling pathway activator to induce in vitro maturation of at least one insulin-positive endocrine cell in the population into the SC-β cell.

[328] Aspects of the invention include the generation of SC-β cells that are similar in form and function to endogenous mature β-cells, but differ from native β-cells. SC-β cells may respond to at least one glucose stimulation. In some embodiments, the SC-β cells exhibit a response to at least two consecutive glucose stimulations. In some embodiments, the SC-β cells demonstrate a response to at least three consecutive glucose stimulations. In some embodiments, the SC-β cells exhibit a response to multiple (eg, sequential) glucose stimulation that resembles the response of endogenous human islets to multiple glucose stimulation. In some embodiments, the SC-β cells are capable of releasing or secreting insulin in response to two successive glucose stimulations. In some embodiments, the SC-β cells are capable of releasing or secreting insulin in response to three successive glucose stimulations. In some embodiments, the SC-β cells are capable of releasing or secreting insulin in response to four successive glucose stimulations. In some embodiments, the SC-β cells are capable of releasing or secreting insulin in response to five successive glucose stimulations. In some embodiments of the invention, the SC-β cells release or secrete insulin in response to continuous sequential stimulations with glucose. In some embodiments of the invention, cells can be analyzed to determine if they respond to successive glucose stimulations by determining the presence of a systematic increase in intracellular Ca ²⁺ as described in the examples provided herein.

[329] In some embodiments of the invention, the morphology of SC-β cells resembles the morphology of endogenous β-cells. In some embodiments, the SC-β cell exhibits an in vitro glucose-stimulated insulin secretion (GSIS) response. In some embodiments, the SC-β cell exhibits a GSIS response in vivo. In some embodiments, the SC-β cell exhibits in vitro and in vivo GSIS responses. In some in vitro and/or in vivo embodiments, the GSIS response resembles the GSIS responses of endogenous mature β-cells. In some embodiments, the SC-β cell exhibits an in vitro response (GSIS) that resembles the GSIS response of endogenous β-cells. In some embodiments, the SC-β cell exhibits an in vivo GSIS response that resembles the GSIS response of endogenous β-cells. The GSIS response can be observed immediately after transplantation into the body of a human or animal subject. In some embodiments, a GSIS response is observed within two weeks of transplantation of the SC-β cell into a human or animal subject. In some embodiments, a GSIS response is observed within two weeks of transplantation of the SC-β cell into a human or animal subject. In some embodiments, the SC-β cell GSIS response is observed up to three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or up to 1 year or more after transplantation of an SC-β cell into a human subject or subject- animal.

[330] In some embodiments, the SC-β cells display at least one marker of mature endogenous pancreatic β cells. Exemplary markers include, without limitation, Pdx1, HNF6, Ptf1a, Sox9, FoxA2, Nkx2.2, Ngn3, and NKX6-1. In some embodiments, expression of a marker selected from the group consisting of HNF6, Ptf1a, Sox9, FoxA2, Nkx2.2, Ngn3, and NKX6-1 is upregulated in SC-β cells by a statistically significant amount compared to pluripotent stem cells. (eg, embryonic stem cells or induced pluripotent cells) from which the SC-β cells were derived.

[331] The invention contemplates the use of any TGF-β signaling inhibitor that induces differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (e.g., alone or in any combination with at least one thyroid hormone signaling pathway activator (TH) or optionally a protein kinase inhibitor). In some embodiments, the TGF-β signaling pathway includes TGF-β type I receptor kinase signaling. In some embodiments, the TGF-β signaling inhibitor comprises an Alk5 II inhibitor.

[332] The invention contemplates the use of any thyroid hormone signaling pathway activator that induces differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (e.g., alone or in any combination with at least one TGF-β signaling inhibitor or optionally a protein kinase inhibitor). In some embodiments, the thyroid hormone signaling pathway activator comprises T3.

[333] In some embodiments, Pdx1 positive, NKX6-1 positive, insulin positive cells are optionally contacted with a protein kinase inhibitor. In some embodiments of the invention Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are not contacted with a protein kinase inhibitor. In some embodiments, Pdx1 positive, NKX6-1 positive, insulin positive endocrine cells are contacted with a protein kinase inhibitor. The invention contemplates the use of any protein kinase inhibitor that induces differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (e.g., alone or in any combination with at least one TGF-β signaling inhibitor and/or signaling pathway activator thyroid hormone). In some embodiments, the protein kinase inhibitor comprises staurosporine.

[334] In some embodiments of the invention, this method includes bringing a population of cells (eg, insulin-positive endocrine cells) in contact with at least one additional factor maturation β-cells.

[335] In some embodiments, the at least one additional β-cell maturation factor comprises a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments of the invention, this method includes bringing Pdx1-positive, NKX6-1-positive, insulin-positive cells into contact with a CFTR inhibitor. The invention contemplates the use of any CFTR inhibitor that induces differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (e.g. alone or in any combination with at least one TGF-β signaling inhibitor and/or signaling pathway activator thyroid hormone, and optionally a protein kinase inhibitor). In some embodiments, the CFTR inhibitor comprises Gly-H101.

[336] In some embodiments, the at least one additional β-cell maturation factor comprises an O-GlcNAcase inhibitor. In some embodiments of the invention, this method includes bringing Pdx1-positive, NKX6-1-positive, insulin-positive cells into contact with an inhibitor of O-GlcNAcase. The invention contemplates the use of any O-GlcNAcase inhibitor that induces differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (e.g. alone or in any combination with at least one TGF-β signaling inhibitor and/or activator thyroid hormone signaling pathway, and optionally a protein kinase inhibitor). In some embodiments, the O-GlcNAcase inhibitor comprises Thiamet-G.

[337] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) used may vary. In some embodiments of the invention, insulin-positive endocrine cells are brought into contact with at least one inhibitor of the TGF-β signaling pathway at a concentration of 100 nm - 100 μm. In some embodiments of the invention, insulin-positive endocrine cells are brought into contact with at least one inhibitor of the TGF-β signaling pathway at a concentration of 10 μm. In some embodiments, insulin-positive endocrine cells are contacted with at least one TGF-β signaling inhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM. In some embodiments, insulin-positive endocrine cells are contacted with at least one TGF-β signaling inhibitor at a concentration of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM. In some embodiments, insulin-positive endocrine cells are contacted with at least one inhibitor of the TGF-β signaling pathway at a concentration of 9.1 μM, 9.2 μM, 9.3 μM, 9.4 μM, 9.5 μM , 9.6 μM, 9.7 μM, 9.8 μM, or 9.9 μM. In some embodiments, insulin-positive endocrine cells are contacted with at least one TGF-β signaling inhibitor at a concentration of 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, or 19 μM. In some embodiments, insulin-positive endocrine cells are contacted with at least one inhibitor of the TGF-β signaling pathway at a concentration of 10.1 μM, 10.2 μM, 10.3 μM, 10.4 μM, 10.5 μM , 10.6 µM, 10.7 µM, 10.8 µM or 10.9 µM.

[338] In some embodiments, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM - 10 μM. In some embodiments of the invention, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments of the invention, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM , 0.8 µM or 0.9 µM. In some embodiments of the invention, insulin-positive endocrine cells are brought into contact with a thyroid hormone signaling pathway activator at a concentration of 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM , 1.7 µM, 1.8 µM or 1.9 µM. In some embodiments, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM.

[339] In some embodiments, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 10 nM - 1 μM. In some embodiments of the invention, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM. In some embodiments, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, or 190 nM. In some embodiments, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM.

[340] In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a CFTR inhibitor at a concentration of 100 nM - 100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a CFTR inhibitor at a concentration of 10 nM - 10 μM.

[341] In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with an O-GlcNAcase inhibitor at a concentration of 100 nM - 100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with an O-GlcNAcase inhibitor at a concentration of 10 nM - 10 μM.

[342] In some embodiments, an SC-β cell can be obtained by differentiating at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by bringing Pdx1-positive, NKX6-1- positive, insulin-positive endocrine cells under conditions that stimulate cell clustering, in contact with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor, every other day for a period of seven to 14 days to induce in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit GSIS response in vitro and/or in vivo. In some embodiments, the GSIS response resembles the endogenous β-cell GSIS response.

[343] In some embodiments, an SC-β cell can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells in a population into SC-β cells, e.g., by converting Pdx1-positive , NKX6-1-positive, insulin-positive endocrine cells in contact with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor to induce in in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-producing endocrine cells into SC-β cells, wherein SC-β cells exhibit an in vitro and/or in vivo GSIS response that resembles the GSIS response endogenous β-cell.

[344] In some aspects, the invention provides a method for producing SC-β cells, comprising: bringing Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into contact under conditions that stimulate cell clustering with i) an inhibitor of the signaling pathway transforming growth factor β (TGF-β), ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor to induce in vitro maturation of at least one of Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo. In some embodiments, the GSIS response resembles the endogenous β-cell GSIS response.

[345] In some aspects, the invention provides a method for generating SC-β cells from pluripotent cells, comprising: a) differentiating the pluripotent cells in the population into Pdx1-positive pancreatic progenitor cells; b) differentiating at least some of the Pdx1 positive pancreatic progenitors into Pdx1 positive, NKX6-1 positive pancreatic progenitors by contacting the Pdx1 positive pancreatic progenitors under conditions that promote cell clustering with i) at least one growth factor from the FGF family, i) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, every other day for a period of five days to induce differentiation of at least some Pdx1- positive pancreatic progenitors in the population into NKX6-1 positive pancreatic progenitors, wherein the NKX6-1 positive pancreatic progenitors express Pdx1 and NKX6-1; c) differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by bringing Pdx1-positive, NKX6-1-positive pancreatic cells - precursors under conditions that promote cell clustering, in contact with i) an inhibitor of the TGF-β signaling pathway, b) an activator of the TH signaling pathway, and optionally c) at least one inhibitor of the SHH pathway, ii) an activator of the RA signaling pathway, iii) an inhibitor γ-secretases and vi) at least one growth factor from the epidermal growth factor (EGF) family, every other day for a period of five to seven days to induce differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1-positive, insulin-positive some endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin; and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by bringing the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells under conditions that stimulate cell clustering, in contact with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor, every other day for a period of seven to 14 days to induce in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo. In some embodiments, the GSIS response resembles the GSIS response of endogenous mature β-cells.

[346] In some aspects, the invention provides a method for generating SC-β cells from pluripotent cells, comprising: a) differentiating at least some of the pluripotent cells in the population into Pdx1-positive pancreatic progenitor cells; b) differentiating at least some of the Pdx1 positive pancreatic progenitors into Pdx1 positive, NKX6-1 positive pancreatic progenitors by contacting the Pdx1 positive pancreatic progenitors under conditions that promote cell clustering with i) KGF, ii) Sant1 and optionally iii) low concentrations of RA, every other day for a period of five days to induce differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into NKX6-1-positive pancreatic cells -progenitors, with NKX6-1-positive pancreatic progenitor cells expressing Pdx1 and NKX6-1; c) differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by bringing Pdx1-positive, NKX6-1-positive pancreatic cells - precursors under conditions that stimulate cell clustering, in contact with i) Alk5 inhibitor II, ii) T3 and optionally iii) Sant1, iv) RA, v) XXI and vi) betacellulin, every other day for a period of time ranging from five to seven days to induce differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1 -positive, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin; and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by bringing the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells under conditions that stimulate cell clustering, in contact with i) inhibitor II Alk5, ii) T3 and optionally iii) staurosporine, every other day for a period of time ranging from seven to 14 days, to induce in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein SC-β cells exhibit an in vitro and in vivo GSIS response that resembles the endogenous β-cell GSIS response.

[347] In general, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are kept in a suitable culture medium for a period of time sufficient to induce in vitro maturation of at least some Pdx1-positive, NKX6-1- positive, insulin-positive endocrine cells into SC-β cells. An exemplary suitable culture medium is shown in Table 4 above and Table 5 below.

[348]

[349] In some embodiments, a suitable culture medium includes 1066 supplemented islet medium from Connought Medical Research Laboratories (CMRLS). In some embodiments of the invention, a suitable culture medium includes a CMRLS component (eg, auxiliary zinc). In some embodiments of the invention, a suitable culture medium is shown in Table 3. In some embodiments of the invention, CMRLS is supplemented with serum (eg, human). In some embodiments, the CMRLS is supplemented with serum substituents (eg, KOSR). In some embodiments of the invention, CMRLS is supplemented with fetal bovine serum. In some embodiments of the invention, CMRLS is supplemented with 10% fetal bovine serum. In some embodiments, a suitable culture medium for differentiating insulin-positive endocrine cells into SC-β cells includes S3 medium. In some embodiments of the invention, the conditions that stimulate cell clustering include suspension culture. In some embodiments of the invention, the time period is at least 7 days. In some embodiments of the invention, the time period is from 7 days to 21 days. In some embodiments of the invention, the time period is from 7 to 14 days. In some embodiments of the invention, the time period is from 10 to 14 days. In some embodiments of the invention, the time period is 14 days. In some embodiments, the suspension culture is replenished every other day (eg, with β-cell maturation factors).

[350] In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% Pdx1 positive, NKX6-1 positive , insulin-positive endocrine cells are induced to mature into SC-β cells. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% Pdx1 positive, NKX6-1 positive, insulin positive endocrine cells are induced to mature into SC-β cells. In some embodiments of the invention, at least 30% of the resulting cells contain SC-β cells. In some embodiments, the β cells express C-peptide, insulin, NKX6-1, Pdx1 and co-express NKX6-1 and C-peptide.

[351] In some embodiments, the SC-β cells include human cells. In some embodiments of the invention, the in vitro generation of SC-β cells is scalable.

[352] Isolated Cell Populations

[353] Aspects of the invention relate to isolated populations of cells obtained in accordance with the methods described herein. In some embodiments, a population of SC-β cells is obtained by bringing at least one insulin-positive endocrine cell or progenitor thereof into contact with at least one β-cell maturation factor described herein. In some embodiments, a population of SC-β cells is obtained by bringing at least one insulin-positive endocrine cell or progenitor thereof into contact with at least two of the β-cell maturation factors described herein. In some embodiments, a population of SC-β cells is obtained by bringing at least one insulin-positive endocrine cell or progenitor thereof into contact with at least three β-cell maturation factors described herein. In some embodiments, a population of SC-β cells is obtained by bringing at least one insulin-positive endocrine cell or progenitor thereof into contact with at least four of the β-cell maturation factors described herein. In some embodiments, a population of SC-β cells is obtained by bringing at least one insulin-positive endocrine cell or progenitor thereof into contact with at least five β-cell maturation factors described herein. In some embodiments, a population of SC-β cells is obtained by bringing at least one insulin-positive endocrine cell or progenitor thereof into contact with at least seven, at least eight, at least nine, or at least ten the β-cell maturation factors described herein.

[354] In some aspects, the invention provides an isolated population of definitive endoderm cells. An isolated population of definitive endoderm cells can be obtained by differentiating at least some of the pluripotent cells in the population into definitive endoderm cells, for example by contacting the population of pluripotent cells with i) at least one growth factor from the TGF-β superfamily and ii) a signaling activator a Wnt pathway to induce differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of the definitive endoderm.

[355] In some aspects, the invention provides an isolated population of primary intestinal tube cells. An isolated population of primary intestinal tube cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primary intestinal tube cells, for example, by contacting the definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family, to induce differentiation of at least some cells of the definitive endoderm into cells of the primary intestinal tube, wherein the cells of the primary intestinal tube express at least one marker characteristic of the definitive endoderm.

[356] In some aspects, the invention provides an isolated population of Pdx1-positive pancreatic progenitor cells. An isolated population of Pdx1-positive pancreatic progenitors can be obtained by differentiating at least some of the primary intestinal tube cells in the population into Pdx1-positive pancreatic progenitors, for example by contacting the primary intestinal tube cells with i) at least one inhibitor bone morphogenetic protein (BMP) signaling pathway, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator, and v) at least at least one protein kinase C activator to induce differentiation of at least some cells of the primary intestinal tube into Pdx1-positive pancreatic progenitors, wherein the Pdx1-positive pancreatic progenitors express Pdx1.

[357] In some aspects, the invention provides an isolated population of NKX6-1 positive pancreatic progenitor cells. An isolated population of NKX6-1 positive pancreatic progenitors can be obtained by differentiating at least some of the Pdx1-positive pancreatic progenitors in the population into Pdx1-positive, NKX6-1-positive pancreatic progenitors, e.g., by converting Pdx1-positive pancreatic progenitor cells in contact with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator to induce differentiation of at least one Pdx1-positive pancreatic progenitor cells in the population into NKX6-1 positive pancreatic progenitors, wherein the NKX6-1 positive pancreatic progenitors express Pdx1 and NKX6-1.

[358] In some aspects, the invention provides an isolated population of insulin-positive endocrine cells. An isolated population of insulin-positive endocrine cells can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells in the population into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, for example, by bringing Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into contact with i) a TGF-β signaling inhibitor, ii) a TH signaling pathway activator, and optionally at least one additional β-cell maturation factor selected from the group , consisting of i) at least one SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) and vi) at least one growth factor from the epidermal growth factor (EGF) family to induce differentiation at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells to Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin .

[359] In some aspects, the invention provides an isolated population of SC-β cells. An isolated population of SC-β cells can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells in the population into SC-β cells, e.g. , insulin-positive endocrine cells in contact with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor to induce in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the β cells exhibit a GSIS response in vitro and/or in vivo. In some embodiments, the GSIS response resembles the endogenous β-cell GSIS response.

[360] Aspects of the invention include microcapsules containing isolated populations of cells described herein (eg, β cells). Microcapsules are well known in the art. Suitable examples of microcapsules are described in the literature (e.g., Jahansouz et al., "Evolution of β-Cell Replacement Therapy in Diabetes Mellitus: Islet Cell Transplantation" Journal of Transplantation 2011; Volume 2011, Article ID 247959; Orive et al., "Application of cell encapsulation for controlled delivery of biological therapeutics", Advanced Drug Delivety Reviews (2013), http://dx.doi.org/10.1016/j.addr.2013.07.009; Hernandez et al., "Microcapsules and microcarriers for in situ cell delivery", Advanced Drug Delivery Reviews 2010; 62:711-730; Murua et al., "Cell microencapsulation technology: Towards clinical application", Journal of Controlled Release 2008; 132:76-83; and Zanin et al., " The development of encapsulated cell technologies as therapies for neurological and sensory diseases", Journal of Controlled Release 2012; 160:3-13). Microcapsules can be prepared in a variety of ways. Typical microcapsules contain an alginate core surrounded by a polycationic layer covered with an outer alginate membrane. The polycationic membrane forms a semi-permeable membrane that provides stability and biocompatibility. Examples of polycations include, without limitation, poly-L-lysine, poly-L-ornithine, chitosan, lactose-modified chitosan, and photopolymerized biomaterials. In some embodiments of the invention, the alginate core is modified, for example, so as to obtain a backbone containing an alginate core containing covalently conjugated oligopeptides with the sequence RGD (arginine, glycine, aspartic acid). In some embodiments of the invention, the alginate core is modified, for example, to obtain a covalently reinforced microcapsule containing a chemoenzymatically engineered alginate with increased stability. In some embodiments of the invention, the alginate core is modified, for example, so as to obtain membrane mimetic films formed by in-situ polymerization of acrylate functionalized phospholipids. In some embodiments, the microcapsules are composed of alginates enzymatically modified with epimerases. In some embodiments of the invention, the microcapsules contain covalent bonds between adjacent layers of the membrane of the microcapsule. In one embodiment of the invention, the microcapsule contains a capsule below the sieving boundary containing alginate coupled with phenolic components. In some embodiments of the invention, the microcapsule contains a backbone containing alginate-agarose. In some embodiments, the SC-β cell is modified with PEG prior to encapsulation in alginate. In some embodiments, isolated cell populations, such as SC-β cells, are encapsulated in photoreactive liposomes and alginate. It should be understood that the alginate used in microcapsules can be replaced by other suitable biomaterials, including, but not limited to, PEG, chitosan, PES hollow fibers, collagen, hyaluronic acid, dextran with RGD, EHD and PEGDA, PMBV and PVA, PGSAS, agarose, agarose with gelatin, PLGA and their multilayer implementations.

[361] In some embodiments of the invention, compositions containing populations of cells obtained in accordance with the methods described herein can also be used as a functional component in a mechanical device designed to obtain one or more endocrine polypeptides of pancreatic islet cells. The simplest form of said device contains a population of pancreatic beta cells (e.g. derived from a population of insulin-positive endocrine cells or their progenitors) delimited by a semi-permeable membrane that impedes the passage of the cell population by keeping them within the device, but does not impede the passage of insulin, glucagon, or somatostatin. secreted by the cell population. The above includes a microencapsulated population of beta cells, typically in the form of cell clusters, to allow interaction that inhibits differentiation. For example, US Pat. weighing >3000, which are cross-linked in such a way that the membrane is permeable to proteins of the size of insulin, but impermeable to molecules with a mol. weighing over 100,000. US Pat. No. 6,023,009 describes islet cells encapsulated in a semi-permeable membrane composed of agarose and agaropectin. Microcapsules of this origin are intended to be inserted into the abdominal cavity of a diabetic patient and are believed to have certain advantages in reducing histocompatibility problems or susceptibility to bacteria.

[362] Also provided are other developed devices containing a population of pancreatic beta cells derived from insulin-positive endocrine cells or their precursors in accordance with the methods described herein, intended for both implantation in patients with diabetes and for extracorporeal therapy. US Pat. No. 4,378,016 describes an artificial endocrine gland comprising an extracorporeal segment, a subcutaneous segment, and a replaceable capsule containing hormone-producing cells. US Pat. No. 5,674,289 describes a biosynthetic pancreas comprising an insular cavity separated by a semi-permeable membrane from one or more vascular cavities with access to surrounding tissue. The devices used typically comprise a cavity adapted to contain the islet cells and a cavity separated from the islet cells by a semi-permeable membrane that collects proteins secreted from the islet cells and which can also transmit a feedback signal to the islet cells, for example regarding circulating glucose levels.

[363] Aspects of the invention include assay methods associated with isolated populations of cells described herein (eg, SC-β cells). In some embodiments, these assay methods can be used to identify one or more candidate agents that promote or inhibit β-cell fate selected from the group consisting of β-cell proliferation, β-cell replication, and β-cell death, β-cell function. -cells, the susceptibility of β-cells to attacks by the immune system, or the susceptibility of β-cells to dedifferentiate or differentiate. In some embodiments of the invention, these methods of analysis can be used to identify one or more candidate agents that stimulate the differentiation of at least one insulin-positive endocrine cell or its progenitor into SC-β cells. In some embodiments of the invention, these methods of analysis can be used to identify one or more candidate agents that stimulate the production of insulin by β-cells or increase the production or secretion of insulin.

[364] The invention provides methods in which SC-β cells are obtained in accordance with the methods described herein from iPS cells obtained from cells extracted or isolated from the body of individuals suffering from a disease (for example, diabetes, obesity, or a disorder, associated with β-cells), and these SC-β cells are compared with normal β-cells from healthy individuals who do not have disease to reveal differences between SC-β cells and normal β-cells that can serve as markers of disease (for example , epigenetic and/or genetic). In some embodiments, β-cells are obtained from an individual with diabetes and compared with normal β-cells, and then β-cells are reprogrammed into iPS cells and the iPS cells are analyzed for epigenetic and/or genetic markers that are present in β-cells derived from an individual with diabetes but not present in normal β-cells to identify these markers (eg, pre-diabetic). In some embodiments, iPS and/or SC-β cells obtained from diabetic patients are used to screen for agents (eg, agents that are capable of modulating genes responsible for the diabetic phenotype).

[365] Methods for differentiating insulin-positive endocrine cells into SC-β cells

[366] Generation of SC-β cells by converting at least one insulin-positive endocrine cell or its progenitor using the methods of the invention has many advantages. First, the methods of the invention enable the generation of autologous SC-β cells that are specific and genetically appropriate for an individual. In general, autologous cells are less likely than non-autologous cells to be subject to immunological rejection. Cells are derived from at least one insulin-positive endocrine cell or progenitor thereof, e.g., a pancreatic progenitor cell, obtained by reprogramming a somatic cell (e.g., fibroblast) from an individual into an induced pluripotent state and then culturing the pluripotent cells to differentiate at least some pluripotent cells into at least one insulin-positive endocrine cell or its precursor, followed by transplantation of at least one insulin-positive endocrine cell or its precursor into the body of the individual so that at least one insulin-positive endocrine cell or its precursor matures in vivo into an SC-β cell, or by inducing in vitro maturation of at least one insulin-positive endocrine cell or its progenitor into an SC-β cell.

[367] In some embodiments, the subject from which at least one insulin-positive endocrine cell or progenitor thereof is obtained is a mammalian subject, such as a human subject. In some embodiments of the invention, the subject suffers from a disorder associated with β-cells. In some embodiments of the invention, the subject suffers from diabetes. In some embodiments of the invention, the subject suffers from prediabetes. In such embodiments, at least one insulin-positive endocrine cell or progenitor thereof can be differentiated into an SC-β cell ex vivo using the methods described herein, and then it can be administered to the subject from whose body the cells were collected to carry out treating a subject for a β-cell related disorder (eg, diabetes).

[368] In some embodiments, at least one insulin-positive endocrine cell, or progenitor thereof, resides in the subject (in vivo) and is converted to become an SC-β cell using the in vivo methods described herein. In some embodiments, conversion of at least one insulin-positive endocrine cell or progenitor thereof to an SC-β cell in vivo can be achieved by administering to a subject a composition comprising at least one, at least two, at least three, at least four, at least five, or at least six β-cell maturation factors, as described herein. In some embodiments, conversion of at least one insulin-positive endocrine cell or progenitor thereof to an SC-β cell in vivo can be achieved by administering to a subject a composition comprising at least one, at least two, at least three, at least four, at least five, or at least six β-cell maturation factors, as described herein.

[369] In some embodiments of the invention, bringing into contact can be carried out by maintaining at least one insulin-positive endocrine cell or its precursor in a culture medium containing one or more β-cell maturation factors. In some embodiments of the invention, at least one insulin-positive endocrine cell or its precursor may be genetically engineered. In some embodiments, at least one insulin-positive endocrine cell or progenitor thereof may be genetically engineered to express one or more β-cell maturation factors as described herein, e.g., to express at least one polypeptide selected from pancreatic and duodenal homeobox 1 polypeptide (PDX-1), insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI/3, B2, Nkx2.2, NKX6-1, GLUT2, PC2, ZnT-8, or a significant the degree of their homologous amino acid sequences, or their functional fragments or functional variants.

[370] In the event that at least one insulin-positive endocrine cell or its progenitor is maintained under in vitro conditions, conventional tissue culture conditions and methods known to those skilled in the art can be used. Methods for isolating and culturing different cells are known to those skilled in the art.

[371] In general, in the methods of the invention, at least one insulin-positive endocrine cell or progenitor thereof can be cultured under standard temperature, pH, and other environmental conditions, e.g., as adherent cells on tissue culture plates at 37°C. C and in an atmosphere containing 5-10% CO2. The cells and/or culture medium are suitably modified to achieve SC-β cell transformation as described herein. In certain embodiments, at least one insulin-positive endocrine cell or progenitor thereof, e.g., a pancreatic progenitor cell, can be cultured on or in the presence of a material that mimics one or more characteristics of the extracellular matrix or contains one or more components of the extracellular matrix or basal membranes. In some embodiments of the invention, Matrigel™ is used. Other materials include proteins or mixtures thereof such as gelatin, collagen, fibronectin, etc. In certain embodiments of the invention, at least one insulin-positive endocrine cell or its precursor can be cultured in the presence of a layer of feeder cells. Such cells may, for example, be of murine or human origin. They may also be irradiated, chemically inactivated by treatment with a chemical inactivator such as mitomycin c, or otherwise treated to inhibit their proliferation if desired. In other embodiments of the invention, at least one insulin-positive endocrine cell or its precursor is cultured without feeding cells. In some embodiments of the invention, insulin-positive endocrine cells or their precursors are cultured under conditions that stimulate cell clustering. As used herein, "conditions that stimulate cell clustering" refers to any conditions that stimulate cell clustering during differentiation into SC-β cells. In some embodiments of the invention, the conditions that stimulate cell clustering include suspension culture. Boretti and Gooch (Tissue Eng. 2006 Apr; 12(4):939-48) report that culturing under less adhesive conditions (low serum medium, low adhesive substrate) stimulated cell clustering in transdifferentiation of adult pancreatic ductal epithelial cells. organism into beta cells in vitro. Accordingly, without being limited by theory, in some embodiments of the invention, the conditions that stimulate cell clustering include minimally adhesive conditions, for example, a medium with low serum content, a substrate with low adhesiveness.

[372] In some examples, β-cell maturation factors can be used to induce differentiation of at least one insulin-positive endocrine cell or progenitor thereof by treating or bringing at least one insulin-positive endocrine cell or progenitor thereof into contact with an effective amount β-cell maturation factor described herein to differentiate at least one insulin-positive endocrine cell or progenitor thereof into at least one SC-β cell (eg, mature pancreatic β-cell).

[373] Accordingly, cells and compositions prepared by the methods described herein are included herein. The exact amount and type of β-cell maturation factor may vary depending on the number of insulin-positive endocrine cells or their progenitors, the necessary stage of differentiation, and the number of stages of differentiation that have passed.

[374] In certain examples, the β-cell maturation factor is present in an effective amount. As used herein, "effective amount" refers to the amount of a compound that must be present for at least 10%, or at least 20%, or at least 30% of the cells in a population of insulin-positive endocrine cells or their progenitors to differentiate into SC cells. -β.

[375] In further examples, β-cell maturation factors may be present in the culture medium of at least one insulin-positive endocrine cell or progenitor thereof, or alternatively, β-cell maturation factors may be added to at least one insulin-positive endocrine cell. cell or its precursor at some stage of growth.

[376] Confirmation of presence and identification of SC-β cells

[377] Any means known to the person skilled in the art can be used to confirm the presence of an SC-β cell, for example, a mature pancreatic β-cell obtained by inducing the differentiation of at least one insulin-positive endocrine cell or its progenitor by treating it with at least one β-cell maturation factor, as described herein.

[378] In some embodiments, the presence of β-cell markers, such as chemically induced SC-β cells, can be confirmed by determining the presence or absence of one or more endogenous β-cell markers. In some embodiments of the invention, this method may include determining the positive expression (eg, presence) of a marker of mature β-cells. In some embodiments of the invention, the marker can be detected using a reagent, for example, a reagent for the detection of NKX6-1 and C-peptide. In particular, SC-β cells according to this document express NKX6-1 and C-peptide and do not significantly express other markers that would be characteristic of immature β-cells (eg, MafB). The reagent for the marker can be, for example, an antibody to this marker or primers for a RT-PCR or PCR reaction, for example, a semi-quantitative or quantitative RT-PCR or PCR reaction. Such markers can be used to determine the production of SC-β cells. The antibody or other detection reagent may be associated with a label, such as a radioactive, fluorescent (eg, GFB), or colorimetric label, for use in detection. If the detection reagent is a primer, it may be provided as a dry preparation, eg lyophilized, or in solution.

[379] The development of at least one insulin-positive endocrine cell or its precursor into an SC-β cell can be monitored by determining the expression of markers characteristic of mature β-cells. In some methods, the expression of certain markers is determined by detecting the presence or absence of the marker. Alternatively, the expression of certain markers can be determined by measuring the level of presence of the marker in the cells of a cell culture or cell population. Some methods determine the expression of markers characteristic of SC-β cells, as well as the absence of significant expression of markers characteristic of insulin-positive endocrine cells or their progenitors, for example, pluripotent stem cells or pancreatic progenitors from which they were derived.

[380] As described in connection with tracking the production of SC-β cells (e.g., mature pancreatic β-cells) from insulin-positive endocrine cells, qualitative or semi-quantitative methods, such as immunoblotting and immunocytochemistry methods known in the art, can be used to determine marker expression. in this field of technology. Alternatively, the expression of the markers can be accurately quantified using a technique such as quantitative PCR, methods well known in the art. In addition, it should be understood that at the polypeptide level, many markers of pancreatic islet hormone-expressing cells are secreted proteins. Therefore, methods for determining the extracellular content of markers, such as ELISA, can be used.

[381] SC-β cells can also be characterized by down-regulation of markers characteristic of the pluripotent stem cells from which SC-β cells were induced. For example, SC-β cells derived from pluripotent stem cells may in the mature state be characterized by statistically significant downregulation of pluripotent stem cell markers - alkaline phosphatase (AP), NANOG, OST-4, SOX-2, SSEA4, TRA-1-60 or TRA-1-81 compared to the pluripotent stem cells from which they were derived. Other markers expressed by pluripotent stem cells include, but are not limited to, alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49/6E; ERas/ECATS, E-cadherin; βIII-tubulin; α-smooth muscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cell-associated transcript 1 (ECAT1); ESG1/DPPAS/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fth117; Sa114; undifferentiated embryonic cell transcription factor (Utf1); Rex1; p53; G3PDH; telomerase, including TERT; X-chromosome silent genes; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; pluripotency factor 2 (DPPA2); breakpoint associated with T-cell lymphoma 1 (Tel1); DPPA3/Stella; DPPA4; Dnmt3L; Sox15; Stat3; Grb2; SV40 large T antigen; HPV16 E6; HPV16 E7, β-catenin and Bmi1 and other common markers of pluripotency, with at least one or more of them downregulated by a statistically significant amount in the mature state compared to pluripotent stem cells from which they were derived.

[382] It is understood that the present invention is not limited to markers referred to herein as markers of mature β-cells, while the present invention also includes markers such as cell surface markers, antigens and other gene products, including EST, RNA (including miRNA and antisense RNA), DNA (including genes and cDNA), as well as their fragments.

[383] Enrichment, isolation and purification of SC-β cells

[384] Another aspect of the present invention relates to the isolation of a population of SC-β cells from a heterogeneous population of cells, wherein such a mixed population of cells contains SC-β cells and insulin-positive endocrine cells or their progenitors from which the SC-β cells were derived. The population of SC-β cells produced by any of the methods described above can be enriched, isolated and/or purified with any cell surface marker present on the SC-β cells that is not present on the insulin-positive endocrine cells or their progenitors from which they were derived. received. Such cell surface markers are also referred to as SC-β cell-specific affinity tags. Examples of affinity tags specific to SC-β cells are antibodies, ligands, or other binding agents specific to a marker molecule, such as a polypeptide present on the cell surface of SC-β cells, that is substantially absent from other cell types (e.g., insulin -positive endocrine cells or their precursors). In some methods, an antibody that binds to a cell surface antigen on an SC-β cell (e.g., a human SC-β cell) is used as an affinity tag to enrich, isolate, or purify chemically induced (e.g., by contacting at least one β-cell maturation factor as described herein) of SC-β cells produced by the methods described herein. Such antibodies are known and commercially available.

[385] Methods for using antibodies to enrich, isolate, and/or purify SC-β cells are known to those skilled in the art. For example, in some embodiments of the invention, a reagent, such as an antibody, is incubated with a cell population containing SC-β cells, while the cell population is treated to reduce intracellular and substrate adhesion. The cell population is then washed, centrifuged and redissolved. In some embodiments, if the antibody is not labeled, the cell suspension is then incubated with a secondary antibody, such as a FITC-conjugated antibody, that is capable of binding to the primary antibody. The SC-β cells are then washed, centrifuged and redissolved in buffer. The SC-β cell suspension is then analyzed and sorted using a Fluorescence Activated Cell Sorter (FACS). Antibody-bound fluorescent reprogrammed cells are harvested separately from unbound non-fluorescent cells (e.g., immature insulin-producing cells), thus separating SC-β cells from other cells present in the cell suspension, e.g., insulin-positive endocrine cells or their progenitors or immature insulin-producing cells (eg, other differentiated cell types).

[386] In another embodiment of the methods described herein, an isolated cell composition containing SC-β cells can be further purified using an alternative method based on affinity or using additional sorting steps using the same or different markers specific for SC cells. -β. For example, in some embodiments, FACS sorting is used to first separate SC-β cells that express NKX6-1, either alone or together with C-peptide expression, or alternatively with the β- marker disclosed herein. cells, from cells that do not express one of these markers (eg, negative cells) in the cell population. A second FAC sort, such as a positive cell sort using FACS to isolate cells that are positive for a marker different from that used in the first sort, allows the cell population to be enriched with reprogrammed cells.

[387] In an alternative embodiment, FACS sorting is used to separate cells by negative sorting for a marker that is present on most insulin-positive endocrine cells or progenitors but not present on SC-β cells.

[388] In some embodiments of the methods described herein, SC-β cells are fluorescently labeled without the use of an antibody and then separated from unlabeled cells using a fluorescence activated cell sorter (FACS). In such embodiments, a nucleic acid encoding a GFB, an VPB, or another nucleic acid encoding an expressible fluorescent marker gene, such as a gene encoding a luciferase, is used to label reprogrammed cells using the methods described above. For example, in some embodiments of the invention, at least one copy of the nucleic acid encoding the GFP or a biologically active fragment thereof is introduced into at least one insulin-positive endocrine cell, which is first chemically induced to an SC-β cell, downstream of the promoter expressed in an SC-β cell, such as an insulin promoter, such that the expression of the GFP gene product or a biologically active fragment thereof is under the control of the insulin promoter.

[389] In addition to the procedures described above, chemically induced SC-β cells can also be isolated using other cell isolation methods. In addition, SC-β cells can also be enriched or isolated by serial subculturing methods under growth conditions that promote selective survival or selective expansion of SC-β cells. Such methods are known to those skilled in the art and may include the use of agents such as, for example, insulin, members of the TGF-beta family including activin A, TGF-beta 2 and 3, bone morphogenetic proteins (BMP-2, -3, - 4, -5, -6, -7, -11, -12 and -13), fibroblast growth factors 1 and 2, platelet-derived growth factor AA and BB, platelet-rich plasma, insulin-like growth factors (IGF-I, II), growth and differentiation factor (GDF-5, -6, -7, -8, -10, -11, -15), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), pleotrophin, endothelium, epidermal growth factor (EGF), beta cellulin. Other pharmaceutical compounds may include, for example, nicotinamide, glucagon-like peptide-I (GLP-1) and II, GLP-1 and 2 mimetitel, exendin-4, retinoic acid, parathyroid hormone.

[390] Using the methods described herein, enriched, isolated and/or purified populations of SC-β cells can be obtained in vitro from insulin-positive endocrine cells or progenitors thereof (that have been differentiated from pluripotent stem cells by the methods described herein). In some embodiments, preferred enrichment, isolation, and/or purification methods relate to the in vitro production of human SC-β cells from human insulin-positive endocrine cells or progenitors thereof that have been differentiated from human pluripotent stem cells or from human induced pluripotent stem cells ( iPS). In an embodiment in which SC-β cells are differentiated from insulin-positive endocrine cells that were previously derived from definitive endoderm cells that were previously derived from iPS cells, the SC-β cells may be autologous to the subject from which the received cells to generate iPS cells.

[391] Using the methods described herein, isolated cell populations of SC-β cells are enriched in terms of content of SC-β cells by at least about 2 to about 1000 times compared with the cell population prior to chemical induction of the population of insulin-positive endocrine cells or their predecessors. In some embodiments of the invention, SC-β cells can be enriched by at least about 5 to about 500 times compared to the population before chemical induction of the population of insulin-positive endocrine cells or their progenitors. In other embodiments, the SC-β cells can be enriched by at least about 10 to about 200 times the population prior to chemical induction of the population of insulin-positive endocrine cells or progenitors thereof. In other embodiments of the invention, SC-β cells can be enriched in from about 20 to about 100 times compared with the population before chemical induction of the population of insulin-positive endocrine cells or their precursors. In other embodiments, the SC-β cells can be enriched by at least about 40 to about 80 times the population prior to chemical induction of the population of insulin-positive endocrine cells or progenitors thereof. In certain embodiments of the invention, SC-β cells can be enriched by at least about 2 to about 20 times compared to the population before chemical induction of the population of insulin-positive endocrine cells or their progenitors.

[392] Compositions containing SC-β cells

[393] Some embodiments of the present invention relate to cell compositions, such as cell cultures or cell populations, containing SC-β cells, wherein the SC-β cells were derived from at least one insulin-positive endocrine cell or its progenitor. In some embodiments of the invention, the cell compositions contain insulin-positive endocrine cells. In some embodiments, cell compositions comprise NKX6-1 pancreatic progenitor cells. In some embodiments, cell compositions comprise Pdx1 pancreatic progenitor cells. In some embodiments, the cell compositions comprise primary intestinal tube cells. In some embodiments of the invention, the cell compositions contain cells of the definitive endoderm.

[394] According to certain embodiments, the chemically induced SC-β cells are mammalian cells, and in a preferred embodiment, such SC-β cells are human SC-β cells. In some embodiments, the insulin-positive endocrine cells are derived from definitive endoderm cells, such as human definitive endoderm stem cells. In accordance with certain embodiments of the invention, the chemically induced Pdx1 positive pancreatic progenitors are mammalian cells, and in a preferred embodiment, such Pdx1 positive pancreatic progenitors are human Pdx1 positive pancreatic progenitors.

[395] Other embodiments of the present invention relate to compositions, such as an isolated cell population or cell culture, containing SC-β cells produced by the methods disclosed herein. In some embodiments, the present invention relates to compositions, such as isolated cell populations or cell cultures, containing chemically induced SC-β cells produced by the methods disclosed herein. In such embodiments, SC-β cells are less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60 %, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of the total number of cells in the SC-β cell population. In some embodiments, the composition comprises a population of SC-β cells that is greater than about 90% of the total number of cells in the cell population, such as at least about 95%, or at least 96%, or at least 97%, or at least 98% or at least about 99% or at least about 100% of the total number of cells in the cell population are SC-β cells.

[396] Certain other embodiments of the present invention relate to compositions, such as isolated cell populations or cell cultures, containing a combination of SC-β cells and insulin-positive endocrine cells or their progenitors from which SC-β cells were derived. In some embodiments, insulin-positive endocrine cells from which SC-β cells were derived are less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%. less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total number of cells in the isolated cell population or culture.

[397] Additional embodiments of the present invention relate to compositions, such as isolated cell populations or cell cultures, obtained by the methods described herein, which contain chemically induced SC-β cells as the main cell type. In some embodiments, the methods and processes described herein make it possible to obtain isolated cell cultures and/or cell populations containing at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89 %, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82% at least about 81%, at least about 80%, at least about 79%, at least about 78%, at least about 77%, at least about 76%, at least about 75%, at least about 74%, at least about 73%, at least about 72%, at least about 71%, at least about 70%, at least about 69%, at least about 68%, at least about 67%, at least about 66%, at least about 65%, at least about 64%, at least about 63%, at least at least about 62%, at least about 61%, at least about 60%, at least about 59%, at least about 58%, at least about 57%, at least about 56%, at least about 55%, at least about 54%, at least about 53%, at least about 52%, at least about 51%, or at least about 50% of SC-β cells.

[398] In another embodiment, the isolated cell populations or cell compositions (or cell cultures) contain human SC-β cells. In other embodiments, the methods and processes described herein make it possible to obtain isolated cell populations containing at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least at least about 30%, at least about 25%, at least about 24%, at least about 23%, at least about 22%, at least about 21%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5% %, at least about 4%, at least about 3%, at least about 2%, or at least about 1% of SC-β cells. In preferred embodiments, the isolated cell populations may contain human SC-β cells. In some embodiments of the invention, the percentage of SC-β cells in cell cultures or populations is calculated without taking into account the feeding cells remaining in the culture.

[399] Other embodiments of the present invention relate to compositions, such as isolated cell populations or cell cultures, containing a mixture of SC-β cells and insulin-positive endocrine cells or their progenitors from which they were differentiated. For example, you can get cell cultures or cell populations containing at least about 5 SC-β cells for every 95 insulin-positive endocrine cells or their precursors. In other embodiments, cell cultures or cell populations can be obtained containing at least about 95 SC-β cells for every 5 insulin-positive endocrine cells or progenitors thereof. In addition, cell cultures or cell populations containing other ratios of SC-β cells and insulin-positive endocrine cells or their precursors are contemplated. For example, compositions containing at least about 1 SC-β cell for every 1,000,000 or at least 100,000 cells, or at least 10,000 cells, or at least 1,000 cells, or 500, or at least 250 cells, or at least 100 or at least 10 insulin-positive endocrine cells or progenitors thereof.

[400] Additional embodiments of the present invention relate to compositions, such as cell cultures or cell populations, containing human cells, including a human SC-β cell, which has at least one characteristic of an endogenous β-cell.

[401] In preferred embodiments of the present invention, cell cultures and/or cell populations of SC-β cells contain human SC-β cells that are non-recombinant cells. In such embodiments, cell cultures and/or cell populations contain no or substantially no recombinant human SC-β cells.

[402] β-cell maturation factors

[403] Aspects of the invention include bringing insulin-positive endocrine cells or their precursors into contact with β-cell maturation factors, for example, to induce maturation of insulin-positive endocrine cells or differentiation of their progenitors into SC-β cells (for example, mature pancreatic β- cells). The term "beta cell maturation factor" refers to an agent that stimulates or facilitates the transformation of at least one insulin-positive endocrine cell or its progenitor into an SC-beta cell. In some embodiments of the invention, the β-cell maturation factor induces the differentiation of pluripotent cells (eg, iPSC or hESC) into definitive endoderm cells, for example, in accordance with the method described herein. In some embodiments of the invention, the β-cell maturation factor induces the differentiation of definitive endoderm cells into cells of the primary intestinal tube, for example, in accordance with the method described herein. In some embodiments, the β-cell maturation factor induces differentiation of primary intestinal tube cells into Pdx1-positive pancreatic progenitor cells, for example, in accordance with the method described herein. In some embodiments, the β cell maturation factor induces the differentiation of Pdx1 positive pancreatic progenitors into NKX6-1 positive pancreatic progenitors, for example, according to the method described herein. In some embodiments, the β cell maturation factor induces the differentiation of NKX6-1 positive pancreatic progenitor cells into insulin positive endocrine cells, for example, in accordance with the method described herein. In some embodiments of the invention, the β-cell maturation factor induces the maturation of insulin-positive endocrine cells into SC-β cells, for example, in accordance with the method described herein.

[404] In general, at least one β-cell maturation factor described herein, alone or in combination with other β-cell maturation factors, can be used to generate SC-β cells in accordance with the methods disclosed herein. In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of the β-cell maturation factors described herein are used in methods for generating SC-β cells.

[405] Transforming Growth Factor β (TGF-β) Superfamily

[406] Aspects of the invention relate to the use of growth factors from the transforming growth factor β (TGF-β) superfamily as β cell maturation factors. The "TGF-β" superfamily refers to proteins having the structural and functional characteristics of known members of the TGFβ family. The characteristics of the TGFβ family proteins are well known, both in terms of structural and functional aspects. It includes the TGFβ protein groups, inhibins (including inhibin A and inhibin B), activins (including activin A, activin B, and activin AB), MIS (Mullerian inhibitory substance), BMP (bone morphogenetic proteins), dpp (decapentaplegic), Vg- 1, MNSF (monoclonal non-specific suppression factor) and others. The activity of this family of proteins is based on specific binding to certain receptors on different cell types. Members of this family contain regions with sequence identity, in particular at the C-terminus, which correspond to their function. The TGFβ family includes more than a hundred different proteins, all of which contain at least one region with amino acid sequence identity. Members of this family include, but are not limited to, the following proteins, identified by their GenBank accession numbers: P07995, P18331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917, P09529, P27093, P04088, Q04999, P17491, P55104 , Q9WUK5, Р55103, O88959, O08717, Р58166, O61643, Р35621, Р09534, Р48970, Q9NR23, Р25703, Р30884, Р12643, Р49001, Р21274, O46564, O19006, Р22004, Р20722, Q04906, Q07104, Р30886, Р18075, Р23359, Р22003 , Р34821, Р49003, Q90751, Р21275, Q06826, Р30885, Р34820, Q29607, Р12644, Q90752, O46576, Р27539, Р48969, Q26974, Р07713, Р91706, Р91699, Р27091, O42222, Q24735, Р20863, O18828, Р55106, Q9PTQ2, O14793 , O08689, O42221, O18830, O18831, O18836, O35312, O42220, Р43026, Р43027, Р43029, O95390, Q9R229, O93449, Q9Z1W4, Q9BDW8, Р43028, Q7Z4P5, Р50414, Р17246, Р54831, Р04202, Р01137, Р09533, Р18341, O19011 , Q9Z1Y6, P07200, Q9Z217, O95393, P55105, P30371, Q9MZE2, Q07258, Q96S42, P97737, AAA97415.1, NP-776788.1, NP-058824.1, EAL24001.1, 1S04Y, NP-09-09 856.1 NP-032406.1 NP-999193.1 XP-519063.1 AAG17260.1 CAA40806.1 NP-001009458.1 AAQ55808.1 AAK40341.1 AAP33019.1 AAK21AI265.1 B40905 AAQ55811.1 AAK40342.1 XP-540364.1 P55102 AAQ55810.1 NP-990727.1 CAA51163.1 AAD50448.1 JC4862 PN0504 BAB17600.1 AAH56742.1 BAB1796 1, CAG05339.1, BAB17601.1, CAB43091.1, A36192, AAA49162.1, AAT42200.1, NP-789822.1, AAA59451.1, AAA59169.1, XP-541000.1, NP-990537.1, NP-002184.17.18 1, AAP83319.1, AAA59170.1, BAB16973.1, AAM66766.1, WFPGBB, 1201278C, AAH30029.1, CAA49326.1, XP-344131.1, AAH48845.1, XP-148966.3,148235, B41385.17.17 AAB26863.1, 1706327A, BAA83804.1, NP-571143.1, CAG00858.1, BAB17599.1, BAB17602.1, AAB61468.1, PN0505, PN0506, CAB43092.1, BAB17598.1, BAA22570.1 BAC81672.1, BAA12694.1, BAA08494.1, B36192, C36192, BAB16971.1, NP-034695.1, AAA49160.1, CAA62347.1, AAA49161.1, AAD30132.1, CAA58290.1, NP-005.1, XP-005522 522443.1, AAM27448.1, XP-538247.1, AAD30133.1, AAC367 41.1 AAH10404.1 AAW30007.1, AAH90232.1, A29619, NP-001007905.1, AAH73508.1, AAD02201.1, NP-999793.1, NP- 990542.1 .1 AAT07302.1 , CAA45018.1 , CAA45019.1 , BAC28247.1 , NP-031581.1 .1 AAB46367.1 CAA05033.1 BAA89012.1 1ES7 AAP20870.1 BAC24087.1 AAG09784.1 BAC06352.1 -571435.1, 1REU, AAC60286.1, BAA24406.1, A36193, AAH55959.1, AAH54647.1, AAH90689.1, CAG09422.1, BAD16743.1, NP-032134.1, XP-532179.1, AAB24876.02, AAB24876.02 , AAA82616.1, CAA40222.1, CAB90273.2, XP-342592.1, XP-534896.1, X P-534462.1 1LXI XP-417496.1 AAF34179.1 AAL73188.1 CAF96266.1 AAB34226.1 AAB33846.1 AAT12415.1 CAA45021.1 AAB27337.1 AAP69917.1 AAT12416.1 NP-571396.1 CAA53513.1 AAO33820.1 AAA48568.1 BAC02605.1 BAC02604.1 BAC02603.1 BAC02602.1 1, BAC02599.1, BAC02598.1, BAC02597.1, BAC02595.1, BAC02593.1, BAC02592.1, BAC02590.1, AAD28039.1, AAP74560.1, AAB94786.1, NP-571417.1 NP-001001557.1 AAH43222.1 AAM33143.1 CAG10381.1 BAA31132.1 EAL39680.1 EAA12482.2 P34820 AAP88972.1 AAP74559.1 XP- 345502.1 NP-038554.1 CAG04089.1 CAD60936.2 NP-031584.1 B55452 AAC60285.1 BAA06410.1 AAH52846.1 NP-031580.1 NP-036959.1 Q29607 AAB27336.1 XP-547817.1 AAT12414.1 AAM54049.1 AAH78901.1 AA025745.1 NP-570912.1 XP-392194.1 AAD20829.1 AAC97113.1 AAC61694.1 AAH1.1 AAR97906.1, BAA32227.1, BAB68395.1, BAC02895.1, AAW51451.1, AAF82188.1, XP-5 AAF99597.1 NP-032133.2 XP-394252.1 XP-224733.2 JH0801 AAP97721.1 NP-989669.1 S43296 P43029 A55452 AAH32495.1 XP-542974.1 NP-032135.1 BAK30827.1 1, EAA12064.2, AAP97720.1, XP-525704.1, AAT07301.1, BAD07014.1, AAR27581.1, AAG13400.1, AAC60127.1, CAF92055.1 CAF97447.1 AAS01764.1 BAD08319.1 CAA10268.1 NP-998140.1 AAR03824.1 AAS48405.1 AAS48403.1 AAK53545.1 AAK84666.1 XP-395420.1 AAK56941.15 AAC4755 1 AAR88255.1 AAK67984.1 AAK67983.1 AAK28706.1 P07713 P91706 P91699 CAG02450.1 AAC47552.1 NP-005802.1 XP-343149.1 AW34055.1 XP-538221.1 AAR27580.1 XP AAF21633.1, AAF21630.1, AAD05267.1, Q9Z1W4, NP-031585.2, NP-57 1094.1 CAD43439.1 CAF99217.1 CAB63584.1 NP-722840.1 CAE46407.1 XP-417667.1 BAC53989.1 BAB19659.1 AAM46922.1 AAA81169.1 AAK28707.13.1 AAB 17573.1, CAH25443.1, CAG10269.1, BAD16731.1, EAA00276.2, AAT07320.1, AAT07300.1, AAN15037.1, CAH25442.1, AAK08152.2, 2009388A, AAR12161.1, CAG01965 .1, CAF94162.1, NP-477340.1, EAL24792.1, NP-001009428.1, AAB86686.1, AAT40572.1, AAT40571.1, AAT40569.1, NP-033886.1, AAB49985.1, AAG3 , Q26974, AAC77461.1, AAC47262.1, BAC05509.1, NP-055297.1, XP-546146.1, XP-525772.1, NP- 060525.2 AAF02773.1 , AAB86692.1, CAB40844.1, BAC38637.1, BAB16046.1, AAN63522.1, NP- 571041.1 .1, AAK18000.1, XP-420540.1, AAL35276.1, AAQ98602.1, CAE71944.1 , AAW50585.1, AAV63982.1, AAW29941.1, AAN87890.1, AAT40568.1, CAD57730.1, AAB81508.1, AAS00534.1, AAC59736.1, BAB79498.1, AAA97392.1, AAP85526.1, NP -999600.2, NP-878293.1, BAC82629.1, CAC60268.1, CAG04919.1, AAN10123.1, CAA07707.1 NP-571951.2 CAC50881.1 AAL99367.1 AAL49502.1 AAB71839.1 AAB65415.1 NP-624359.1 NP-990153.1 AAF78069.1 AAK49790.1 NP-919367.2 NP-001192 544948.1 AAQ18013.1 AAV38739.1 NP-851298.1 CAA67685.1 AAT67171.1 AAT37502.1 AAD27804.1 AAN76665.1 BAC11909.1 XP-421648.1 CAB63704.1 A55706 AAF02780.1 CAG09623.1 NP-067589.1 NP-035707.1 AAV30547.1 AAP49817.1 BAC77407.1 AAL87199.1 CAG07172.1 B36193 CAA33024.1 1, XP-512687.1, XP-510080.1, AAH05513.1, 1KTZ, AAH14690.1, AAA31526.1.

[407] It is contemplated that any growth factor from the TGF-β superfamily that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC cell -β can be used in the methods, compositions and kits described herein.

[408] The growth factor from TGF-β can be obtained naturally or recombinantly. In some embodiments, a growth factor from the TGF-β superfamily includes activin A. The term "activin A" includes activin A fragments and derivatives. publication 218”). Other non-limiting examples of activin A are shown in SEQ ID nos: 2-16 of Publication 218, and non-limiting examples of nucleic acids encoding Activin A are shown in SEQ ID nos: 33-34 of Publication 218. In some embodiments, a growth factor from the TGF- β contains a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to SEQ ID NO: 1 of Publication 218.

[409] In some embodiments, the growth factor from the TGF-β superfamily includes growth and differentiation factor 8 (GDF8). The term "GDF8" includes fragments and derivatives of GDF8. The sequences of the GDF8 polypeptides are available to those skilled in the art. In some embodiments, a growth factor from the TGF-β superfamily comprises a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human GDF8 polypeptide sequence (GenBank accession number EAX10880).

[410] In some embodiments, a growth factor from the TGF-β superfamily includes a growth factor closely related to GDF8, such as growth and differentiation factor 11 (GDF11). GDF11 polypeptide sequences are available to those skilled in the art. In some embodiments, a growth factor from the TGF-β superfamily comprises a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human GDF11 polypeptide sequence (GenBank accession number AAF21630).

[411] In certain embodiments, the methods, compositions, and kits disclosed herein do not include at least one growth factor from the TGF-β superfamily.

[412] In some embodiments, at least one growth factor from the TGF-β superfamily may be replaced by an agent that mimics at least one growth factor from the TGF-β superfamily. Exemplary agents that mimic at least one growth factor from the superfamily TGF-β include, without limitation, IDE1 and IDE2.

[413] TGF-β signaling inhibitors

[414] Aspects of the invention relate to the use of inhibitors of the TGF-β signaling pathway as factors in maturation of β-cells. It is contemplated that any inhibitor of the TGF-β signaling pathway that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC-β cell can be use in the methods, compositions and kits described herein. In certain embodiments of the invention, the methods, compositions and kits disclosed herein do not include an inhibitor of the TGF-β signaling pathway.

[415] In some embodiments, the TGF-β signaling pathway includes TGF-β type I receptor kinase signaling (TGF-β RI). In some embodiments, the TGF-β signaling inhibitor comprises an ALK5 inhibitor II (CAS 446859-33-2, ATP-competitive TGF-B RI kinase inhibitor, also known as RepSox, IUPAC name: 2-[5-(6- methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine In some embodiments, the TGF-β signaling inhibitor is an analog or derivative of an ALK5 inhibitor II.

[416] In some embodiments, the ALK5 inhibitor II analog or derivative is a compound of Formula I as described in US Patent Publication No. 2012/0021519, incorporated herein by reference in its entirety.

[417] In some embodiments, the TGF-β signaling inhibitor is a TGF-β receptor inhibitor as described in US Patent Publication No. 2010/0267731. In some embodiments, the TGF-β signaling inhibitor includes an ALK5 inhibitor as described in US Pat. Nos. 2009/0186076 and 2007/0142376.

[418] In some embodiments, the TGF-β signaling inhibitor is A 83-01. In some embodiments, the TGF-β signaling inhibitor is not A 83-01. In some embodiments, the compositions and methods described herein do not include A 83-01.

[419] In some embodiments, the TGF-β signaling inhibitor is SB 431542. In some embodiments, the TGF-β signaling inhibitor is not SB 431542. In some embodiments, the compositions and methods described herein are not includes SB 431542.

[420] In some embodiments, the TGF-β signaling inhibitor is D 4476. In some embodiments, the TGF-β signaling inhibitor is not D 4476. In some embodiments, the compositions and methods described herein are not includes D 4476.

[421] In some embodiments, the TGF-β signaling inhibitor is GW 788388. In some embodiments, the TGF-β signaling inhibitor is not GW 788388. In some embodiments, the compositions and methods described herein are not includes GW 788388.

[422] In some embodiments, the TGF-β signaling inhibitor is LY 364947. In some embodiments, the TGF-β signaling inhibitor is not LY 364947. In some embodiments, the compositions and methods described herein are not includes LY 364947.

[423] In some embodiments, the TGF-β signaling inhibitor is LY 580276. In some embodiments, the TGF-β signaling inhibitor is not LY 580276. In some embodiments, the compositions and methods described herein are not includes LY 580276.

[424] In some embodiments, the TGF-β signaling inhibitor is SB 525334. In some embodiments, the TGF-β signaling inhibitor is not SB 525334. In some embodiments, the compositions and methods described herein are not includes SB 525334.

[425] In some embodiments, the TGF-β signaling inhibitor is SB 505124. In some embodiments, the TGF-β signaling inhibitor is not SB 505124. In some embodiments, the compositions and methods described herein are not includes SB 505124.

[426] In some embodiments, the TGF-β signaling inhibitor is SD 208. In some embodiments, the TGF-β signaling inhibitor is not SD 208. In some embodiments, the compositions and methods described herein are not includes SD 208.

[427] In some embodiments, the TGF-β signaling inhibitor is GW 6604. In some embodiments, the TGF-β signaling inhibitor is not GW 6604. In some embodiments, the compositions and methods described herein are not includes GW 6604.

[428] In some embodiments, the TGF-β signaling inhibitor is GW 788388. In some embodiments, the TGF-β signaling inhibitor is not GW 788388. In some embodiments, the compositions and methods described herein are not includes GW 788388.

[429] The following compounds from the set of compounds described above can be obtained from various sources: LY-364947, SB-525334, SD-208 and SB-505124 are available from Sigma, P.O. Box 14508, St. Louis, Mo., 63178-9916; 616452 and 616453 are available from Calbiochem (EMD Chemicals, Inc.), 480 S. Democrat Road, Gibbstown, N.J., 08027; GW788388 and GW6604 available from GlaxoSmithKline, 980 Great West Road, Brentford, Middlesex, TW8 9GS, United Kingdom; LY580276 is available from Lilly Research, Indianapolis, Ind. 46285; and SM16 available from Biogen Idea, P.O. Box 14627, 5000 Davis Drive, Research Triangle Park, N.C., 27709-4627.

[430] WNT Signaling Pathway

[431] Aspects of the invention relate to the use of WNT signaling pathway activators as β-cell maturation factors. It is contemplated that any WNT signaling activator that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell may be used in the methods, compositions and kits described herein.

[432] In some embodiments, the WNT signaling pathway activator includes CHIR99021. In some embodiments, the WNT signaling pathway activator comprises a derivative of CHIR99021, eg, a salt of CHIR99021, eg, trihydrochloride - hydrochloride salt of CHIR99021. In some embodiments, the WNT signaling pathway activator comprises a recombinant Wnt3a protein. In some embodiments, the WNT signaling pathway activator comprises a glycogen synthase kinase 3 (GSK3) inhibitor. Exemplary GSK3 inhibitors include, without limitation, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRAT-ide, 10Z-hymenialdizin, indirubin-3-oxime, kenpaullon, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, as well as analogues and derivatives of any of these compounds. In certain embodiments of the invention, the methods, compositions, and kits disclosed herein do not include a WNT signaling pathway activator.

[433] Fibroblast Growth Factor (FGF) Family

[434] Aspects of the invention relate to the use of growth factors from the FGF family as β-cell maturation factors. It is contemplated that any growth factor from the FGF family that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell may be used. in the methods, compositions and kits described herein. In certain embodiments of the invention, the methods, compositions and kits disclosed herein do not include a growth factor from the FGF family.

[435] In some embodiments of the invention, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). The KGF polypeptide sequences are available to those skilled in the art. In some embodiments of the invention, a growth factor from the FGF family contains a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human KGF polypeptide sequence (GenBank accession number AAB21431).

[436] In some embodiments of the invention, at least one growth factor from the FGF family includes FGF2. FGF2 polypeptide sequences are available to those skilled in the art. In some embodiments of the invention, a growth factor from the FGF family contains a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human FGF2 polypeptide sequence (GenBank accession number NP 001997).

[437] In some embodiments of the invention, at least one growth factor from the FGF family includes FGF8B. FGF8B polypeptide sequences are available to those skilled in the art. In some embodiments of the invention, a growth factor from the FGF family contains a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human FGF8B polypeptide sequence (GenBank accession number AAB40954).

[438] In some embodiments of the invention, at least one growth factor from the FGF family includes FGF10. FGF10 polypeptide sequences are available to those skilled in the art. In some embodiments of the invention, a growth factor from the FGF family contains a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human FGF10 polypeptide sequence (GenBank accession number CAG46489).

[439] In some embodiments of the invention, at least one growth factor from the FGF family includes FGF21. FGF21 polypeptide sequences are available to those skilled in the art. In some embodiments of the invention, a growth factor from the FGF family contains a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human FGF21 polypeptide sequence (GenBank accession number AAQ89444.1).

[440] Bone morphogenetic protein (BMP) signaling pathway inhibitors

[441] Aspects of the invention relate to the use of inhibitors of the BMP signaling pathway as β-cell maturation factors. The BMP signaling family is a heterogeneous subgroup of the TGF-β superfamily (Sebald et al. Biol. Chem. 385: 697-710, 2004). More than twenty known BMP ligands are for three different type II receptors (BMPRII, ActRIIa and ActRIIb) and at least three type I receptors (ALK2, ALK3 and ALK6). Dimeric ligands facilitate the assembly of receptor heteromers by allowing the constitutively active type II receptor serine threonine kinases to phosphorylate the type I receptor serine threonine kinases. facilitate nuclear translocation in conjunction with SMAD4, a co-SMAD that also facilitates TGF signaling. In addition, BMP signals can activate intracellular effectors such as MAPK p38 in an SMAD-independent manner (Nohe et al. Cell Signal 16:291-299, 2004). Soluble BMP antagonists such as noggin, chordin, gremlin, and follistatin limit BMP signaling by ligand sequestration.

[442] It is contemplated that any inhibitor of the BMP signaling pathway that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC-β cell, can be used in the methods, compositions and kits described herein. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a BMP signaling pathway inhibitor.

[443] In some embodiments, the BMP signaling inhibitor comprises LDN 193189 (also known as LDN193189, 1062368-24-4, LDN-193189, DM 3189, DM-3189, IUPAC name: 4-[6-(4- piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinolone).

[444] In some embodiments, the BMP signaling inhibitor comprises an analog or derivative of LDN 193189, such as a salt, hydrate, solvent, ester, or prodrug of LDN 193189. In some embodiments, the derivative (eg, salt) of LDN 193189 includes the hydrochloride of LDN 193189 .

[445] In some embodiments, the BMP signaling pathway inhibitor comprises a compound of Formula I of US Patent Publication No. 2011/0053930.

[446] Sonic hedgehog signaling pathway (SHH)

[447] Aspects of the invention relate to the use of inhibitors of the SHH signaling pathway as β-cell maturation factors. It is contemplated that any inhibitor of the SHH signaling pathway that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC-β cell may be used in the methods, compositions and kits described herein.

[448] In some embodiments, the SHH signaling inhibitor comprises Sant1. In some embodiments, the SHH signaling inhibitor comprises SANT2. In some embodiments, the SHH signaling inhibitor comprises SANT3. In some embodiments, the SHH signaling pathway inhibitor comprises SANT4. In some embodiments, the SHH signaling inhibitor comprises Cur61414. In some embodiments, the SHH signaling inhibitor comprises forskolin. In some embodiments, the SHH signaling inhibitor comprises tomatidine. In some embodiments, the SHH signaling inhibitor comprises AY9944. In some embodiments, the SHH signaling inhibitor comprises triparanol. In some embodiments, the SHH signaling pathway inhibitor comprises Compound A or Compound B (as disclosed in US Pat. Publication No. 2004/0060568). In some embodiments, the SHH signaling inhibitor comprises a steroidal alkaloid that antagonizes hedgehog signaling (eg, cyclopamine or a derivative thereof), as disclosed in US Pat. publ. USA No. 2006/0276391. In certain embodiments, the methods, compositions, and kits disclosed herein do not include an inhibitor of the SHH signaling pathway.

[449] Retinoic acid signaling pathway

[450] Aspects of the invention relate to the use of retinoic acid signaling modulators as β-cell maturation factors. It is contemplated that any retinoic acid signaling modulator that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC-β cell may be used in the methods, compositions and kits described herein.

[451] In some embodiments, the retinoic acid signaling modulator includes a retinoic acid signaling activator. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some embodiments, the RA signaling pathway activator comprises a retinoic acid receptor agonist. Exemplary retinoic acid receptor agonists include, without limitation, CD 1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80, BMS 753, tazarotene, adapalene, and CD 2314.

[452] In some embodiments, the retinoic acid signaling modulator comprises a retinoic acid signaling inhibitor. In some embodiments, the retinoic acid signaling inhibitor comprises DEAB (IUPAC name: 2-[2-(diethylamino)ethoxy]-3-prop-2-enylbenzaldehyde). In some embodiments, the inhibitor of the retinoic acid signaling pathway includes an analog or derivative of DEAB.

[453] In some embodiments, the inhibitor of the retinoic acid signaling pathway comprises a retinoic acid receptor antagonist. In some embodiments, the retinoic acid receptor antagonist comprises (E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]benzoic acid, (E)-4 -[[(5,6-dihydro-5,5-dimethyl-8-phenylethynyl)-2-naphthalenyl]ethenyl]benzoic acid, (E)-4-[2-[5,6-dihydro-5,5- dimethyl-8-(2-naphthalenyl)-2-naphthalenyl]ethenyl]benzoic acid and (E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2 -naphthalenyl]ethenyl]benzoic acid. In some embodiments, the retinoic acid receptor antagonist comprises BMS 195614 (CAS#253310-42-8), ER 50891 (CAS#187400-85-7), BMS 493 (CAS#170355-78-9), CD 2665 (CAS No. 170355-78-9), LE 135 (CAS No. 155877-83-1), BMS 453 (CAS No. 166977-43-1) or MM 11253 (CAS No. 345952-44-5).

[454] In certain embodiments, the methods, compositions, and kits disclosed herein do not include a retinoic acid signaling modulator. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a retinoic acid signaling pathway activator. In certain embodiments, the methods, compositions, and kits disclosed herein do not include an inhibitor of the retinoic acid signaling pathway.

[455] Protein kinase C

[456] Aspects of the invention relate to the use of protein kinase C activators as β-cell maturation factors. Protein kinase C belongs to one of the largest families of protein kinase enzymes and has many isoforms. Traditional isoforms include a, βI, βII, γ; new isoforms include δ, ε, η, Θ; and atypical isoforms include ζ, and ι/λ. PKC enzymes are predominantly cytosolic, but when activated, they translocate to the membrane. In the cytoplasm, PKC is phosphorylated by other kinases or autophosphorylated. Activation of some PKC isoforms (eg, PKC-ε) requires a molecule to bind to a diacylglycerol ("DAG") or phosphatidylserine ("PS") binding site. Others are able to activate without the involvement of any secondary binding messengers. PKC activators that bind to the DAG site include, but are not limited to, bryostatin, picologists, phorbol esters, aplisiatoxin, and gnidimacrin. PKC activators that bind to the PS site include, but are not limited to, polyunsaturated fatty acids and their derivatives. It is contemplated that any protein kinase C activator that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC-β cell may be used in the described applications. herein, methods, compositions and kits.

[457] In some embodiments, the PKC activator includes PdbU. In some embodiments of the invention, the PKC activator includes TRV. In some embodiments, the PKC activator includes cyclopropanated polyunsaturated fatty acids, cyclopropanated monounsaturated fatty acids, cyclopropanated polyunsaturated fatty alcohols, cyclopropanated monounsaturated fatty alcohols, cyclopropanated polyunsaturated fatty acid esters, cyclopropanated monounsaturated fatty acid esters, acids, cyclopropanated polyunsaturated fatty acid phosphates, cyclopropanated monounsaturated fatty acid phosphates, macrocyclic lactones, DAG derivatives, isoprenoids, octylindolactam V, gnidimacrine, iripallidal, ingenol, naphthalenesulfonamides, diacylglycerol kinase inhibitors, fibroblast growth factor 18 (FGF-18), insulin growth factor, hormones and growth factor activators as described in WIPO Publication No. WO/2013/071282. In some embodiments, bryostatin includes bryostatin-1, bryostatin-2, bryostatin-3, bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9, bryostatin-10, bryostatin-11 , bryostatin-12, bryostatin-13, bryostatin-14, bryostatin-15, bryostatin-16, bryostatin-17 or bryostatin-18. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a protein kinase C activator.

[458] γ-Secretase Inhibitors

[459] Aspects of the invention relate to the use of γ-secretase inhibitors as β-cell maturation factors. It is contemplated that any γ-secretase inhibitor that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC-β cell may be used in the methods, compositions and kits described herein. There are many inhibitors of γ-secretase. In some embodiments, the γ-secretase inhibitor comprises XXI. In some embodiments, the γ-secretase inhibitor comprises DAPT. Additional exemplary γ-secretase inhibitors include, but are not limited to, the γ-secretase inhibitors described in US Pat. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a γ-secretase inhibitor.

[460] Thyroid hormone signaling pathway activators

[461] Aspects of the invention relate to the use of thyroid hormone signaling pathway activators as β-cell maturation factors. It is contemplated that any thyroid hormone signaling pathway activator that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC-β cell may be used. in the methods, compositions and kits described herein. In certain embodiments of any aspect described herein, the methods, compositions, and kits disclosed herein do not include a thyroid hormone signaling pathway activator. In certain embodiments of any aspect described herein, the methods, compositions, and kits disclosed herein do not include T3 or the T3 analog described herein.

[462] In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator comprises a T3 analog or derivative. Exemplary T3 analogs include, but are not limited to, selective and non-selective thyromimetics, selective agonist-GC-1 TRβ, CC-24,4-hydroxy-RSV 106, MB07811, MB07344,3,5-diiodotyropropionic acid (DITPA); selective agonist GC-1 TR-β; 3-iodothyronamine (T(1)AM) and 3,3',5-triiodothyroacetic acid (Triac) (bioactive metabolites of thyroxine hormone (T(4)); KB-2115 and KB-141; thyronamines; SKF L-94901; DIBIT; 3'-AC-T2; tetraiodothyroacetic acid (Tetrac) and triiodothyroacetic acid (Triac) (by oxidative deamination and decarboxylation of the alanine chain of thyroxine [T4] and triiodothyronine [T3]), 3,3',5'-triiodothyronine (rT3 ) (by deiodination of T4 and T3), 3,3'-diiodothyronine (3,3'-T2) and 3,5-diiodothyronine (T2) (by deiodination of T4, T3 and rT3) and 3-iodothyronamine (T1AM) and thyronamine (T0AM) (by deiodination and amino acid decarboxylation of T4 and T3), as well as structural analogs of TH, such as 3,5,3'-triiodotyropropionic acid (Triprop), 3,5-dibromo-3-pyridazinone-1-thyronine (L -940901), N-[3,5-dimethyl-4-(4'-hydroxy-3'-isopropylphenoxy)-phenyl]-oxamic acid (CGS 23425), 3,5-dimethyl-4-[(4'- hydroxy-3'-isopropylbenzyl)phenoxy]acetic acid (GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetate acetic acid (KB-141); and 3,5-diiodotyropropionic acid (DITPA).

[463] In some embodiments, the thyroid hormone signaling pathway activator includes a T3 prodrug or prohormone, such as T4 thyroid hormone (eg, thyroxine or L-3,5,3',5'-tetraiodothyronine).

[464] In some embodiments, the thyroid hormone signaling pathway activator is the iodothyronine composition described in US Pat. No. 7,163,918.

[465] Epidermal growth factors (EGF) family

[466] Aspects of the invention relate to the use of growth factors from the EGF family as β-cell maturation factors. It is contemplated that any growth factor from the EGF family that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell may be used. in the methods, compositions and kits described herein. In some embodiments of the invention, at least one growth factor from the EGF family includes betacellulin. In some embodiments of the invention, at least one growth factor from the EGF family includes EGF. Epidermal growth factor (EGF) is a 53-amino acid cytokine that is proteolytically cleaved from a large integral membrane protein precursor. In some embodiments, a growth factor from the EGF family comprises a variant EGF polypeptide, e.g., an isolated epidermal growth factor polypeptide having at least 90% amino acid identity with a wild-type human EGF polypeptide sequence as disclosed in US Pat. No. 7,084,246. In some embodiments, a growth factor from the EGF family comprises an engineered EGF mutant that binds to and agonizes the EGF receptor as disclosed in US Pat. No. 8,247,531. In some embodiments of the invention, at least one growth factor from the EGF family is replaced by an agent that activates the signaling pathway of the EGF family. In some embodiments, a growth factor from the EGF family includes a compound that mimics EGF. In certain embodiments of the invention, the methods, compositions and kits disclosed herein do not include a growth factor from the EGF family.

[467] Protein kinase inhibitors

[468] Aspects of the invention relate to the use of protein kinase inhibitors as β-cell maturation factors. It is contemplated that any protein kinase inhibitor that is capable, alone or in combination with other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or progenitor thereof into an SC-β cell may be used in the methods described herein. , compositions and sets.

[469] In some embodiments, the protein kinase inhibitor comprises staurosporine. In some embodiments, the protein kinase inhibitor comprises a staurosporine analogue. Exemplary staurosporine analogs include, but are not limited to, Ro-31-8220, a bisindolylmaleimide (Bis) compound, 10'-{5''-[(methoxycarbonyl)amino]-2''-methyl}-phenylaminocarbonyl staurosporine, staralog (see e.g., Lopez et al., "Staurosporine-derived inhibitors broaden the scope of analog-sensitive kinase technology", J. Am. Chem. Soc. Soc. 2013; 135(48): 18153-18159) and cgp41251.

[470] In some embodiments, the protein kinase inhibitor is a PKCβ inhibitor. In some embodiments, the protein kinase inhibitor is a PKCβ inhibitor with the following structure, or a derivative, analog, or variant of the compound below:

[471] In some embodiments, the PKCβ inhibitor is a GSK-2 compound with the following structure, or a derivative, analog, or variant of the compound below:

[472] In some embodiments, the PKC inhibitor is bisindolylmaleimide. Exemplary bisindolylmaleimides include, without limitation, bisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide III, hydrochloride or derivative, analogue or variant. In some embodiments, the derivative, analog, or variant is selected from a derivative, analog, or variant of a compound selected from:

[473] In some embodiments, the PKC inhibitor is pseudohypericin or a derivative, analog, or variant of the compound below:

[474] In some embodiments, the PKC inhibitor is indorublin-3-monoxime, 5-iodo, or a derivative, analog, or variant of the compound below:

[475] In certain embodiments, the methods, compositions, and kits disclosed herein do not include a protein kinase inhibitor.

[476] Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor

[477] Aspects of the invention relate to the use of CFTR inhibitors as β-cell maturation factors. It is contemplated that any CFTR inhibitor that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC-β cell may be used in the procedures described in herein, methods, compositions and kits.

[478] Numerous CFTR inhibitors used herein are available to those skilled in the art. Exemplary CFTR inhibitors include, without limitation, LPA2 receptor agonist CFTR inhibitors as disclosed in US Pat. publ. No. 2007/0078111, hydrazine-containing CFTR inhibitors disclosed in US Pat. publ. USA No. 2008/0269206. In some embodiments, the CFTR inhibitor comprises a pore glycine hydrazine uptake CFTR inhibitor. In some embodiments, the CFTR inhibitor comprises Gly-H101. In some embodiments, the CFTR inhibitor comprises a derivative or analogue of Gly-H101 (see, for example, Muanprasat et al., "Discovery of Glyciine Hydrazide Pore-occluding CFTR Inhibitors", J. Gen. PHysiol 2004; 124(2): 125- 137). In certain embodiments of the invention, the methods, compositions, and kits disclosed herein do not include a CFTR inhibitor.

[479] O-GlcNAse inhibitor

[480] Aspects of the invention relate to the use of O-GlcNAcase inhibitors as β-cell maturation factors. It is contemplated that any inhibitor of the O-GlcNAcase that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing differentiation of at least one insulin-producing endocrine cell or its progenitor into an SC-β cell may be used in the methods, compositions and kits described herein. Numerous O-GlcNAse inhibitors used herein are available to those skilled in the art. Exemplary O-GlcNAse inhibitors include, without limitation, permeable glycosidase inhibitors (see, for example, WIPO Publication No. WO/2013/169576 and WO/2013/166654), and selective glycosidase inhibitors (see, for example, WIPO Publication No. WO/2013/ 000084 and WO/2013/000085). In some embodiments, the O-GlcNAse inhibitor comprises Thiamet-G. In certain embodiments, the methods, compositions, and kits disclosed herein do not include an O-GlcNase inhibitor.

[481] Mixed compositions

[482] Another aspect of the present invention relates to mixing insulin-positive endocrine cells or their precursors and at least one β-cell maturation factor, for example, to induce the differentiation of at least one insulin-positive endocrine cell or its precursor into SC- β.

[483] In another aspect, the present invention provides a composition, such as a reaction mixture, comprising at least one insulin-positive endocrine cell or progenitor thereof (e.g., a population of insulin-positive endocrine cells or progenitors thereof for differentiation into SC-β cells) and at least one β-cell maturation factor. In an alternative embodiment, the present invention relates to a reaction mixture containing (i) a population of SC-β cells obtained by chemically inducing differentiation of a population of insulin-positive endocrine cells or their progenitors to differentiate into an SC-β cell, and (ii) at least one factor of maturation of β-cells.

[484] In some embodiments, the concentration of at least one β-cell maturation factor added to the reaction mixture is a sufficient dose to induce differentiation of at least one insulin-positive endocrine cell or its progenitor into an SC-β cell, as described in this document.

[485] In some embodiments, the composition comprises a concentration of at least one β-cell maturation factor of about 25 nM to 10 µM, or about 25 nM to 50 nM, or about 50 nM to 100 nM, or about 100 nM to 200 nM, or about 200 nM to about 500 nM, or about 500 nM to about 1 µM, or about 1 µM to 2 µM, or about 2 µM to 5 µM, or about 5 µM to 10 µM.

[486] In some embodiments, the composition or mixture comprises a concentration of at least one β-cell maturation factor of at least about 5 nM, at least about 7 nM, at least about 10 nM, at least about 12 nM, at least about 15 nM, at least about 17 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM , at least about 45 nM, at least about 50 nM, at least about 100 nM, or at least about 200 nM, or at least about 300 nM, or at least about 400 nM, or at least about 500 nM, or greater than 500 nM, or any integer in the range 10-500 nM, or any integer in the range 5-50 nM, or any integer in the range 50-100 nM, or any integer in the range 100 nM -200 nM, or any integer in the range 200 nM-500 nM. In some embodiments, the composition or mixture contains a concentration of at least one β-cell maturation factor of at least about 0.1 μM, or at least about 0.2 μM, or at least about 0.3 μM, or at least about 0.4 µM, or at least about 0.5 µM, or at least about 1 µM, at least about 1.5 µM, at least about 2 µM, at least about 2.5 μM, at least about 3 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 6 μM, at least at least about 7 μM, at least about 8 μM, at least about 9 μM, or at least about 10 μM, or greater than 10 μM, or any integer in the range of 0.1-0.5 μM, or any an integer in the range 0.5-10 µM, or any integer in the range 0.1-10 µM, or any integer in the range 0.5-5 µM, or any integer in the range 5 µM-10 µM.

[487] Compositions and sets

[488] This document describes compositions that contain the cell described herein (eg, SC-β cell or mature pancreatic β-cell). In some embodiments of the invention, the composition also contains the β-cell maturation factor described herein and/or cell culture media. Also described herein are compositions containing the compounds described herein (eg, cell culture media containing one or more of the compounds described herein). This document describes the kits.

[489] Another aspect of the present invention relates to kits for practicing the methods disclosed herein and for producing SC-β cells or mature pancreatic β-cells disclosed herein. In one aspect, the kit contains at least one insulin-positive endocrine cell or progenitor thereof and at least one β-cell maturation factor as described herein, and optionally, the kit may further contain instructions for converting at least one insulin α-positive endocrine cell or its progenitor into the SC-β cell population using the methods described herein. In some embodiments, the kit contains at least two β-cell maturation factors. In some embodiments, the kit contains at least three β-cell maturation factors. In some embodiments, the kit contains at least four β-cell maturation factors. In some embodiments, the kit contains at least five β-cell maturation factors. In some embodiments, the kit contains at least six β-cell maturation factors. In some embodiments, the kit contains at least seven β-cell maturation factors. In some embodiments, the kit contains at least eight β-cell maturation factors. In some embodiments, the kit contains at least nine β-cell maturation factors. In some embodiments, the kit contains at least ten β-cell maturation factors. In some embodiments, the kit contains β-cell maturation factors for differentiating pluripotent stem cells into definitive endoderm cells. In some embodiments, the kit contains β-cell maturation factors for differentiating definitive endoderm cells into primary intestinal tube cells. In some embodiments, the kit contains β-cell maturation factors for differentiating primary intestinal tube cells into Pdx1-positive pancreatic progenitor cells. In some embodiments, the kit contains β cell maturation factors for differentiating NKX6-1 positive pancreatic progenitor cells into insulin positive endocrine cells. In some embodiments, the kit contains β-cell maturation factors for differentiating insulin-positive endocrine cells into SC-β cells.

[490] In some embodiments, the kit contains any combination of β-cell maturation factors, for example, to differentiate pluripotent stem cells into definitive endoderm cells, differentiate definitive endoderm cells into primary intestinal tube cells, differentiate primary intestinal tube cells from Pdx1-positive pancreatic cells -progenitors, differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells, and differentiation of insulin-positive endocrine cells into SC-β cells.

[491] In one embodiment of the invention, the kit may contain a pluripotent stem cell for use as a positive control, for example, to evaluate or monitor the effectiveness or ability of a compound of formula (I) to chemically induce differentiation of a pluripotent stem cell into at least one insulin-positive an endocrine cell or its precursor and subsequently into an SC-β cell. Accordingly, the kit may contain a sufficient amount of at least one β-cell maturation factor to induce differentiation of a control population of pluripotent stem cells (positive control) as well as to induce differentiation of a population of pluripotent stem cells of interest (e.g., a user's preferred pluripotent stem cell, e.g. , iPS cells) into at least one insulin-positive endocrine cell or progenitor cell or SC-β cell.

[492] In some embodiment, the compound in the kit may be provided in a waterproof or gas-tight container, which, in some embodiments, contains substantially no other components of the kit. The compound may be supplied in more than one container, for example, it may be supplied in a container containing sufficient reagents for a predetermined number of reactions, for example 1, 2, 3 or more individual reactions to induce pluripotent stem cells into definitive endoderm cells and subsequently into insulin-positive endocrine cells, and subsequently into SC-β cells. The β-cell maturation factor may be provided in any form, such as liquid, dry or lyophilized form. It is preferred that the compound(s) described herein (eg, β-cell maturation factors) be substantially purified and/or sterile. When the compound(s) described herein is provided as a liquid solution, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being preferred. When the compound(s) described herein is provided in dry form, recovery is generally carried out by adding a suitable solvent. A solvent, such as sterile water or a buffer, may optionally be provided in a kit.

[493] In some embodiments of the invention, the kit optionally additionally contains information material. Information material may be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the compound(s) described herein in the methods described herein.

[494] The information material of the kits is not limited in its instructive or informative material. In one embodiment of the invention, the information material may include information about the production of the compound, the molecular weight of the compound, concentration, expiration date, information about the lot number and place of production, and so on. In one embodiment of the invention, the informational material relates to methods of administering the compound. In addition, the information material of the kits is not limited in its form. In many cases, the information material, such as instructions, is provided in printed form, such as printed text, an image, and/or a photograph, such as a label or printed sheet. However, the information material may also be provided in other formats such as Braille, machine-readable material, videotape or audiotape. In another embodiment of the invention, the information material from the kits contains contact information, for example, a physical address, an email address, a website or a telephone number, where the user of the kit can obtain comprehensive information about the connection described herein and / or its use in those described in methods in this document. Of course, the information material may also be provided in any combination of formats.

[495] In one embodiment, the communication material may contain instructions for administering the compound(s) described herein (e.g., β-cell maturation factor) in an appropriate manner to carry out the methods described herein, e.g., in a suitable dosage, dosage form or by a suitable route of administration (eg, in the dosage, dosage form, or route of administration described herein) (eg, into an in vitro cell or an in vivo cell). In another embodiment, the communication material may contain instructions for administering the compound(s) described herein to a suitable subject, eg, a human, eg, a human at risk of having the disorder described herein, or into a cell in vitro.

[496] In addition to the compound(s) described herein, a kit composition may contain other ingredients such as a solvent or buffer, stabilizer, preservative, flavoring agent (e.g., bittering agent or sweetener), flavoring or other cosmetic ingredient, and/or an additional agent, for example, for the induction of pluripotent stem cells (eg, in vitro) or for the treatment of a pathological condition or disorder described herein. Alternatively, other ingredients may be included in the kit, but in different compositions or in different containers than the compound described herein. In such embodiments, the kit may contain instructions for mixing the compound(s) described herein and other ingredients, or for using the compound(s) described herein with other ingredients, such as instructions for mixing the two agents prior to administration.

[497] The β-cell maturation factor described herein may be provided in any form, such as liquid, dry, or lyophilized form. It is preferred that the compound(s) described herein be substantially purified and/or sterile. When the compound(s) described herein is provided as a liquid solution, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being preferred. When the compound(s) described herein is provided in dry form, recovery is generally carried out by adding a suitable solvent. A solvent, such as sterile water or a buffer, may optionally be provided in a kit.

[498] The kit may contain one or more containers for a composition containing at least one β-cell maturation factor described herein. In some embodiments of the invention, the kit contains separate containers (for example, two separate containers for two agents), partitions or compartments for composition(s) and informational material. For example, the composition may be in a bottle, vial or syringe, and the communication material may be in a plastic file or bag. In other embodiments of the invention, the individual elements of the set are in the same non-separated container. For example, the composition is in a bottle, vial or syringe with attached information material in the form of a label. In some embodiments, a kit comprises a plurality (eg, a package) of individual containers, each containing one or more unit dosage forms (eg, a dosage form described herein) of a compound described herein. For example, a kit contains a plurality of syringes, ampoules, foil packs, or blister packs, each containing a single dose of a compound described herein. The containers of the kits may be airtight, waterproof (eg, resistant to changes in moisture or evaporation), and/or light-tight.

[499] The kit optionally comprises a device suitable for administering the composition, such as a syringe, inhaler, pipette, tongs, measuring spoon, dropper (eg, eye dropper), swab (eg, cotton swab or wood swab), or any similar device. For delivery. In a preferred embodiment of the invention, the device is a medical implant device, for example, packaged for surgery.

[500] Also, the kit may also contain a component for detecting a marker of SC-β cells, for example, the marker described herein, for example, a reagent for detecting positive SC-β cells. Or, in some embodiments, the kit may also contain reagents for detecting negative markers of SC-β cells, for negative selection of SC-β cells, or for detecting cells that do not express negative markers (eg, SC-β cells). Reagents can be, for example, an antibody to a marker or primers for a RT-PCR or PCR reaction, for example, a semi-quantitative or quantitative RT-PCR or PCR reaction. Such markers can be used to determine iPS cell production. If the detection reagent is an antibody, it may be provided as a dry preparation, eg lyophilized, or in solution. The antibody or other detection reagent may be associated with a label, such as a radioactive, fluorescent (eg, GFB), or colorimetric label, for use in detection. If the detection reagent is a primer, it may be provided as a dry preparation, eg lyophilized, or in solution.

[501] There may be a need to analyze the karyotype of SC-β cells. Accordingly, the kit may contain a component for karyotyping, for example, a probe, dye, substrate, enzyme, antibody, or other reagents used to obtain a karyotype of the cell.

[502] The kit may contain SC-β cells, eg, mature pancreatic β cells, derived from insulin-positive endocrine cells or their precursors of the same type, eg, for use as a positive control cell type.

[503] Also, the kit may contain informational materials, such as instructions, for the use of two or more components included in the kit.

[504] Information material may be descriptive, instructional, marketing, or other material that relates to the methods described herein and/or the use of the compound(s) described herein to differentiate a pluripotent stem cell in accordance with the methods described herein. In one embodiment of the invention, the information material may include information about the production of the compound, the molecular weight of the compound, concentration, expiration date, information about the lot number and place of production, and so on. In one embodiment, the information material relates to methods for culturing a population of insulin-positive endocrine cells in the presence of at least one β-cell maturation factor described herein.

[505] Methods for administering cells

[506] In one embodiment, the cells described herein, for example, a population of SC-β cells, are transplantable, for example, a population of SC-β cells can be administered to a subject. In some embodiment, the subject to which the SC-β cell population is administered is the same subject from which the pluripotent stem cell was derived for differentiation into an SC-β cell (eg, for autologous cell therapy). In some embodiments of the invention, the subject is another subject. In some embodiments of the invention, the subject suffers from diabetes, for example, type I diabetes, or is a normal subject. For example, cells for transplantation (eg, a composition containing a population of SC-β cells) may be in a form suitable for transplantation, for example, organ transplantation.

[507] Said method may further comprise administering the cells to a subject in need, such as a mammalian subject, such as a human subject. The source of the cells may be a mammal, preferably a human. The source or recipient of the cells can also be a non-human subject, such as an animal model. The term "mammal" includes organisms that include mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and preferably humans. Likewise, transplantable cells can be obtained from any of these organisms, including a non-human transgenic organism. In one embodiment, the transplanted cells are genetically engineered, for example, the cells contain an exogenous gene or have been genetically engineered to inactivate or alter an endogenous gene.

[508] A composition containing a population of SC-β cells can be administered to a subject using an implantable device. Implantable devices and related technologies are known in the art and are useful as delivery systems when sustained or time-limited delivery of the compounds or compositions described herein is required. In addition, an implantable device-based delivery system is useful for targeting specific sites for delivery of a compound or composition (eg, specific sites, organs). Negrin et al., Biomaterials, 22(6):563 (2001). The present invention may also employ time-limited release technology that utilizes alternative delivery methods. For example, time-limited release formulations based on polymeric technologies, sustained release technologies, and encapsulation (eg, polymeric, liposomal) technologies can also be used to deliver the compounds or compositions described herein.

[509] Pharmaceutical compositions containing a population of insulin-producing glucose-responsive cells

[510] For administration to a subject, a population of cells obtained by the methods disclosed herein, for example, a population of SC-β cells (obtained by bringing at least one insulin-positive endocrine cell into contact with at least one β-cell maturation factor (for example , one, two, three, four, five or more of the β-cell maturation factors described herein)) can be administered to a subject, for example, in pharmaceutically acceptable compositions. These pharmaceutically acceptable compositions contain a therapeutically effective amount of a population of SC-β cells, as described above, admixed together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.

[511] As described in more detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, for example, liquid dosage forms (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (eg intended for buccal, sublingual or systemic absorption), boluses, powders, granules, tongue pastes; (2) parenteral administration, for example by subcutaneous, intramuscular, intravenous or epidural injection such as a sterile solution or suspension or sustained release preparation; (3) topical administration, eg, a controlled release cream, ointment, or skin patch or spray; (4) intravaginal or rectal, for example, in the form of a pessary, cream or foam; (5) sublingual; (6) ophthalmic; (7) transdermal; (8) transmucosal; or (9) nasal administration. In addition, the compounds can be implanted or injected into a patient's body using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24:199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); US patent No. 3773919; and US Patent No. 353270960.

[512] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms that are medically suitable for use in contact with human and animal tissues without undue toxicity, irritation, or allergic reactions. or other problems or complications and are characterized by a rational ratio of beneficial effect/risk.

[513] As used herein, the term “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or medium such as a liquid or solid excipient, diluent, excipient, manufacturing aid (e.g., lubricant, talc, calcium or zinc stearate). or steric acid) or a solvent-encapsulating material that is involved in the transfer of a specific compound from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and harmless to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as glucose, lactose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients such as coconut oil and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents such as polypeptides and amino acids (23) serum components such as serum albumin, HDL and LDL; (22) C ₂ -C ₁₂ alcohols such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical preparations. Wetting agents, colorants, separating agents, coating agents, sweeteners, flavors, flavors, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" and the like are used interchangeably herein.

[514] The expression "therapeutically effective amount" as used herein in relation to a population of cells means that the number of appropriate cells in a population of cells, for example, SC-β cells or mature pancreatic β-cells, or a composition containing SC-β cells according to the present invention, is effective in the sense of producing the desired therapeutic effect in at least a subpopulation of cells in the body of the animal with a rational benefit/risk ratio applicable to any type of treatment. For example, this is an amount of a population of SC-β cells administered to a subject that is sufficient to result in a statistically significant, detectable change in at least one symptom of type 1, type 1.5, or type 2 diabetes, such as glycated hemoglobin level, glucose level fasting blood, hypoinsulinemia, etc. Methods for determining a therapeutically effective amount are well known to those skilled in the art. In general, the therapeutically effective amount may vary depending on the subject's medical history, age, condition, sex, as well as the severity and type of the subject's condition and the administration of other pharmaceutically active agents.

[515] As used herein, the term "administer" refers to introducing a composition into a subject's body in a manner or manner that results in at least partial localization of the composition at the desired location in the body so that the desired effect is produced. The compound or composition described herein may be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), inhalation, nasal, rectal and topical (including buccal and sublingual) administration.

[516] Typical modes of administration include, but are not limited to, injection, infusion, installation, inhalation, or ingestion. "Injection" includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In preferred embodiments, the compositions are administered by intravenous infusion or injection.

[517] By "treating", "preventing" or "alleviating" a disease or disorder is meant delaying or preventing the onset of such disease or disorder, reversing, alleviating, alleviating, suppressing, slowing or stopping the progression, aggravating or aggravating the progression or severity of a condition, associated with such disease or disorder. In one embodiment, the symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least fifty%.

[518] Treatment of diabetes is determined by standard medical methods. The goal of diabetes treatment is to bring your blood sugar levels as close to normal as possible within safety limits. Typically goals are 80-120 milligrams per deciliter (mg/dL) on an empty stomach and 100-140 mg/dL at bedtime. A particular physician may set different goals for the patient depending on other factors, such as how often the patient has a hypoglycemic reaction. Medical tests used include testing the patient's blood and urine to determine blood sugar levels, testing glycated hemoglobin (HbA1c; measuring the average blood glucose level over the past 2-3 months, with a normal range of 4-6%), testing cholesterol levels, and fat, as well as a study of the level of protein in the urine. Such studies are standard studies known to those skilled in the art (see, for example, American Diabetes Association, 1998). A successful treatment program can also be determined by reducing the number of patients in the program who have complications associated with diabetes, such as eye disease, kidney disease, or nerve disease.

[519] Delaying the onset of diabetes in a subject refers to delaying the onset of at least one symptom of diabetes, for example, hyperglycemia, hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, kidney failure, cardiovascular disease (including coronary heart disease, peripheral arterial disease, cerebrovascular disease, atherosclerosis, and high blood pressure), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, or combinations thereof, for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months At least 6 months At least 1 year At least 2 years At least 5 years At least 10 years At least 20 years At least 30 years, at least 40 years or more, and may include the entire lifespan of the subject.

[520] In certain embodiments of the invention, the subject is a mammal, such as a primate, such as a human. The terms "patient" and "subject" are used interchangeably herein. Preferably the subject is a mammal. The mammal may be a human, monkey, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Non-human mammals can advantageously be used as subjects that represent animal models of type 1 diabetes, type 2 diabetes, diabetes mellitus, or pre-diabetic conditions. In addition, the methods described herein can be used to treat domesticated animals and/or companion animals. The subject may be male or female. The subject may be someone who has previously been diagnosed or diagnosed with diabetes (e.g., type 1 or type 2), one or more complications associated with diabetes, or a pre-diabetic condition, and optionally who has previously been treated for diabetes, one or more complications. associated with diabetes, or a pre-diabetic condition. The subject may also be someone who is not suffering from diabetes or a pre-diabetic condition. A subject may also be someone who has been diagnosed or identified with diabetes, one or more diabetes-related complications, or a pre-diabetic condition, but who has demonstrated improvement in known risk factors for diabetes as a result of receiving one or more diabetes treatments, one or more complications associated with diabetes, or a pre-diabetic condition. Alternatively, the subject may also be one who has not previously been diagnosed with diabetes, one or more complications associated with diabetes, or a pre-diabetic condition. For example, a subject may be one who demonstrates the presence of one or more risk factors for diabetes, complications associated with diabetes, or a pre-diabetic condition, or a subject who does not demonstrate risk factors for diabetes, or a subject asymptomatic for diabetes, one or more complications, associated with diabetes, or pre-diabetic condition. The subject may also be one who suffers from or is at risk of developing diabetes or a pre-diabetic condition. A subject may also be one who has been diagnosed or identified with one or more diabetes-related complications or a pre-diabetic condition as defined herein, or alternatively, a subject may be one who has not previously been diagnosed or identified with one or more more complications associated with diabetes, or a pre-diabetic condition.

[521] As used herein, "a subject in need of SC-β cells" refers to a subject who is diagnosed or identified with or at risk of developing diabetes (e.g., type 1, type 1.5, or type 2), one or more complications associated with diabetes, or a pre-diabetic condition.

[522] A subject in need of a population of SC-β cells can be determined using any of the methods used to diagnose diabetes. For example, type 1 diabetes can be diagnosed with a glycosylated hemoglobin (A1C) test, a random blood glucose test, and/or a fasting blood glucose test. Diagnosis parameters for diabetes are known in the art and readily available to those skilled in the art.

[523] In some embodiments, the methods of the invention further comprise selecting a subject in need of additional SC-β cells. A subject in need of a population of SC-β cells can be selected based on the symptoms presented, such as symptoms of type 1, type 1.5, or type 2 diabetes. Typical symptoms of diabetes include, but are not limited to, increased thirst (polydipsia), frequent urination (polyuria), increased hunger (polyphagia), increased fatigue, weight loss, hyperglycemia, low insulin levels, high blood sugar (e.g., levels sugars greater than 250 mg, greater than 300 mg), urinary ketones, fatigue, dry and/or irritated skin, blurred vision, slow-healing cuts and wounds, more infections than usual, numbness and tingling in the feet, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including ischemic heart disease, peripheral arterial disease, cerebrovascular disease, atherosclerosis, and high blood pressure), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, or combinations thereof.

[524] In some embodiments, a composition comprising a population of SC-β cells to be administered to a subject may further comprise a pharmaceutically active agent, such as those known in the art for the treatment of diabetes or agents having hypoglycemic activity, such as inhibitors dipeptidyl peptidases 4 (DPP-4) (eg, alogliptin, linagliptin, saxagliptin, sitagliptin, vildagliptin, and berberine), biguanides (eg, metformin, buformin, and phenformin), peroxisome proliferator-activated receptor (PPAR) modulators such as thiazolidinediones (TZD) (eg, pioglitazone, rivoglitazone, rosiglitazone, and troglitazone), dual PPAR agonists (eg, aleglitazar, muraglitazar, and tezaglitazar), sulfonylureas (eg, acetohexamide, carbutamide, chlorpropamide, gliclazide, tolbutamide, tolazamide, glibenclamide (glyburide), glipizide, gliquidone, glycopyramide and glimepiride), meglitinides (“glinides”) (for example, nateglinide, repaglinide, and miti glinide), glucagon-like peptide-1 (GLP-1) and its analogs (eg, exendin-4, exenatide, liraglutide, albiglutide), insulin and insulin analogs (eg, insulin lispro, insulin aspart, insulin glulisine, insulin glargine, insulin detemir, Exubera, and NPH insulin), alpha-glucosidase inhibitors (eg, acarbose, miglitol, and voglibose), amylin analogs (eg, pramlintide), sodium-dependent glucose cotransporter T2 (SGLT T2) inhibitors (eg, dapagliflozin, remogliflozin, and sergliflozin ) and others (for example, benfluorex and tolrestat).

[525] In type 1 diabetes, unwanted destruction of β-cells occurs due to a continuous autoimmune response. Therefore, this autoimmune response can be attenuated by the use of compounds that suppress or block such an autoimmune response. In some embodiments, a composition comprising a population of SC-β cells to be administered to a subject may further comprise a pharmaceutically active agent that is an immune response modulator. As used herein, the term "immune response modulator" refers to a compound (eg, small molecule, antibody, peptide, nucleic acid, or gene therapy reagent) that suppresses an autoimmune response in a subject. Without being limited by theory, it can be said that the immune response modulator suppresses the autoimmune response by suppressing the activity, activation or expression of inflammatory cytokines (for example, IL-12, IL-23 or IL-27) or STAT-4. Exemplary immune response modulators include, but are not limited to, members of the group consisting of lysophyllin (LSF) and LSF analogues and derivatives described in US Pat. No. 6,774,130, the contents of which are incorporated herein by reference in their entirety.

[526] A composition containing SC-β cells can be administered to a subject at the same time or at different times with the introduction of a pharmaceutically active agent or composition containing it. When administered at different times, compositions containing a population of SC-β cells and/or a pharmaceutically active agent for administration to a subject may be administered over 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 8 hours. hours, 12 hours, 24 hours after the introduction of another. If the compositions containing the SC-β cell population and the composition containing the pharmaceutically active agent are administered in different pharmaceutical compositions, the route of administration may be different. In some embodiments of the invention, the subject is administered a composition containing SC-β cells. In other embodiments of the invention, the subject is administered a composition containing a pharmaceutically active agent. In another embodiment, the subject is administered compositions containing a population of SC-β cells admixed with a pharmaceutically active agent. In another embodiment of the invention, a composition containing a population of SC-β cells and a composition containing a pharmaceutically active agent are administered to the subject, with the introduction occurring at approximately the same time or sequentially one after the other.

[527] The toxicity and therapeutic efficacy of administration of compositions containing a population of SC-β cells can be determined using standard pharmaceutical procedures in cell cultures or in experimental animals, for example, to determine the LD50 (dose lethal to 50% of the population) and ED50 (dose , therapeutically effective for 50% of the population). Compositions containing a population of SC-β cells that exhibit high therapeutic indices are preferred.

[528] The number of compositions containing a population of SC-β cells can be tested in several well-known animal models.

[529] Non-obese diabetic (NOD) mice carry a genetic defect that results in insulitis manifesting at a few weeks of age (Yoshida et al., Rev. Immunogenet. 2:140, 2000). In 60-90% of females, clinical diabetes mellitus develops after 20-30 weeks. Immune pathology is similar to that observed in type I diabetes in humans. Other models of type I diabetes are mice carrying transgenic and knockout mutations (Wong et al., Immunol. Rev. 169:93, 1999). Recently in Lenzen et al. (Diabetologia 44:1189, 2001) a rat model of spontaneous type I diabetes has been reported. Hyperglycemia (>500 mg glucose/dl) can also be induced in mice by a single intraperitoneal injection of streptozotocin (Soria et al., Diabetes 49:157, 2000) or by sequential application of low doses of streptozotocin (Ito et al., Environ. Toxicol. Pharmacol 9:71, 2001). To investigate the efficacy of implanted islets, mice are monitored for glucose to return to normal levels (<200 mg/dl).

[530] Larger animals are a good model for studying the complications of chronic hyperglycemia. Dogs can be made insulin dependent by pancreas removal (J. Endocrinol. 158:49, 2001) or by feeding galactose (Kador et al., Arch. Opthalmol. 113:352, 1995). There is also a hereditary model for type I diabetes in Keeshond dogs (Am. J. Pathol. 105:194, 1981). One of the earliest works with the canine model (Banting et al., Can. Med. Assoc. J. 22:141, 1922) resulted in two Canadians making a long ocean voyage to Stockholm in February 1925.

[531] By way of illustration, a pilot study can be carried out by implanting SC-β cells in the body of the following animals: a) non-diabetic athymic (deficient in relation to T-cells) mice; b) athymic mice with streptozotocin treatment-induced diabetes; and c) Nude mice during islet regeneration after partial pancreatic resection. The number of transplanted cells is equivalent to ^~ 1000-2000 normal human β-cells implanted under the renal capsule, in the liver or in the pancreas. In the case of non-diabetic mice, endpoints may be graft survival (by histology) and insulin production by chemistry, RIA, ELISA, and immunohistochemistry. For streptozotocin-treated animals and animals with partial pancreatic resection, survival, metabolic regulation (blood glucose) and weight gain can also be assessed.

[532] In some embodiments of the invention, data obtained after analysis of cell culture and in animal studies can be used in the selection of a range of dosages for use in humans. The dosage of such compounds preferably corresponds to a range of circulating concentrations that includes an ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration.

[533] A therapeutically effective dose of a composition containing a population of SC-β cells can also be initially estimated using cell culture analysis. The dose can be determined in in vivo animal models to achieve insulin secretion at an appropriate concentration in response to circulating plasma glucose levels. Alternatively, the effect of any particular dosage can be monitored using an appropriate bioassay method.

[534] To make decisions regarding the duration and frequency of treatment, medical specialists typically monitor the condition of the subjects to determine whether the treatment has a beneficial therapeutic effect, and to determine whether to reduce or increase the dosage, reduce or increase the frequency of administration, interrupt treatment, continue treatment, or make other changes to the treatment regimen. The dosage regimen may vary from once a week to daily, depending on a number of clinical factors such as the subject's sensitivity to the polypeptides. The required amount can be administered at one time or divided into fractional doses, such as 2-4 fractional doses, and administered over a period of time, such as at appropriate intervals throughout the day or according to another regimen. Such fractional doses may be administered as unit dosage forms. In some embodiments of the invention, the introduction is constant, for example, one or more doses daily over a period of weeks or months. Examples of dosing regimens are daily, twice a day, three times a day, or four or more times a day over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months. , 5 months or 6 months or more.

[535] In another aspect of the invention, the methods provide for the use of a population of SC-β cells as disclosed herein. In one embodiment of the invention, the isolated population of SC-β cells disclosed herein can be used to manufacture a pharmaceutical composition for transplantation to subjects in need of treatment, for example, a subject who is ill or at risk of developing diabetes, for example, without limitation, subjects with hereditary or acquired diabetes. In one embodiment, the isolated population of SC-β cells may be genetically modified. In another aspect, the subject may be ill or at risk for diabetes and/or a metabolic disorder. In some embodiments, the isolated population of SC-β cells disclosed herein may be autologous and/or allogeneic. In some embodiments, the subject is a mammal, and in other embodiments, the mammal is a human.

[536] The use of the isolated population of SC-β cells disclosed herein has advantages over existing methods, since the population of SC-β cells can be differentiated from insulin-positive endocrine cells or their progenitors derived from stem cells, e.g. iPS obtained or collected from a subject who is injected with an isolated population of SC-β cells. This is a huge advantage as it provides a renewable source of SC-β cells that can be differentiated from stem cells into insulin-positive endocrine cells by methods known to those skilled in the art, and then further differentiated by methods described herein into pancreatic β-like cells. cells or cells having the characteristics of pancreatic β-cells for transplantation into a subject, in particular a substantially pure population of mature pancreatic β-like cells that do not bear the risks and limitations of cells derived from other systems.

[537] In another embodiment, an isolated population of SC-β cells (e.g., mature pancreatic β-cells or β-like cells) can be used as models to study differentiation properties into insulin-producing cells, such as pancreatic β-cells. or pancreatic β-like cells, or developmental pathways of cells of endodermal origin into pancreatic β-cells.

[538] In some embodiments, insulin-positive endocrine cells or SC-β cells can be genetically engineered to contain markers operably linked to promoters that are expressed when the marker is expressed or secreted, for example, the marker can be operably linked to insulin promoter and thus marker expression occurs when insulin-positive endocrine cells or their progenitors differentiate into SC-β cells that express and secrete insulin. In some embodiments, a population of SC-β cells can be used as a model for studying the differentiation pathway of cells that differentiate into islet β cells or pancreatic β-like cells.

[539] In other embodiments of the invention, insulin-producing glucose-responsive cells can be used as models to study the role of islet β-cells in the pancreas and in the development of diabetes and metabolic disorders. In some embodiments, the SC-β cells may be from a normal subject or a subject that carries a mutation and/or polymorphism (e.g., in the Pdx1 gene that results in non-insulin-dependent early-onset diabetes mellitus (NIDDM) as well as adult-onset diabetes mellitus in juvenile type 4 (MODY4), which can be used to identify small molecules and other therapeutic agents that can be used to treat subjects with diabetes carrying a mutation or polymorphism in Pdx1.In some embodiments, SC-β cells can be genetically engineered so to correct a polymorphism in the Pdx1 gene prior to administration to a subject for therapeutic treatment of a subject with diabetes In some embodiments, SC-β cells can be genetically engineered to carry the mutation and/or polymorphism.

[540] In one embodiment, the invention relates to a method of treating diabetes or a metabolic disorder in a subject, comprising administering an effective amount of a composition comprising the SC-β cell population disclosed herein to a subject with diabetes and/or a metabolic disorder. In a further embodiment, the invention provides a method of treating diabetes, comprising administering a composition comprising a population of SC-β cells disclosed herein to a subject who is ill or at increased risk of developing diabetes, in an effective amount sufficient to produce insulin in response to elevated levels. blood glucose.

[541] In one embodiment of the above methods, the subject is a human, and the population of SC-β cells disclosed herein is human cells. In some embodiments, the invention contemplates that the population of SC-β cells disclosed herein is administered directly to the subject's pancreas or is administered systemically. In some embodiments, the SC-β cell population disclosed herein may be administered at any suitable site in the subject's body, such as in a capsule in a blood vessel or liver, or any suitable site where the administered SC-β cell population can secrete insulin into response to elevated blood glucose levels.

[542] The present invention also relates to a method of treating a subject with diabetes or a metabolic disorder that results from a genetic defect, physical injury, environmental or air pollution, ill health, obesity, and other risk factors for diabetes known to those skilled in the art. technology. Treatment efficacy in a subject administered a composition comprising a population of SC-β cells can be monitored by clinically accepted criteria and tests that include, for example, (i) the glycated hemoglobin (A1C) test, which provides information on the subject's mean blood sugar level. over the past two to three months based on the determination of the percentage of blood sugar attached to hemoglobin, the oxygen carrier protein in red blood cells. The higher the blood sugar level, the more sugar attached to hemoglobin. An A1C level of 6.5 percent or more in two separate samples indicates that the subject has diabetes. A value of 6-6.5% suggests that the subject has prediabetes. (ii) Random blood sugar. A blood sample is taken from the subject at a random time, and a random blood sugar level of 200 milligrams per deciliter (mg/dL) - 11.1 millimoles per liter (mmol/L) or higher is indicative of the subject having diabetes, ( iii) Fasting blood sugar test. A blood sample is taken from the subject after an overnight fast. Fasting blood sugar levels of 70-99 mg/dL (3.9 and 5.5 mmol/L) are normal. If the subject's fasting blood sugar is 126 mg/dL (7 mmol/L) or higher on two separate samples, the subject has diabetes. A fasting blood sugar level of 100 to 125 mg/dL (5.6 to 6.9 mmol/L) indicates that the subject has prediabetes. (iv) Oral glucose tolerance test. A blood sample is taken from the subject after the subject has been fasting for at least eight hours or overnight and has subsequently drunk a sugar solution, and the blood sugar level is determined two hours later. A blood sugar level of less than 140 mg/dL (7.8 mmol/L) is normal. A blood sugar level of 140 to 199 mg/dL (7.8 to 11 mmol/L) is considered pre-diabetic. This is sometimes called impaired glucose tolerance (IGT). A blood sugar level of 200 mg/dL (11.1 mmol/L) or higher indicates that the subject has diabetes.

[543] In some embodiments, the effect of administering the population of SC-β cells disclosed herein to a subject in need thereof is associated with improved exercise tolerance or other quality of life measures, as well as reduced mortality. The effect of cell therapy with a population of SC-β cells can be manifested within a time ranging from days to weeks after the procedure. At the same time, beneficial effects can appear within a few hours after the procedure and can last for several years. In some embodiments of the invention, the effect of cell therapy with a population of SC-β cells is manifested within two weeks after the procedure.

[544] In some embodiments, the SC-β cell population disclosed herein can be used to repair or regenerate tissues in a human patient or other subject in need of such treatment. In some embodiments of the invention, compositions containing populations of SC-β cells can be administered in a manner that allows engraftment or migration to a desired tissue site and repair or regeneration of a functionally deficient area. Special devices are available that are adapted to deliver cells capable of repopulating the β-cell population in the pancreas or other desired site. Accordingly, SC-β cells can be injected into the recipient's pancreas or administered by intramuscular injection.

[545] In some embodiments of the invention, compositions containing the population of SC-β cells disclosed herein have many applications in clinical therapy, research, development, and commercial purposes. For example, for therapeutic purposes, the population of SC-β cells disclosed herein can be administered to increase insulin production in response to an increase in blood glucose in case of any obvious need, such as an inborn metabolic disorder, a consequence of a pathological condition (for example, diabetes) or the result of a characteristic injury (ie, damage to the pancreas or loss or damage to islet β-cells). In some embodiments, the population of SC-β cells disclosed herein is administered to a subject not only to help restore the function of damaged or otherwise unhealthy tissues, but also to facilitate the remodeling of damaged tissues.

[546] To determine whether cell compositions are suitable for therapeutic administration, a population of SC-β cells can first be examined in a suitable animal model. At one level, cells are evaluated for their ability to survive and maintain their phenotype in vivo. Cell compositions containing SC-β cells can be administered to immunodeficient animals (such as nude mice, or animals with chemically or radiation-induced immunodeficiency). Tissues are harvested during regrowth and assessed for the presence of injected cells or their progeny.

[547] This can be done by introducing cells that express a detectable label (such as green fluorescent protein or β-galactosidase); that have been previously labeled (eg, with BrdU or [3H]thymidine), or by subsequent detection of a constitutive cell marker (eg, using a human-specific antibody). The presence and phenotype of an introduced SC-β cell population can be assessed by immunohistochemistry or ELISA using a human-specific antibody, or by RT-PCR analysis using primers and hybridization conditions that result in amplification specific to human polynucleotides consistent with published sequence data. .

[548] A large number of animal models for the study of diabetes are available and known in the art, for example, as disclosed in US Pat. No. 6,187,991, which is incorporated herein by reference, as well as rodent-based models; NOD (non-obese mice), BB_DB mice, KDP rats, and TCR mice and other animal models of diabetes as described in Rees et al, Diabet Med. April 2005; 22(4):359-70; Srinivasan K, et al., Indian J Med. Res. March 2007; 125(3):451-7; Chatzigeorgiou A, et al., In Vivo. 2009 March-April; 23(2):245-58, which are incorporated herein by reference.

[549] In some embodiments, the population of SC-β cells disclosed herein can be administered in any physiologically acceptable adjuvant, wherein the SC-β cells can find an appropriate site for replication, proliferation and/or engraftment. In some embodiments, the SC-β cell population disclosed herein can be introduced by injection, catheter, or the like. In some embodiments, the SC-β cell population disclosed herein can be frozen at liquid nitrogen temperature and stored for an extended period of time, usable after thawing. If frozen, the SC-β cell population is usually stored in a medium containing 10% DMSO, 50% FBS, 40% RPMI 1640. After thawing, the cells can be expanded by the application of growth factors and/or nurse cells relevant to culturing SC-β cells. as described in this document.

[550] In some embodiments, the SC-β cell population disclosed herein may be provided in the form of a pharmaceutical composition containing an isotonic excipient prepared under sufficiently sterile conditions for use in humans. For general principles of drug preparation, see Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. The choice of cellular excipient and any accompanying elements for a composition containing the SC-β cell population disclosed herein is adapted according to the route and device used for administration. In some embodiments, a composition comprising a population of SC-β cells may also contain or be supplemented with one or more other ingredients that facilitate engraftment or functional mobilization of SC-β cells. Suitable ingredients include matrix proteins that promote or stimulate adhesion of SC-β cells or complementary cell types, in particular epithelial cells. In another embodiment of the invention, the composition may contain resorbable or biodegradable matrix scaffolds.

[551] In some embodiments of the invention, the population of SC-β cells disclosed herein can be genetically modified to introduce genes useful for insulin-producing cells, such as pancreatic β cells, for example, to correct a genetic defect in an individual, introduce a selectable a marker, etc., or genes useful for selection against non-insulin producing cells differentiated from at least one insulin-positive endocrine cell or progenitor thereof, or for selective suicide of implanted SC-β cells. In some embodiments of the invention, the population of SC-β cells can also be genetically modified to increase survival, regulate proliferation, and the like. In some embodiments, the SC-β cell population disclosed herein can be genetically altered by transfection or transduction with an appropriate vector, homologous recombination, or other appropriate method such that the cells express a gene of interest. In one embodiment, a population of SC-β cells is transfected with genes encoding the telomerase catalytic component (TERT), typically under the control of a heterologous promoter, which increases telomerase expression over that seen with an endogenous promoter (see International Patent Application WO 98 /14592, which is incorporated herein by reference). In other embodiments of the invention, a selectable marker is introduced to provide a greater degree of purification of the SC-β cell population. In some embodiments, the SC-β cell population can be genetically modified with vector-containing supernatants over a period of 8-16 hours and then exchanged for growth medium for 1-2 days. Selection of genetically modified SC-β cells can be carried out using a drug susceptibility selection agent such as puromycin, G418 or blasticidin, and then culture is repeated.

[552] Gene therapy can be used to modify a cell to replace a gene product, to facilitate tissue regeneration, to treat a disease, or to improve cell survival after implantation in a subject (i.e., prevent rejection).

[553] In an alternative embodiment of the invention, the population of SC-β cells disclosed herein can also be genetically modified to increase their ability to participate in tissue regeneration or to deliver a therapeutic gene to the site of administration. The vector is constructed using the known coding sequence of the desired gene, operably linked to a promoter that is polyspecific or specifically active in a differentiated cell type. In particular, of interest are cells that have been genetically modified to express one or more different types of growth factors such as somatostatin, glucagon, and other factors.

[554] Many vectors are available for transferring exogenous genes to the SC-β target cells disclosed herein. These vectors may be episomal, such as plasmids, vectors derived from viruses such as cytomegalovirus, adenovirus, etc., or may be integrated into the genome of the target cell by homologous recombination or random integration, such as vectors derived from retroviruses. such as MMLV, HIV-1, ALV, etc. In some embodiments, combinations of retroviruses and appropriate packaging cell lines can also be used, wherein the capsid proteins remain functional to infect the SC-β cells disclosed herein. Typically, the SC-β cells and the virus are incubated for at least 24 hours in the culture medium. In some embodiments, the SC-β cells are allowed to grow in the culture medium for short intervals, in some applications 24-73 hours, or for at least two weeks, and may be allowed to grow for five weeks or more before being analysis. Commonly used retroviral vectors are "defective", ie. unable to produce viral proteins necessary for productive infection. Growth in a packaging cell line is required for vector replication.

[555] The specificity of the retrovirus to the host cell is determined by the envelope protein env (p120). The envelope protein is provided by the packaging cell line. Shell proteins are divided into at least three types - ecotropic, amphotropic and xenotropic. Retroviruses packaged with an ecotropic envelope protein, such as MMLV, are capable of infecting most mouse and rat cell types. Ecotropic packaging cell lines include BOSC23 (Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses carrying an amphotropic envelope protein such as 4070A (Danos et al, supra.) are capable of infecting most mammalian cell types, including humans, dogs and mice. Amphotropic packaging cell lines include PAl2 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with a xenotropic envelope protein, such as AKR env, are capable of infecting most mammalian cell types with the exception of mouse cells. In some embodiments of the invention, the vectors may contain genes that subsequently need to be removed, for example, using a recombinase system, such as Cre/Lox, or destroy the cells that express them, for example, by including genes that allow selective toxicity, such as herpes virus TK genes, Bc1-Xs, etc.

[556] Appropriate inducible promoters are activated in the desired target cell type, both in transfected cells and in their progeny. By transcriptional activation is meant that transcription in target cells is at least about 100-fold higher than baseline, more often at least about 1000-fold. Various promoters are known to be induced in different cell types.

[557] In one aspect of the present invention, the population of SC-β cells disclosed herein is suitable for systemic administration or for targeting to an anatomical site. A population of SC-β cells can, for example, be engrafted in or near the pancreas of a subject, or can be administered systemically, for example, without limitation, by intra-arterial or intravenous administration. In alternative embodiments of the invention, the population of SC-β cells according to the present invention can be administered by various routes suitable for implantation into the pancreatic or secretory system, including, but not limited to, parenteral, including intravenous and intra-arterial administration, intrathecal administration, intraventricular administration, intraparenchymal, intracranial, intracisternal, intrastriatal and intragigral administration. Optionally, the SC-β cell population is administered together with an immunosuppressive agent.

[558] In some embodiments, the population of SC-β cells can be administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, the mode of administration, the age, sex and body weight of the patient, as well as other factors known to medical practitioners. Thus, in the context of this document, a pharmaceutically "effective amount" is defined based on considerations known in the art. The amount must be effective in order to achieve an improvement, including, but not limited to, increased survival or faster recovery, or improvement or disappearance of symptoms and other indicators, as selected as appropriate indicators by those skilled in the art. The SC-β cell population can be administered to a subject in the following locations: clinic, clinical room, emergency department, hospital ward, intensive care unit, operating room, cath ward, and radiology ward.

[559] In other embodiments, the SC-β cell population is maintained for further implantation/infusion. The SC-β cell population can be divided into more than one aliquot or unit so that a portion of the SC-β cell population is retained for further use and a portion immediately administered to the subject. Medium or long-term storage of all or part of the cells in a cell bank is also within the scope of this invention, as disclosed in US Patent Application No. 20030054331 and Patent Application No. WO 03024215, which are incorporated herein by reference in their entirety. At the end of the treatment, the concentrated cells can be loaded into a delivery device, such as a syringe, for administration to the recipient by any means known to those skilled in the art.

[560] In some embodiments, a population of SC-β cells can be used alone or in combination with other cells, tissues, tissue fragments, growth factors such as VEGF and other known angiogenic or arteriogenic growth factors, biologically active or inert compounds, resorbable plastic matrices or other additives designed to improve delivery, efficacy, tolerability, or population performance. In some embodiments, the SC-β cell population can also be modified by insertion of DNA or by placement in cell culture in such a way as to alter, enhance, or supplement the function of the cells to achieve structural or therapeutic goals. For example, gene transfer methods for stem cells are known to those skilled in the art as disclosed in (Morizono et al., 2003; Mosca et al., 2000), which may include viral transfection methods and, in particular, gene transfer methods using adeno-associated viruses as disclosed in (Walther and Stein, 2000) and (Athanasopoulos et al., 2000). It is also possible to carry out methods on a non-viral basis, as disclosed in (Murarnatsu et al., 1998).

[561] In another aspect, in some embodiments of the invention, a population of SC-β cells can be combined with a gene encoding proangiogenic(e) growth factor(s). You can also use genes encoding anti-apoptotic factors or agents. The addition of a gene (or combination of genes) can be carried out using any method known in the art, including, but not limited to, adenoviral transduction, gene guns, liposome mediated transduction and retrovirus or lentivirus mediated transduction, plasmid adeno-associated virus. The cells can be implanted together with a carrier material carrying a gene delivery agent capable of releasing and/or presenting the genes in the cells over time so that transduction can continue or be initiated. In particular, when cells and/or tissue containing cells are administered to a patient other than the patient from whom the cells and/or tissue were obtained, one or more immunosuppressive agents may be administered to the patient receiving the cells and/or tissue to reduce and preferably prevent graft rejection. As used herein, the term "immunosuppressive drug or agent" includes pharmaceutical agents that suppress or interfere with immune function. Examples of immunosuppressive agents suitable for the methods disclosed herein include agents that inhibit T cell/B cell costimulation pathways, such as agents that interfere with T cell and B cell mating via the CTLA4 and B7 pathways as disclosed in US Patent Publication No. 2002/0182211, which is incorporated herein by reference. In one embodiment, the immunosuppressive agent is cyclosporin A. Other examples include mycophenolate mofetil, rapamycin, and antithymocyte globulin. In one embodiment, the immunosuppressive drug is administered with at least one therapeutic agent. The immunosuppressive drug is administered in a form that is compatible with the route of administration and administered at a dosage sufficient to achieve the desired therapeutic effect. In another embodiment of the invention, an immunosuppressive drug is administered temporarily for a sufficient time to induce tolerance in cardiovascular stem cells according to the invention.

[562] Pharmaceutical compositions containing effective amounts of a population of SC-β cells are also provided by the present invention. These compositions contain an effective amount of SC-β cells, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient. In some aspects of the present invention, a population of SC-β cells is administered to a subject in need of a transplant in a sterile saline solution. In other aspects of the present invention, the SC-β cell population is administered in Hank's Balanced Salt Solution (HBSS) or Isolate S, pH 7.4. Other approaches may also be used, including the use of a serum-free cell medium. In one embodiment, the SC-β cell population is administered in plasma or fetal bovine serum and DMSO. In some indications, systemic administration of a population of SC-β cells to a subject may be preferred, while direct administration at or near diseased and/or damaged tissue may be preferred in other indications.

[563] In some embodiments, the SC-β cell population may optionally be packaged in a suitable container with instructions for the necessary actions, such as reconstituting or thawing (in the case of a frozen product) the SC-β cell population prior to administration to a subject.

[564] In one embodiment, the isolated population of SC-β cells disclosed herein is administered together with a differentiation promoting agent. In one embodiment, the SC-β cells are combined with a differentiation promoting agent for administration to a subject. In another embodiment, the cells are administered to the subject separately from the differentiation promoting agent. Optionally, if the cells are administered separately from the differentiation promoting agent, there is a temporal separation between the administration of the cells and the differentiation promoting agent. The time division can range from less than a minute to hours or days. Determining the optimal time and order of administration is a simple and routine procedure for a person skilled in the art.

[565] Diagnosing Diabetes

[566] Type 1 diabetes is an autoimmune disease that results in the destruction of insulin-producing β-cells in the pancreas. The lack of insulin leads to an increase in fasting blood glucose (70-120 mg/dl in people without diabetes), which begins to appear in the urine above the elimination threshold (about 190-200 mg/dl for most people). According to the definition of the World Health Organization in the case of diabetes mellitus, the diagnostic value of fasting plasma glucose concentration corresponds to 7.0 mmol / l (126 mg / dl) and above (for whole blood 6.1 mmol / l or 110 mg / dl) or the level glucose after 2 hours is 11.1 mmol/L or higher (200 mg/dL or higher).

[567] Type 1 diabetes can be diagnosed using various diagnostic tests, which include, but are not limited to, the following: (1) a glycated hemoglobin (A1C) test, (2) a random blood glucose test, and/or (3 ) fasting blood glucose test.

[568] The glycated hemoglobin (A1C) test is a blood test that displays the average blood glucose level of a subject over the previous two to three months. This test determines the percentage of blood glucose attached to hemoglobin that correlates with blood glucose levels (for example, the higher the blood glucose level, the more glycated hemoglobin). An A1C level of 6.5 percent or more in two separate samples indicates the presence of diabetes. A result of 6-6.5% is considered pre-diabetic, indicating a high risk of developing diabetes.

[569] A random blood glucose test involves obtaining a blood sample at a random time point from a subject suspected of having diabetes. Blood glucose can be expressed in milligrams per deciliter (mg/dL) or millimoles per liter (mmol/L). An occasional blood glucose level of 200 mg/dL (11.1 mmol/L) or higher indicates the likelihood of the subject having diabetes, especially in combination with any signs and symptoms of diabetes such as frequent urination and increased thirst.

[570] To perform a fasting blood glucose test, a sample is obtained after an overnight fast. A fasting blood glucose level of less than 100 mg/dL (5.6 mmol/L) is considered normal. A fasting blood glucose level of 100 to 125 mg/dL (5.6 to 6.9 mmol/L) is considered prediabetic, and a level of 126 mg/dL (7 mmol/L) or higher in two separate samples indicates the presence of diabetes.

[571] Type 1 diabetes can be distinguished from type 2 diabetes using a C-peptide assay that measures endogenous insulin production. The presence of anti-islet antibodies (to glutamic acid decarboxylase, insulin-associated peptide-2, or insulin) or lack of insulin resistance, as determined by a glucose resistance test, also indicates type 1, while many type 2 diabetics continue to produce insulin internally and observe some degree of insulin resistance.

[572] Testing for GAD 65 antibodies has been proposed as an improved test to distinguish between type 1 and type 2 diabetes, as the immune system appears to be relevant to the etiology of type 1 diabetes.

[573] In some embodiments, the present invention provides compositions for using a population of SC-β cells obtained by the methods disclosed herein to restore islet function in a subject in need of such therapy. Any pathological condition associated with impaired production of pancreatic hormones (insulin, glucagon or somatostatin) or inability to properly regulate secretion may be considered an indication for treatment with cells (for example, a population of SC-β cells) obtained in accordance with this invention. Of particular interest is the treatment of type I diabetes mellitus (insulin-dependent).

[574] Subjects in need of treatment may be selected for treatment based on proven long-term dependence on exogenous insulin administration and an acceptable risk profile. The subject receives approximately 10,000 SC-β cells or cell equivalents per kg of body weight. If the cells are not autologous, the subject may be treated with an immunosuppressive agent such as FK506 and rapamycin (oral) and daclizumab (intravenous) before surgery to overcome the allotype mismatch. A composition containing a population of SC-β cells can be administered through a catheter in the portal vein. An ultrasound of the subject's abdomen can then be done and blood tests done to determine liver function. The daily insulin requirement is monitored and, if necessary, a second graft is administered to the subject. Follow-up monitoring includes frequent blood tests to determine drug levels, immune function, general health, and whether the patient remains insulin independent.

[575] General approaches to the care of diabetic patients are given in standard textbooks such as the Textbook of Internal Medicine, 3rd Edition by W.N. Kelley ed., Lippincott-Raven, 1997; and in specialist literature such as Diabetes Mellitus: A Fundamental and Clinical Text 2nd Edition by D. Leroith ed., Lippincott Williams & Wilkins 2000; Diabetes (Atlas of Clinical Endocrinology Vol.2) by C.R. Kahn et al. eds., Blackwell Science 1999; and Medical Management of Type 1 Diabetes 3rd Edition, McGraw Hill 1998. The use of islet cells for the treatment of type I diabetes is discussed in Cellular Inter-Relationships in the Pancreas: Implications for Islet Transplantation by L. Rosenberg et al., Chapman & Hall 1999; and Fetal Islet Transplantation by C.M. Peterson et al. eds., Kluwer 1995.

[576] As always, the primary responsibility for the selection of the subject, the mode of administration, and the dosage of the SC-β cell population lies with the attending physician. For purposes of commercial distribution, the SC-β cell populations disclosed herein are typically provided in the form of a pharmaceutical composition containing an isotonic excipient prepared under sufficiently sterile conditions for use in humans. Also included in the invention are sets of SC-β cell populations that exist at any time during their manufacture, distribution, or use. SC-β cell population sets comprise any combination of two or more of the cell populations described herein, exemplified by, but not limited to, differentiation of definitive endoderm cells into pdx1-positive pancreatic progenitor cells and their subsequent differentiation, e.g., into insulin-producing cells, such as mature pancreatic β-cells or mature pancreatic β-like cells as defined herein. In some embodiments, cell compositions containing populations of SC-β cells can be administered (eg, implanted in a subject) in combination with other cell types, eg, other differentiated cell types, which sometimes share the same genome. All cell types in a kit may be packaged together or in separate containers at the same location, or at different locations operated by the same organization or by different organizations in a business relationship.

[577] For general principles of drug preparation, the reader may refer to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996. The composition is optionally packaged in suitable container with instructions for use, e.g. for the treatment of diabetes.

[578] In some embodiments, compositions containing populations of SC-β cells can also be used as a functional component in a mechanical device designed to produce one or more pancreatic islet cell endocrine polypeptides. The simplest form of the device contains a population of SC-β cells behind a poppy-permeable membrane that impedes the passage of the population of cells, leaving them inside the device, but does not interfere with the passage of insulin, glucagon, or somatostatin secreted by the cell population. The above includes a microencapsulated population of SC-β cells, typically in the form of cell clusters, to allow an interaction that inhibits dedifferentiation. For example, US Pat. weighing >3000, which are cross-linked in such a way that the membrane is permeable to proteins of the size of insulin, but impermeable to molecules with a mol. weighing over 100,000. US Pat. No. 6,023,009 describes islet cells encapsulated in a semi-permeable membrane composed of agarose and agaropectin. Microcapsules of this origin are intended to be inserted into the abdominal cavity of a diabetic patient and are believed to have certain advantages in reducing histocompatibility problems or susceptibility to bacteria.

[579] The use of other developed devices containing a population of SC-β cells, intended both for implantation in patients with diabetes and for extracorporeal therapy, is also envisaged. US Pat. No. 4,378,016 describes an artificial endocrine gland comprising an extracorporeal segment, a subcutaneous segment, and a replaceable capsule containing hormone-producing cells. US Pat. No. 5,674,289 describes a biosynthetic pancreas comprising an insular cavity separated by a semi-permeable membrane from one or more vascular cavities with access to surrounding tissue. The devices used typically comprise a cavity adapted to contain the islet cells and a cavity separated from the islet cells by a semi-permeable membrane that collects proteins secreted from the islet cells and which can also transmit a feedback signal to the islet cells, for example regarding circulating glucose levels.

[580] Methods for determining β-cell maturation factors that increase the production of SC-β cells or pancreatic β-cells

[581] This document describes a method for determining a β-cell maturation factor or an agent that enhances the production of SC-β cells (eg, mature pancreatic β-cells). In certain examples, a multivariable and/or high throughput screening method is provided. Said method comprises treating at least one insulin-positive endocrine cell or progenitor thereof with at least one compound (e.g., a compound from a library or a compound described herein) and determining whether the compound enhances SC-β cell formation, e.g., mature pancreatic β-cells, from at least one insulin-positive endocrine cell or its precursor. A cell can be defined as an SC-β cell (eg, mature pancreatic β cell) using one or more of the markers described herein. In some examples, at least one insulin-positive endocrine cell or progenitor thereof may be differentiated prior to treatment with a library compound. In other examples, two or more compounds can be used in a screening assay, either alone or together. In further examples, at least one insulin-positive endocrine cell or progenitor thereof may be placed in a multiwell plate and a compound library screened by adding different members of the library to different wells of the multiwell plate. Such library screening can quickly identify compounds that result in the generation of SC-β cells, eg, mature pancreatic β cells, from at least one insulin-positive endocrine cell or progenitor thereof.

[582] In some embodiments of the invention, this method further includes isolating a population of SC-β cells, for example, pancreatic β-cells (for example, when at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or more of a given cell type).

[583] In some embodiments, the method further comprises implanting the SC-β cells produced by the methods described herein into a subject (e.g., a subject with diabetes, e.g., type I, type II, or type 1.5 diabetes). In some embodiments, the SC-β cell is derived from a stem cell obtained from a subject. In some embodiments of the invention, the SC-β cell is derived from a stem cell obtained from a donor other than the subject, for example, related to the subject.

[584] In one aspect, the invention relates to a SC-β cell, eg, a mature pancreatic β cell, obtained by the method described herein. In another aspect, the invention relates to a composition containing an SC-β cell obtained as described herein.

[585] In another aspect, the invention relates to a kit containing insulin-positive endocrine cells or their precursors; at least one β-cell maturation factor described herein; and instructions for using the insulin-positive endocrine cells or progenitors thereof and at least one β-cell maturation factor to produce an SC-β cell (eg, a mature pancreatic β-cell). In some embodiments of the invention, the kit further comprises: a component for detecting a marker of mature β-cells, for example, the marker described herein, for example, a reagent for detecting a marker of maturation of β-cells, for example, an antibody to the marker; and mature pancreatic β-cell, for example, for use as a control.

[586] In some embodiments, the kit further comprises: a component for differentiating an endoderm cell, eg, a definitive endoderm cell, into a second cell type cell, eg, at least one insulin-positive endocrine cell or progenitor thereof; and instructions for using an endoderm cell (eg, a definitive endoderm cell) as described herein and a component to produce a second type of cell, eg, at least one insulin-positive endocrine cell or a precursor thereof. In some embodiments, the kit further comprises: a component for detecting a second cell type cell marker, eg, a marker described herein, eg, a Pdx1 detection reagent, eg, an antibody to the marker; and a cell of a second cell type, eg at least one insulin-positive endocrine cell or a precursor thereof, eg for use as a control.

[587] In one aspect, the invention relates to a method of stimulating the differentiation of insulin-positive endocrine cells or progenitors thereof into SC-β cells, comprising providing at least one insulin-positive endocrine cell or progenitor thereof and providing at least one maturation factor β- cells (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the β-cell maturation factors described herein) to differentiate at least one insulin-positive endocrine cell or progenitor thereof into an SC-β cell (eg, mature pancreatic β-cell) after treatment of the stem cell with at least one β-cell maturation factor. In some embodiments of the invention, at least one insulin-positive endocrine cell or its precursor is obtained from a mammal. In some embodiments of the invention, at least one insulin-positive endocrine cell or its precursor is obtained from a mouse or a human. In some embodiments, at least one insulin-positive endocrine cell or precursor thereof is obtained by culturing an embryonic stem cell (eg, a mammalian embryonic stem cell, such as a mouse or human embryonic stem cell). In some embodiments, at least one insulin-positive endocrine cell or precursor thereof is obtained by culturing an induced pluripotent stem cell (eg, a mammalian iPs cell, such as a mouse or human iPs cell).

[588] In some embodiments, a plurality of insulin-positive endocrine cells, or precursors thereof, differentiate into a plurality of mature pancreatic β cells or SC-β cells, for example, by bringing the plurality of insulin-positive endocrine cells, or precursors thereof, into contact with at least one, at least two, at least three or more of the β-cell maturation factors described herein.

[589] In some embodiments, a plurality of insulin-positive endocrine cells or progenitors thereof are treated with β-cell maturation factors for about 1, 2, 4, 6, 8, 10, 12, 14, 16 or more days. In some embodiments, a plurality of insulin-positive endocrine cells or progenitors thereof are treated with β-cell maturation factors at a concentration of about 25 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM or 10 µM. In some embodiments, a plurality of insulin-positive endocrine cells or progenitors thereof are treated with β-cell maturation factors at a concentration of about 250 nM, 400 nM, 500 nM, 600 nM, 700 nM, or 800 nM. In some embodiments, greater than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the insulin-positive endocrine cells or progenitors thereof differentiate into mature pancreatic β cells or SC- β.

[590] In some aspects, the invention provides artificial islets constructed using the SC-β cells described herein. In some aspects, the artificial islet contains one or more SC-β cells differentiated in vitro from pluripotent stem cells, for example, in accordance with the method described herein.

[591] In some aspects, the invention provides an artificial pancreas comprising SC-β cells differentiated in vitro from pluripotent stem cells.

[592] It should be understood that the above detailed description and the following examples are illustrative only and do not limit the scope of the invention. Various changes and modifications to the disclosed embodiments of the invention, obvious to those skilled in the art, may be made without departing from the spirit and scope of the invention. In addition, all referenced patents, patent applications, and publications are expressly incorporated herein by reference for the purposes of describing and disclosing, for example, the methodologies described in such publications that may be applied in connection with the present invention. These publications are presented in respect of their content solely prior to the filing date of this application. In this regard, nothing should be taken as an admission that the inventors are not entitled to backdate such disclosure of the invention on the basis of a previous invention or for any other reason. All statements regarding the date or content of such documents are based on information available to the authors of the application and do not constitute an endorsement of the correctness of the dates or content of these documents.

* * *

[593] Examples

[594] Example 1 - Generation of functional pancreatic β-cells in vitro

[595] Brief Description

[596] Generation of insulin-producing pancreatic β-cells from stem cells in vitro would provide an unparalleled source of cells for drug development and transplantation therapy in diabetes. However, insulin-producing cells previously derived from human pluripotent stem cells (hPSCs) lack many of the characteristics of true β-cells, including in vitro and/or in vivo function. The work described herein demonstrates an exemplary, scalable differentiation protocol that allows the generation of SC-β cells from hPSCs in vitro. Surprisingly and unexpectedly, these SC-β cells secrete comparable amounts of insulin to adult β-cells in response to multiple sequential glucose stimulation, provide Ca2+ flux, express markers found in β-cells, and package insulin into secretory granules. As a proof of concept, SC-β cells also respond to known antidiabetic drugs and proliferative stimulants in vitro. In addition, SC-β cells secrete high levels of human insulin into the serum of mice immediately after transplantation, and transplantation of these cells immediately alleviates the symptoms of hyperglycemia in diabetic mice. The work described herein is an important step forward in the use of stem cell-derived β-cells (ie, SC-β cells) for the treatment of diabetes and for in vitro β-cell studies and drug screening.

[597] The following work demonstrates several advantages of SC-β cells produced according to the methods described herein, for example, SC-β cells exhibit glucose-stimulated insulin secretion in vitro, have similarities to human islet β cells in terms of gene expression, and ultrastructures, secrete human insulin and alleviate symptoms of hyperglycemia when transplanted into mice, provide a new platform for cell therapy (e.g., transplantation into a subject in need of additional and/or functional β-cells), drug screening (e.g., insulin production/secretion, survival , dedifferentiation, etc.), research (eg, determining the difference in functionality between normal and diabetic β-cells), and tissue engineering (eg, using SC-β cells as the first cell type in islet repair).

[598] Introduction

[599] The discovery of human pluripotent stem cells (hPSCs) has raised the possibility that replacement cells and tissues could someday be generated for disease treatment and drug screening. Research in the last ten years has brought this area closer to the goal by developing strategies for generating cells that are difficult to obtain in any other way, such as neurons or cardiomyocytes (Kriks et al., 2011; Shiba et al., 2012). These cells have also been transplanted into animal models and are able to take root in the host, in some cases with beneficial effects such as suppression of arrhythmias with stem cell-derived cardiomyocytes (Shiba et al., 2012), restoration of ambulation after injury spine with oligodendrocyte progenitor cells (Keirstead et al., 2005) or improved vision after transplantation of retinal epithelial cells in rat models of blindness (Lu et al., 2009).

[600] One of the fastest growing diseases that can be treated with stem cell-derived tissue is diabetes, which affects more than 300 million people worldwide according to the International Diabetes Federation. Type 1 diabetes results from autoimmune destruction of β-cells in the pancreatic islets, while the more common type 2 diabetes results from peripheral tissue insulin resistance and β-cell dysfunction. Such patients, in particular those suffering from type 1 diabetes, could potentially be treated by transplantation of new functional β-cells. Cadaveric human islet transplantation has shown that patients can become insulin independent for five years or more with this strategy, but this approach is very limited due to the lack of donor human islets (Bellin et al., 2012). Generating an unlimited supply of human β-cells from stem cells could make this therapy available to millions of new patients. β-cells represent an ideal option for regenerative medicine as only one type of cell needs to be generated and these cells can be placed anywhere in the body in an immunoprotective device (e.g. the microcapsule described herein) or material with access to the vascular system.

[601] Pharmaceutical screening for new drugs that can improve β-cell function or proliferation is also hampered by the limited supply of cadaveric islets and their high variability due to differences in cause of death, donor genetic background, and other factors in their isolation. Therefore, the systematic continuous supply of SC-β cells may provide a unique and extremely valuable platform for the discovery of new drugs against diabetes.

[602] Significant progress has been made to date in the generation of a β-cell lineage in vitro from hPSCs. Now cells from the definitive endoderm and subsequent pancreatic progenitors can be differentiated with high efficiency (Kroon et al., 2008; D'Amour et al., 2006; D'Amour et al., 2005; Rezania et al., 2012) . Importantly, these cells can further differentiate into functional β-cells within three to four months after transplantation into rodents (Kroon et al., 2008; Rezania et al., 2012), indicating that they have a differentiation potential for development into β-cells, given sufficient time and appropriate stimulation. Unfortunately, the month-long process that occurs with cells in vivo remains a black box and it is not clear whether this process will work in the case of human patients. Other work has focused on generating insulin-producing cells from human pancreatic progenitors in vitro. However, the cells obtained to date are not genuine β-cells. These cells either do not show glucose-stimulated insulin secretion in vitro, or do not express appropriate β-cell markers such as NKX6-1 or PDX1, or abnormally express other hormones such as glucagon, or do not function after in vivo transplantation, or show a combination of these abnormalities. traits (D'Amour et al., 2006; Cheng et al., 2012; Narayanan et al., 2013; Xie et al., 2013; Nostro et al., 2011).

[603] The work described herein proposes a strategy for virtually unlimited large-scale production of functional human β-cells from hPSCs in vitro. By applying sequential modulation of signaling pathways in combination with a 3-dimensional cell culture system, monohormonal insulin-producing cells (SC-β cells) can be generated that co-express key β-cell markers and exhibit ultrastructural features of β-cells. In addition, these cells mimic the function of human islets both in vitro and in vivo. Finally, the cells show proof of concept for their dual purpose of in vitro drug screening and in vivo transplantation therapy for diabetes.

[604] Results

[605] In Vitro Generation of Glucose-Sensitive Insulin-Secreting β Cells from hPSC

[606] An exemplary method for generating functional β-cells from hPSCs in vitro is shown in FIG. 1A. To obtain a large number of β-cells, a scalable suspension-based culture system was used, which makes it possible to generate >10 ⁸ hPSC and cell types at later stages of differentiation (Schulz et al., 2012). Clusters of human embryonic stem cells HUES8 approximately 100-200 µm in diameter were induced to high purity definitive endoderm (>95% Sox17+) and further to early pancreatic progenitors (>90% PDX1+) using protocols based on past publications (FIG. 1B ) (Schulz et al., 2012; Rezania et al., 2012). A method was further defined in which extended time in culture with a member of the FGF family, KGF, the sonic hedgehog inhibitor Sant1, and a low concentration of retinoic acid was used to generate high levels of pancreatic progenitor cell clusters co-expressing NKX6-1+/PDX1+ (>60 % NKX6-1+/PDX1+ cells) (FIG. 1A.) Transplantation of these pancreatic progenitor cells into mice has been reported to result in functional β-cells in vivo after 3-4 months (Rezania et al., 2012). This was used as a starting point to develop a protocol to replicate the generation of functional β-cells in vitro.

[607] NKX6-1+/PDX1+ pancreatic progenitor cells were then differentiated into C-peptide-expressing endocrine cells using a previously published protocol (control differentiation) or a newly developed protocol (new differentiation). The protocol of control differentiation allowed for several months to obtain cells that were monohormonal ENS+ and INS+/GCG+ and INS+/SST+ polyhormonal (PH) cells. The abbreviation PH was used to refer to these cells. On the other hand, the new differentiation protocol included 2-3 weeks of unique series of culture steps including sonic hedgehog signaling inhibition, retinoic acid signaling, gamma-secretase inhibition, TGFβ signaling inhibition, EGF signaling, thyroid hormone signaling, and CMRL 1066 islet medium. (Nostro et al., 2011; Rezania et al., 2012; Thowfeequ et al., 2007; Aguayo-Mazzucato et al., 2013; D'Amour et al., 2006). It was hypothesized that the C-peptide + cells produced according to this new differentiation protocol were analogous to primary adult β-cells (1° β) and hence referred to as β-stem cells (SC-β) (i.e. SC-β cells).

[608] A key functional feature of the β-cell is its ability to systematically perform glucose-stimulated insulin secretion (GSIS). Virtually all existing regulated differentiation protocols generate insulin-expressing cells from hPSCs that do not exhibit GSIS in vitro (D'Amour et al., 2006). One protocol has been reported using endodermal progenitor cell lines as a starting population that produces cells that can secrete some insulin in response to a single glucose challenge (Cheng et al., 2012). In contrast, SC-β cells produced by the methods described herein can respond to at least three consecutive stimulations with high glucose concentrations. These cells secreted high levels of insulin in a manner similar to primary adult β-cells, while the PH cells from the control protocol did not respond to glucose (FIG. 2A-2C and FIG. 3A-2C). The stimulation index, calculated from the ratio between insulin secreted in response to high glucose concentration (20 mM) and low glucose concentration (2 mM), was similar for SC-β cells and primary adult β-cells, 2.3 ± 0.9 and 2.3±1.4, respectively. In addition, there was a small percentage of stimulation with high concentrations of glucose to which neither SC-β cells nor primary adult β-cells responded. In addition, the amount of insulin secreted per cell in response to 20 mM glucose for SC-β cells was indistinguishable from that secreted by primary adult β-cells, 2.6 ± 1.6 and 2.5 ± 1.2 µIU/10 ³ cells, respectively. Together, these data suggest that the in vitro function of SC-β cells produced according to the new differentiation protocol is very similar to equivalent genuine primary adult β-cells.

[609] In vitro functionality of SC-β cells obtained according to the new protocol of differentiation was later confirmed by determining the change in intracellular Ca ²⁺ . β-cells sense changes in glucose levels through calcium signaling; an increase in glucose levels leads to membrane depolarization, which leads to an influx of calcium ions, which is responsible for triggering the release of insulin (Mohammed et al., 2009). This influx of calcium in cell clusters stained with Fluo-4 AM, a fluorescent dye that is an indicator of calcium, was monitored in real time using fluorescence microscopy (FIG. 4A). This method allowed calcium influx to be analyzed both at the population level and at the single cell level, and showed that both SC-β cells and primary adult β-cells responded to sequential glucose stimulation by systematically increasing intracellular Ca ²⁺ in a similar way, consistent with normal GSIS, while PH cells generated according to the differentiation control protocol exhibited an abnormal calcium response consistent with their abnormal GSIS (FIG. 4B). In single cell analysis, most individual SC-β cells and primary adult β cells responded to 2-3 consecutive glucose stimulations via calcium flux, while most PH cells responded to 0 stimulations (FIGS. 4C-4E). In contrast to rodent β-cells, human β-cells are known to exhibit some degree of dyssynchrony in response to high glucose concentrations (Rutter and Hodson, 2013). These data show that both the entire population and individual cells in SC-β cell clusters function similarly to β-cells in isolated islets and further support the conclusion that SC-β cells generated according to the new differentiation protocol function in vitro.

[610] Stem cell-derived β-cells from hPSCs have similarities to primary human β-cells

[611] After revealing that SC-β cells function like primary adult β-cells in vitro, the two cell populations were analyzed for protein expression, gene expression and ultrastructure. Unlike most previously reported hPSC-derived insulin-producing cells, these SC-β cells expressed both normal β-cell markers, PDX1 and NKX6-1 (FIGS. 5A and 5B). Rare non-β-cell hormones were observed, but they did not colocalize with cells positive for NKX6-1/C-peptide (FIG. 5C). SC-β stained positively for both insulin and C-peptide, a stoichiometric by-product of proinsulin processing, indicating that insulin staining is associated with endogenous cellular insulin production (FIG. 6), and staining for ISL1, MAFA and MAFB (FIGS. 7A-7C). Flow cytometry quantification revealed that the methods described herein yielded 40% NKX6-1/C peptide, which is similar to the percentage of these components in cadaveric human islets (FIG. 5D). In addition, only 8% of all C-peptide+ cells co-express glucagon and 4% co-express somatostatin (FIGS. 8A-8C). a key NKX6-1 marker for β-cells or functioned in vivo (Cheng 2012.) One recent study has shown that guided differentiation protocols that generate higher levels of NKX6-1 lead to better transplant outcomes in the case of transplants pancreatic progenitor cells (Rezania et al., 2013). In addition, conditional gene knockout studies have shown that NKX6-1 expression is required for β-cell function in adult mouse islets, suggesting that co-expression of these factors in our cells may help explain their functionality (Taylor et al., 2013).

[612] Improved protein expression of several key markers of β-cells indicates that the transcriptional network of these cells better matches the network of human islet β-cells. Recent studies have shown that INS+ PH cells obtained according to previously described protocols bear no resemblance to adult islet INS+ β cells (Hrvatin et al., 2014; Xie et al., 2013). Microarray analysis of sorted INS+ cells obtained according to previously published protocols showed that they clustered with fetal β-cells and not with functional adult human β-cells sorted in the same way.

[613] In order to compare the SC-β cells of the invention with adult human islets, INS+/NKX6-1+ cells were sorted in the same manner and global microarray gene expression analysis was performed. In contrast to the previously described stem cell-derived INS+ PH cells, the SC-β cells described herein clustered more closely with human adult β cells than with fetal β cells or INS+ PH cells (FIG. 5E). In addition, these data show that the expression of canonical β-cell genes such as PDX1, MNX1 and NKX2-2 was more similar in SC-β cells and adult human β-cells than previously described INS+ PH cells. Analysis of the 100 most differentially expressed genes in this dataset also showed that SC-β cells and adult human β-cells had more similarity to each other than previously described PH cells or fetal β-cells (FIG. 5F). The remaining differences between SC-β cells and human adult β-cells can be corrected by making small changes in the composition of the culture media during late stages of differentiation.

[614] Since the gene and protein expression profiles of SC-β cells are similar to those of primary human β-cells, it has been hypothesized that SC-β cells should also package insulin protein into appropriate secretory granules, as is the case with genuine β-cells. -cells. β-cells package insulin into secretory granules that are initially pale gray with a halo around them and then mature into dark, crystallized polygonal granules with a light halo (FIG. 9A). Previous studies of INS+ cells derived from stem cells revealed that these cells contain only polyhormonal and alpha-like granules, which are distinct round granules with a dark halo (D'Amour et al., 2006; Kroon et al., 2008) . These results were replicated according to a control protocol that yielded INS+ cells that contained only abnormal polyhormonal and alpha-like granules and only a few β-cell granules (FIG. 9A). On the other hand, the methods described herein allow the generation of SC-β cells that package and crystallize insulin protein into prototypical β-cell granules (FIG. 9A). Both developing insulin granules and mature crystallized insulin granules were observed in both primary human β-cells and SC-β cells (FIG. 9B). To confirm the protein content of these granules, immunolabeling with gold was performed using particles against insulin and glucagon. Whereas RN cell granules were characterized by abnormal levels of both insulin and glucagon, granules of primary human β-cells and SC-β cells contained only insulin (FIG. 9C). Thus, the ultrastructure of SC-β cells closely mirrors that of adult human β-cells.

[615] Generation of glucose-sensitive insulin-secreting β-cells from hiPSC In Vitro

[616] The new differentiation protocol used to develop functional β-cells from hPSCs discussed above was reused to generate functional β-cells from hiPSC lines in vitro. hiPSC lines were generated in a Harvard iPSC Core using dermal tissue fibroblasts grown from cells obtained from non-diabetic or type 1 diabetic patients. Unlike the hPSC line, which was generated from embryos, the hiPSC line was generated from human tissue to show that functional β-cells can be developed directly from cells of sick patients, ie. patients with type 1 diabetes.

[617] The resulting β-cells were stimulated with glucose to determine their ability to systematically carry out glucose-stimulated insulin secretion (GSIS). It has been found that multiple β-cells produced by the methods described herein can respond to at least three consecutive stimulations with high glucose concentrations (FIGS. 10A-10B). In this particular experiment, hiPSC-β cells derived from a non-diabetic cell line (1013-3FA) were compared with hiPSC-β cells derived from a type 1 diabetic cell line (1031SeVA). The stimulation index, calculated from the ratio between insulin secreted in response to high glucose concentration (20 mM) and low glucose concentration (2 mM), was greater for β-cells derived from a non-diabetic cell line compared to β-cells derived from type 1 diabetic cell line. In addition, the amount of insulin secreted per cell in response to stimulation with 2 mM or 20 mM glucose was greater for β-cells derived from a type 1 diabetic cell line than for β-cells derived from a non-diabetic cell line. Together, these data suggest that in vitro functioning of hiPSC-β cells derived from a type 1 diabetic cell line is similar to that of their corresponding non-diabetic cells.

[618] After identifying that hiPSC-β cells respond to glucose in vitro, the inventors analyzed how similar non-diabetic populations and type 1 diabetic populations are in terms of protein expression, gene expression and ultrastructure. Three different non-diabetic cell lines and three different type 1 diabetic cell lines were used to determine NKX6-1/C peptide expression. As with hPSC, these hiPSC-β cells expressed both normal β-cell markers PDX1 and NKX6-1 (FIGS. 11A-11F). Flow cytometry quantification revealed that the methods described herein yielded about 40% NKX6-1/C peptide, which is similar to the percentage of these components in cadaveric human islets (FIG. 5D).

[619] Stem cell-derived β-cells function in vivo after transplantation

[620] The data described up to this point relate to the generation of functional human β-cells in vitro. To further explore their ability to function in vivo, these cells were transplanted under the renal capsule of immunocompromised mice (FIGS. 12A-12D and Table S1).

[621] When transplanting adult human islets, human insulin was detected in the serum of these mice for two weeks. In contrast, when previously described pancreatic progenitor cells were transplanted into mice, no insulin was detected two weeks after transplantation (Kroon et al., 2008; Schulz et al., 2012; Rezania et al., 2012). Instead, the cells required a 3-4 month poorly understood in vivo maturation phase. On the other hand, SC-β cells, like human islets, secreted high levels of insulin into the host circulation in response to glucose stimulation for two weeks after transplantation (FIG. 12A). PH cells prepared according to previously described protocols were also transplanted as controls and these cells were found not to secrete significant levels of insulin in response to glucose in vivo (FIG. 12A). In addition, it was also confirmed that the obtained pancreatic progenitor cells did not produce a detectable level of insulin in vivo for two weeks, as previously described (FIG. 12C).

[622] Some insulin-producing cells that are not true β-cells can uncontrollably secrete insulin into the animal's bloodstream, functioning more like an insulin granule than a responsive β-cell. To verify that SC-β cells not only secrete high levels of insulin, but also do so in a functionally sensitive manner, the amount of human insulin in the bloodstream of mice was measured both before and after strong glucose stimulation. In both human islet transplants and SC-β cell transplants, 9 of 12 mice studied demonstrated functional glucose-stimulated insulin secretion in vivo two weeks after transplantation (FIG. 12A).

[623] After the final in vivo GSIS stimulation, the animals were sacrificed and the transplanted kidneys were removed for analysis. Histological sections of these kidneys revealed the presence of large grafted areas of human cells under the renal capsule. Immunofluorescent staining of these areas showed that both SC-β cells and grafted human islets contained high levels of insulin-expressing cells (FIG. 12B). These INS+ cells also coexpressed the canonical β-cell transcription factor PDX1, as expected for functional β-cells. Staining analysis for insulin and glucagon further revealed that SC-β cells remained monohormonal after transplantation (FIGS. 13A-13B). Also, a small population of GCG+ cells was observed when generated by this protocol by immunofluorescence and flow cytometry (FIG. 7 and FIG. 10), and these cells were also observed in vivo after transplantation (FIG. 13).

[624] Additionally, in vitro insulin secretion was found to increase with time when supplemented CMRL media containing the Alk5 inhibitor and T3 hormone were used in the last stage of differentiation. Thus, SC-β cells were cultured in vitro for an additional week (two weeks instead of one week in the medium for the last stage) and tested whether they would also secrete more insulin in vivo. After transplantation of these older cells, unexpectedly ten times higher levels of insulin were observed than for the same number of transplanted younger SC-β cells (FIG. 12D). Thus, the levels of insulin function achieved in vivo with human SC-β cells were similar to those achieved with human islet transplantation. Together, these transplantation data suggest that SC-β cells may become an alternative clinical option for cell transplantation that is not dependent on unstable and limited supplies of donor cadaveric islets or a poorly understood and poorly controlled in vivo maturation period from transplanted stem-derived cells. pancreatic progenitor cells.

[625] Culture media

[626] In order to induce in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells contain in suitable culture medium for a sufficient period of time. In the present invention, as mentioned above, it has been found that the use of CMRL media containing the Alk5 inhibitor and T3 hormone allows in vitro cultured SC-β cells to secrete more insulin in vivo. It was found that the addition of additional factors to CMRL media at the last stage of differentiation (stage 6) (FIG. 14A) leads to an improvement in the response, consisting of glucose-stimulated insulin secretion (GSIS), by SC-β cells, according to both the value of the stimulation index , determined by the ratio between high and low glucose stimulation, and the amount of insulin released. Such additional factors may include, but are not limited to, Sant1, XXI, and SSP. The stimulation index, calculated from the ratio between insulin secreted in response to high glucose concentration (15 mM) and low glucose concentration (2.5 mM), was large for SC-β cells that were kept in CMRL medium containing the Alk5 inhibitor, the hormone T3 and any component from Sant1, XXI, or SSP, compared to SC-β cells maintained in CMRL medium alone, CMRL medium containing an Alk5 inhibitor and T3 hormone, or S3 medium containing an Alk5 inhibitor and T3 hormone (FIG. .14V). In addition, the amount of insulin secreted was greater for SC-β cells that were kept in CMRL containing the Alk5 inhibitor, T3 hormone, and any component from Sant1, XXI, or SSP, compared to SC-β cells that were kept only in CMRL medium, CMRL medium containing an Alk5 inhibitor and T3 hormone, or S3 medium containing an Alk5 inhibitor and T3 hormone (FIG. 14C). Together, these data suggest that it is not only the maintenance of SC-β cells in CMRL medium at the end of differentiation that is important to induce the maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells. , but the addition of certain factors such as Sant1, XXI, or SSP to CMRL medium further enhances glucose-stimulated insulin secretion (GSIS) in these cells.

[627] Increasing Cell Survival and Functionality

[628] One of the challenges in stem cell technology has been to generate enough SC-β cells that can be used for drug screening, disease modeling, or therapeutic purposes. For example, hES cells are technically difficult to culture, prone to apoptosis due to cell decay and dissociation, undergo massive cell death, in particular after complete dissociation, and have low cloning efficiency. (Watanabe, K. et al., Nature Biotechnology 25, 681-686 (2007)). As a result, the number of viable SC-β cells at the output may be low. By modifying the protocol between steps 3 and 6 as shown in FIG. 15A, more pure NKX6.1+ endocrine cassettes can be generated (FIG. 15B), resulting in more SC-β cells for possible therapeutic use.

[629] In steps 3-5, for example, cell survival can be improved by adding a rho-associated protein kinase inhibitor or a Rock inhibitor. It is believed that the addition of a Rock inhibitor improves the survival of ES cells by preventing dissociation-induced apoptosis and thus increasing cloning efficiency. (Watanabe et al.) Examples of Rock inhibitors include, but are not limited to, Y-27632, Fasudil/HA1077 and H-1152. The present invention has also shown that treatment of cells with a Rock inhibitor improves cell survival (FIG. 15C).

[630] In addition to treating the cells with the Rock inhibitor, in steps 3-4, cells can be treated either with activin A alone or in combination with nicotinamide. Both activin A and nicotinamide have been shown to improve cell survival. One way they improve cell survival is by down-regulating SOX2, a marker of pluripotent stem cells, from about 81% to about 47% (FIG. 15D). Since SOX2 and NKX6-1 are mutually exclusive (FIG. 15E), downregulation of SOX2 leads to upregulation of the NKX6-1 marker of mature pancreatic β cells.

[631] In steps 5-6, cell survival can be further improved by treating the cells with staurosporine. Staurosporine is known to promote the differentiation of neuronal, glial, and neurosphere cells from stem-like cells and is also considered to be an inducer of apoptosis. (Schumacher, et al., Mol. Cell. Neurosci. 2003; 23(4): 669-680). In the present invention, the addition of staurosporine resulted in a substantially pure endocrine population by increasing the percentage of chromogranin A, a marker for pancreatic endocrine cells, (FIG. 15F) and increasing the percentage of NKX6-1. C-peptide+ cells (FIG. 15G).

[632] Finally, cell survival can be further enhanced by treating cells with culture media containing Alk5i, T3 in combination with XXI, a γ-secretase inhibitor that enhances β-cell maturation. As shown in FIG. 15H and 15I, addition of XXI to the medium in steps 5 and 6 can increase the NeuroD1+ population.

[633] Together, these data suggest that the addition of certain components to cells at various stages of the process is important to improve cell survival as well as to enrich the population of cells that can differentiate into therapeutically useful SC-β cells.

[634] Application of SC-β cells in translational biology

[635] A major challenge in the field of stem cell technology has been the generation of differentiated cell types that are close enough to their normally developed equivalent cells to be used for drug screening, disease modeling, or therapeutic purposes. It turned out that SC-β cells obtained according to the new protocol of differentiation, as discussed in this document, function both in vitro and in vivo in a manner similar to primary human β-cells. Therefore, it has been suggested that these cells can be used in translational medicine (FIG. 16A).

[636] One of the most common therapeutic strategies for the treatment of type 2 diabetes is the administration of oral antidiabetic drugs. Many of these pharmaceutical agents act directly on β-cells to increase insulin secretion. For example, sulfonylurea drugs increase endogenous insulin secretion by blocking potassium channels (Modi, 2007). To date, β-cell drug screening has been limited to rodent islets, transformed cell lines, or the highly variable and limited supply of cadaveric human islets. Given that rodent metabolism bears only partial resemblance to human metabolism, a reliable continuous supply of human β-cells for analysis would be very useful. In this case, the inventors tested whether SC-β cells can be used for in vitro therapeutic screening.

[637] First, the inventors investigated whether SC-β cells can respond to existing antidiabetic drugs of several different classes (FIG. 16B). LY2608204 is a glucokinase activator in clinical trials, while liraglutide, tolbutamide, and nateglinide are examples of GLP-1 agonists and secretagogues (sulfonylureas and meglitinides), respectively, that are used in clinical settings (Matschinsky, 2009; Modi, 2007; Vetere et al., 2014). Indeed, SC-β cells showed a tendency to respond to treatment with these drugs, increasing insulin secretion in vitro after glucose stimulation (FIG. 16C). These data provide initial evidence that SC-β cells may provide a new platform for drug screening in the future.

[638] After that, the inventors investigated whether SC-β cells can model the replication of β-cells. Increasing a patient's β-cell mass can improve glycemic control by producing more total insulin without the need to increase the amount of insulin produced per β-cell. To date, literally a few drugs that have stimulated β-cell replication in rodent or cell line models have been translated into human β-cells. Studies of islet mass during human pregnancy and studies in rodents using prolactin treatment have suggested the presence of hormonal regulation of β-cell replication (Parsons et al., 1992; Labriola et al., 2007; Brelje et al., 2004) . The inventors investigated whether prolactin treatment could increase β-cell replication in a population of SC-β cells. After culturing SC-β cells with prolactin, the cells were stained and fixed for C-peptide and the proliferation marker Ki67 (FIGS. 16D and 16E). As with primary β cells, the initial number of Ki67+ proliferating SC-β cells was very low (0.20±0.08% Ki67+/C-peptide+) (FIG. 16F). Quantification of untreated and prolactin-treated cells revealed a trend towards increased proliferation in the case of prolactin (0.39±0.08% Ki67+/C-peptide+), suggesting that SC-β cells may be able to respond to proliferative stimuli in studies replication (FIG. 16F).

[639] Finally, the inventors investigated whether SC-β cells can be directly used as cell therapy for the treatment of diabetes. In contrast to type 2 diabetes, the enhancement of β-cell function or β-cell replication by new pharmaceuticals is likely to have little therapeutic effect in patients with type 1 diabetes, in which pancreatic β-cells are destroyed by autoimmune attack. These patients can be effectively treated by replacing lost β-cells with donor allogeneic islets according to the Edmonton transplant protocol (Shapiro et al., 2006).

[640] One useful model for severe diabetes is the Akita mouse. Akita mice carry a mutation in the insulin gene that results in protein misfolding, complete and irreversible destruction of β-cells, and progressive severe hyperglycemia. Recovery of Akita mice to normoglycemia can be achieved by transplantation of mouse or human islets into the renal capsule (Pearson et al., 2008). Therefore, SC-β cells produced according to the methods described herein were investigated to see if they could regulate diabetic hyperglycemia. The results showed that transplantation of SC-β cells, but not INS+ PH cells from the previously described protocols, into young Akita mice resulted in a rapid reversal of the progressive hyperglycemia observed in these animals (FIG. 16G). Fasting blood glucose measurements in mice transplanted with SC-β cells averaged less than 200 mg/dL, while those transplanted with control PH cells showed significantly higher blood glucose levels. over 400 mg/dl, also seen in non-transplanted Akita mice (FIG. 16G) (Pearson et al., 2008). As expected, human insulin levels were high in mice transplanted with SC-β cells and virtually undetectable in control animals transplanted with PH cells (FIG. 16H). Mice transplanted with SC-β cells also showed better body weight maintenance than control mice, indicative of normal insulin function (FIG. 17). Thus, SC-β cells are able to secrete insulin and arrest and reverse progressive hyperglycemia in a diabetic mouse model almost immediately after transplantation.

[641] Discussion

[642] The work described herein demonstrates that functional β-cells can be generated directly from hPSC and hiPSC in vitro. Together, the data described herein demonstrate that these cells (ie, SC-β cells) function similarly to primary human β-cells both in vitro and in vivo after transplantation. Cells SC-β, obtained in accordance with the methods described in this document, represent new opportunities for β-cell research and therapy. To date, the limited supply of donor cadaveric islets, high inter-sample variability due to differences in patient characteristics or isolation, and the notoriously low replication of human β-cells in vitro have severely limited the supply of human β-cells. This limitation has limited transplant options for patients and for high-throughput drug screening and disease modeling. One 68 kg (150 lb) patient needs approximately 340-748 million islet transplants to effectively treat type 1 diabetes with islet transplantation (McCall and Shapiro, 2012). The strategy described herein is efficient and highly scalable, making it practical for a single laboratory to grow hundreds of millions to billions of SC-β cells at a time. The main clinical advantage of SC-β cells compared to previously described stem cell based therapies for diabetes is that these cells represent the first opportunity for cell therapy that does not require a potentially unpredictable black box period of additional differentiation in vivo. .

[643] Unlike primary human β-cells, SC-β cells can also be obtained from cells with any desired genetic background. iPS cells derived from patients with diabetes or other metabolic syndromes can be differentiated into SC-β cells to model diseases and study β-cell function or susceptibility to stress or immune attack. Technologies such as TALEN and CRISPR make it possible to edit the genome of ES or iPS cells to introduce and explore variants identified by genetic analysis such as genome-wide association scanning (GWAS). Similarly, it is now possible to compare β-cell drug responses between matched pairs of mutant and non-mutant β-cells (Ding et al., 2013). The identification and investigation of new biomarkers for β-cell function or pharmacogenetics can also be carried out using a combination of these technologies. Thus, SC-β cells provide a novel, human-centric platform for in vitro drug discovery and characterization of metabolism and diabetes.

[644] Generation of SC-β cells also represents a potentially important step towards the generation of islets and pancreatic organs in the future. The incorporation of pancreatic niche cells, such as mesenchymal or endothelial cells, into stem cell-derived pancreatic cell cultures may be beneficial (Sneddon et al., 2012; Lammert et al., 2001). Other evidence suggests that the presence of alpha and delta cells may be important for the fine regulation of β-cell function (Rodriguez-Diaz et al., 2011). In addition, tissue engineering of pancreatic organs requires the introduction of functional exocrine and ductal tissues, if possible, with carefully designed architecture. The generation of SC-β cells represents a step forward towards the clinical application of stem cell biology.

[645] Experimental Procedures

[646] Cell culture

[647] hPSC lines were maintained undifferentiated in mTESR1 (StemCell Technologies Inc.; 05850) in 500 ml roller bottles (Coming, VWR; 89089-814) placed on a 9-position magnetic stir bar at 70 rpm in incubator at 37°C, 5% CO ₂ and 100% humidity. The main line used was the HUES8 line. Cells were dispersed with accutase and plated in single wells at a density of 0.5 million/ml in mTESR with 10 μM Y27632 (Abcam; ab120129). The mTESR1 medium was changed (m/w Y27632) after 48 hours. 24 hours after medium change, the cultures were separated to maintain a cluster diameter of <300 µm. Cultures were regularly examined for pathogens, karyotype, and content of pluripotency markers.

[648]

[649] Typical environments used for controlled differentiation were as follows:

[650] Medium S1: MCDB131 (Cellgro; 15-100-CV) + 8 mM D-(+)-Glucose (Sigma; G7528) + 2.46 g/l NaHCO ₃ (Sigma; S3817) + 2% FAF- BSA (Proliant; 68700) + ITS-X (Invitrogen; 51500056) 1:50,000 + 2 mM Glutamax (Invitrogen; 35050079) + 0.25 mM Vitamin C (Sigma Aldrich; A4544) + 1% Pen/Strep (Cellgro; 30 -002-CI).

[651] Medium S2: MCDB131 + 8 mM D-Glucose + 1.23 g/l NaHCO ₃ + 2% FAF-BSA + ITS-X 1:50,000 + 2 mM Glutamax + 0.25 mM Vitamin C + 1% Pen / Strep.

[652] Medium S3: MCDB131 + 8 mM D-Glucose + 1.23 g/l NaHCO ₃ + 2% FAF-BSA + ITS-X 1:200 + 2 mM Glutamax + 0.25 mM Vitamin C + 1% Pen / Strep.

[653] BE5 medium: MCDB131 + 20 mM D-Glucose + 1.754 g/L NaHCO ₃ + 2% FAF-BSA + ITS-X 1:200 + 2 mM Glutamax + 0.25 mM Vitamin C + 1% Pen/Strep + Heparin 10 µg/ml (Sigma; H3149).

[654] CMRLS: Augmented CMRL 1066 (Mediatech; 99-603-CV) + 10% FBS (HyClone™, VWR; 16777) + 1% Foam/Strep.

[655] All media were sterilized by filtration through a 0.22 µm bottle neck filter (Corning). For all subsequent media changes, small molecules and growth factors were added to the base media immediately prior to media change in a fume hood under low light.

[656] To initiate differentiation of SC-β cells, cells were seeded at a density of 0.5 million/ml, and differentiation began after 48 hours. The replacement of environments took place as follows -

[657] Day 1: S1 + 100 ng/ml Activin A (R&D Systems; 338-AC) + 3 μM Chir99021 (Stemgent; 04-0004-10)).

[658] Day 2: S1 + 100 ng/ml Activin A. Days 4, 6: S2 + 50 ng/ml KGF (Peprotech; AF-100-19)).

[659] Days 7, 8: S3 + 50 ng/mL KGF + 0.25 µM Sant1 (Sigma; S4572) + 2 µM Retinoic Acid (RA) (Sigma; SML0559) + 200 nM LDN193189 (Day 7 only) (Sigma ; SML0559) + 500 nM PdBU (EMD Millipore; 524390)).

[660] Days 9, 11, 13: S3 + 50 ng/ml KGF + 0.25 μM Sant1 + 100 nM RA.

[661] Days 14, 16: BE5 + 0.25 μM Sant1 + 50 nM RA + 1 μM XXI (EMD Millipore; 565790) + 10 μM Alk5i II (Axxora; ALX-270-445) + 1 μM L-3, 3',5-Triiodothyronine (T3) (EMD Millipore; 64245) + 20 ng/ml Betacellulin (Thermo Fisher Scientific; 50932345)).

[662] Days 18, 20: BE5 + 25 nM RA + 1 μM XXI + 10 μM Alk5i II + 1 μM T3 + 20 ng/ml Betacellulin.

[663] Days 21-35 (media change every two days): CMRLS + 10 μM Alk5i II + 1 μM T3. Cells were examined by in vitro assays between 28 and 32 days. Cells were transplanted on day 28 unless otherwise noted.

[664] Alternate, Days 21-35: CMRLS + 10 μM Alk5i II + 1 μM T3 + Sant1.

[665] Alternate, Days 21-35: CMRLS + 10 μM Alk5i II + 1 μM T3 + XXI.

[666] Alternate, Days 21-35: CMRLS + 10 μM Alk5i II + 1 μM T3 + SSP.

[667] In order to obtain cells according to the PH protocol that would mimic previous publications, the same differentiation protocol was used up to day 14. On days 14 and 16, cells were fed with S3 + 1 µM Alk5i II, 200 nM LDN193189, and 100 nM RA (Rezania et al., 2012). On day 18 and beyond, cells were fed every other day with S3 + 100 nM RA. The cells were kept in this medium until the experimental analysis. Cells were maintained in culture and examined after the same number of days in differentiation medium as SC-β cells to account for the effect of time.

[668] Flow Cytometry

[669] Cells were dispersed into a suspension containing single cells by incubation in TrypLE Express at 37° C. for 10-30 minutes in a 15 ml Falcon tube. Starting from stage 5, the clusters took longer to dissociate into single cells. Clusters were to be incubated in TrypLE Express until they dissociated into single cells after mixing by gentle pipetting with P1000. TrypLE Express was blocked with 3-4 times the volume of the medium, and the cells were centrifuged for 5 min at 1000 rpm. Cells were washed twice in PBS and transferred to a 1.7 ml securely sealed microcentrifuge tube (Bioscience Inc.; 11510). After making sure that the number of cells is 1-10 million cells per tube, volumes of 0.5-1 ml were used as follows. Cells were resuspended in 4% paraformaldehyde (PFA) (Electron Microscopy Scienc Nm; 15710) and incubated on ice for 30 min. Cells were then washed 3 times in PBS followed by incubation in blocking buffer (PBS + 0.1% TritonX100 (VWR; EM-9400) + 5% donkey serum (Jackson Immunoresearch; 017-000-121)) on ice for 1 hour . After fixation, cells become more resistant to centrifugation; therefore, after fixation, all centrifugations were performed at 3000g for 3 min. The cells were then resuspended in blocking buffer with primary antibodies and incubated at 4° C. overnight.

[670] Primary antibodies were used at a ratio of 1:300 unless otherwise indicated: goat anti-human PDX-1/IPF1 (R&D Systems; AF2419), mouse anti-NKX6-1 (University of Iowa, Developmental Hybridoma Bank; F55A12-supernatant) ( 1:100), rabbit to chromogranin A (Abcam; ab15160), rat to insulin (pro-)/C-peptide (Developmental Studies Hybridoma Bank at the University of Iowa; GN-ID4) (must add glucagon and somatostatin). Cells were washed 2 times in blocking buffer and then incubated in blocking buffer along with secondary antibodies on ice for 2 hours (without light). Secondary antibodies conjugated to Alexa Fluor 488, 647 (Life Technologies) or PE (Thermo Fisher Scientific; NC9774252) were used to visualize the primary antibodies. Cells were then washed 3 times in sorting buffer (PBS + 0.5% BSA (Sigma; A8412)) and resuspended one last time in 500-700 µl of sorting buffer, filtered through a 40 µm nylon mesh into FACS tubes (BD Falcon; 352235 ) and analyzed with an LSR-II FACS device (BD Biosciences) recording at least 30,000 events. The results were analyzed using the FlowJo software.

[671] Immunofluorescence

[672] Cells were dispersed and plated in 96-well plates. After a day in culture, the cells were washed in PBS and fixed in 4% PFA for 20 min at RT. After 3 washes in PBS, cells were blocked for 1 hour at RT in PBS + 0.1% Triton X-100 + 5% donkey serum. All incubations with primary antibodies were performed overnight at 4°C in blocking solution at a dilution of 1:500: goat anti-human PDX-1/IPF1, mouse anti-NKX6-1, rabbit anti-chromogranin A, rat insulin (pro-)/ C-peptide, Ki67. The next day, the cells were washed 2 times in PBS, followed by incubation with secondary antibodies for 1 hour at RT at a dilution of 1:500 (without light). Secondary antibodies conjugated to Alexa Fluor 488, 594 or 647 (Life Technologies) were used to visualize the primary antibodies. After 3 washes in PBS, all nuclei were visualized by DAPI staining (Invitrogen; D1306). Sample images were obtained using an Olympus IX51 microscope or a Zeiss LSC 700 confocal microscope. The percentage of target cell types was assessed using the Array Scan Cellomics multiparametric analysis system. In this case, 30 images were obtained and quantified for each well. Cells labeled with antibody staining and the total number of cells (based on nuclear DAPI staining) were quantified to obtain a percentage of target cell types.

[673] Immunohistochemistry

[674] Cell clusters or islets were fixed by immersion in 4% PFA for 1 hour at CT. The samples were then washed 3 times in PBS, immersed in Histogel™ and sectioned for histological analysis. The 10 μm sections were then deparaffinized with Histoclear (Thermoscientific; C78-2-G) and rehydrated. For antigen unmasking, preparations were removed from 0.1 M EDTA (Ambion; AM9261) and placed in an autoclave (Proteogenix; 2100 Retriever) for two hours. The preparations were then blocked with PBS + 0.1% Triton X-100 + 5% donkey serum for 1 hour at RT, followed by overnight incubation in blocking solution with primary antibodies at 4°C. The following primary antibodies were used in a ratio of 1:100, unless otherwise indicated: goat anti-human PDX-1/IPF1, mouse anti-NKX6-1, rabbit anti-chromogranin A, rat anti-insulin (pro-)/C-peptide, glucagon, somatostatin. The next day, the cells were washed 2 times in PBS, followed by incubation with secondary antibodies for 2 hours at RT (without light). Secondary antibodies conjugated to Alexa Fluor 488 or 594 were used to visualize the primary antibodies. After washing with PBS, the histological slides were embedded in Vectashield DAPI embedding medium (Vector Laboratories; H-1200), covered with coverslips, and sealed with nail polish. Typical images were obtained using an Olympus IX51 microscope or a Zeiss LSC 510 or 710 confocal microscope.

[675] Glucose-stimulated insulin secretion

[676] Krebs buffer (Krb): Di H ₂ O with 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl ₂ , 1.2 mM MgCl ₂ , 1 mM Na ₂ HPO ₄ , 1.2 mM KH ₂ PO ₄ , 5 mM NaHCO ₃ , 10 mM HEPES (Life Technologies; 15630080), 0.1% BSA (Proliant; 68700). The solutions were equilibrated to 37° C. in an incubator and 1 ml volumes were used in the following protocols. Approximately 0.5 million cells were transferred in clusters to autoclaved 1.7 ml securely sealed microfuge tubes and washed 2 times in Krb. The clusters were then pre-incubated in Krb with 2 mM glucose for 2 hours in an incubator to remove residual insulin. It should be noted that during all incubations, the lids of the tubes were left open and covered with a lid, which allowed air exchange. Clusters were washed 2 times in Krb and then incubated in Krb containing 2 mM glucose for exactly 30 minutes, and after incubation, 200 μl of supernatant (low glucose sample) was collected. Clusters were washed 2 times in Krb and then incubated in Krb with 20 mM glucose for exactly 30 minutes, and after incubation, 200 μl of supernatant (high glucose sample) was collected. Stimulation with low and high glucose concentrations was repeated two additional times (a total of 3 paired stimulations). Finally, the clusters were washed 2 times in Krb and then incubated in Krb with 2 mM glucose + 30 mM KCl for exactly 30 min, and after incubation, 200 μl of the supernatant (sample with KCl polarization stimulation) was collected. After KCl stimulation, the clusters were dispersed with Tryple and counted with Viacell (manufacturer) to normalize the amount of insulin secreted relative to the number of cells.

[677] Samples of supernatants containing secreted insulin were examined using Human Ultrasensitive Insulin ELISA (ALPCO Diagnostics; 80-INSHUU-E01.1) and samples were measured on a FLUOstar optima spectrophotometer (BMG lantech) at 450 nm. If the ELISA was not performed on the same day, the samples were stored at -80°C. In order to obtain insulin concentrations corresponding to the ELISA range, a dilution of the samples from 1:100 to 1:200 in PBS was usually sufficient.

[678] Electron microscopy

[679] Cell clusters were fixed with a mixture containing 1.25% PFA, 2.5% glutaraldehyde and 0.03% picric acid in 0.1 M sodium cacodylate buffer (pH 7.4) for at least 2 hours at CT. Cell clusters were then washed in 0.1 M cacodylate buffer, then fixed in a mixture of 1% osmium tetroxide (OsO4) and 1.5% potassium ferrocyanide (KFeCN6) for at least 2 hours at RT, washed in 0.1 M cacodylate buffer , then fixed in 1% osmium tetroxide (OsO4)/1.5% potassium ferrocyanide (KFeCN6) for 1 hour, washed in water 3x and stained in 1% aqueous uranyl acetate for 1 hour followed by 2 washes in water and subsequent dehydration in alcohol of varying degrees of purification (10 min each; 50%, 70%, 90%, 2×10 min 100%). The samples were then incubated in propylene oxide for 1 hour and filtered with ON in a 1:1 mixture of propylene oxide and Epon TAAB (Marivac Canada Inc. St. Laurent, Canada). The next day, the samples were immersed in TAAB Epon and polymerized at 60°C for 48 hours. Using a Reichert Ultracut-S microtome, ultrathin sections (about 60 nm) were obtained, placed on a copper grid, stained with 0.2% lead citrate and examined using a transmission electron microscope JEOL 1200EX or TecnaiG ² Spirit BioTWIN. Images were recorded using an AMT 2k CCD camera and analyzed using ImageJ software.

[680] Transplantation studies in SCID mice lacking natural killer cells

" (Terumo; SB*S23BL), соединенную с 2 мл шприцом, - промывали 1 мл среды RPMI с добавлением 5% сыворотки ФБС (Coming; 35-011-CV), затем загружали 0,6 мл среды RPMI без добавления сыворотки. Кластеры загружали через наконечник иглы катетера, который помещали вертикально на 5 минут при комнатной температуре, чтобы под воздействием гравитации они оказались внизу трубки катетера и около окончания иглы. Во время этапа приготовления клеток иммунодефицитных (ТКИН/отсутствие естественных клеток-киллеров) мышей (возраст?) анестезировали авертином 1,25% (250 мг/кг; 0,5 мл/25 г 1,25% авертина/массу тела), а операционное поле с левой стороны обривали и дезинфицировали бетадином и спиртом. Делали разрез длиной около 1 см, чтобы открыть почку и инъецировали кластеры под почечную капсулу путем введения иглы катетера под почечную капсулу и инъекции объема около 100 мкл, содержащего 5 миллионов эквивалентных клеток, собранных в кластеры. Брюшную полость зашивали рассасывающимися ПДС нитками (POLY-DOX; 2016-06)), а кожу зажимали хирургическими зажимами (Kent Scientific Corp; INS750346-2). Во время операции и восстановительного периода мышей размещали на оборудованном микроциркулирующим насосом одеяле (~37°С), чтобы способствовать быстрому восстановлению мышей после анестезии, и давали дозу 5 мг/мкг карпрофена непосредственно после операции и через 24 часа после первой дозы. Среднее время трансплантации составляло приблизительно 3 минуты на мышь. Через 14 дней после операции убирали зажимы и проводили осмотр мышей дважды в неделю.[681] 5 million clustered hPSC-derived cells were resuspended in RPMI1640 medium (Life technologies; 11875-093), aliquoted into 200 µl PCR tubes, and kept on ice for 5-10 minutes before loading into the catheter . To prepare for cell loading, each catheter is a 23G × infusion set

"(Terumo; SB*S23BL) connected to a 2 ml syringe, washed with 1 ml of RPMI medium with the addition of 5% PBS serum (Coming; 35-011-CV), then loaded with 0.6 ml of RPMI medium without addition of serum. Clusters were loaded through the tip of the catheter needle, which was placed vertically for 5 minutes at room temperature, so that under the influence of gravity they were at the bottom of the catheter tube and near the tip of the needle.During the cell preparation step of immunodeficient (SCID/lack of natural killer cells) mice (age?) anesthetized with avertin 1.25% (250 mg/kg; 0.5 ml/25 g 1.25% avertin/body weight) and the surgical field on the left side was shaved and disinfected with betadine and alcohol.A cut about 1 cm long was made to open the kidney and injected the clusters under the renal capsule by inserting a catheter needle under the renal capsule and injecting a volume of about 100 μl containing 5 million equivalent cells collected in clusters.The abdominal cavity was sutured with absorbable PDS sutures (POLY-DOX; 2016-06)), and the skin was clamped with surgical clamps (Kent Scientific Corp; INS750346-2). During the operation and recovery period, mice were placed on a microcirculatory pump-equipped blanket (~37°C) to promote rapid recovery of mice from anesthesia, and were given a dose of 5 mg/μg of carprofen immediately after surgery and 24 hours after the first dose. The average transplant time was approximately 3 minutes per mouse. The clamps were removed 14 days after the operation and the mice were examined twice a week.

[682] After two weeks of recovery from surgery, human insulin availability and glucose sensitivity of transplanted cells were assessed by performing a glucose tolerance test (GTT). After an overnight fast of mice for 16 hours, GTT was performed by IP (intraperitoneal) injection of 2 g D-(+)-glucose/1 kg body weight and blood sampling before and/or after injection by puncture of the facial vein using a lancet (Feather; 2017 -01). Thereafter, human insulin was quantified using a human insulin ELISA kit (ALPCO Diagnostics; 80-INSHUU-E01.1). Implants derived from SCID mice were dissected, fixed in 4% PFA (Electron Microscopy Scienc Nm; 15710), embedded in paraffin, and sectioned for histological analysis.

[683] Calcium Imaging

[684] About 10-20 hPSC-derived clusters were seeded onto 96-well Matrigel coated plates and allowed to establish in an incubator at 37° C., 5% CO ₂ and 100% humidity. Clusters were washed with prewarmed (37°C) Krebs buffer supplemented with 2.5 mM glucose, then incubated with 50 μM Ca ²⁺ sensitive fluorescent probe Fluo4-AM (Life Technologies; F14217) in Krb buffer containing 2.5 mM glucose on for 45 minutes in an incubator at 37°C. The clusters were washed with 2.5 mM glucose buffer Krb, then further incubated in an incubator at 37° C. for an additional 15 minutes. The clusters were then immediately placed under an AxioZoom V16 microscope (Carl Zeiss) for high resolution dynamic visualization of different groups of clusters in different wells. Fluo-4 excitation occurred at 488 nm and emission was recorded between 490 and 560 nm. Dynamic series of images were acquired at single cell resolution corresponding to 80x magnification every 17 seconds, imaging up to 10 wells per image. The increase in glucose stimulation and stimulation time during imaging was as follows. Imaging was started with a 5 min stimulation with 2 mM glucose in Krb followed by a 5 min stimulation with 20 mM glucose in Krb buffer. These low and high glucose stimulations were repeated two more times, then imaging was terminated with a 5 minute stimulation with 30 mM KCl in Krb buffer for a total imaging time of 35 minutes. Between stimulations, imaging was stopped and the clusters were washed with 2 mM glucose Krb buffer, and then the next glucose buffer Krb was added. The change in fluorescence intensity over 35 minutes of imaging was analyzed at single cell resolution using Imagej/Fiji, using StackReg to correct for cluster movement during imaging and an ROI system to measure cell fluorescence intensity during imaging. All 7 stimulations, recorded as 5-minute clips, were combined into one VirtualDub video and uploaded to youtube for collective use.

[685] Intracellular flow cytometry and gene expression analysis

[686] MARIS was performed as described in Hrvatin et al., 2014. Cells were collected in single cell suspension, fixed in 4% PFA on ice, incubated with primary antibodies and then with secondary antibodies in RNase-containing buffer. The cells were then sorted by FACS to obtain at least 100,000 cells per sample. Thereafter, the samples were incubated in 50° C. digestion buffer for 3 hours before RNA isolation. RNA concentration was quantified using a Nanodrop 1000. Double-stranded cDNA was obtained by reverse transcription from at least 100 ng of total RNA. Then amplified mRNA (cRNA) was obtained by In vitro transcription overnight with biotin-labeled nucleotides and concentrated by vacuum centrifugation at 30°C. At least 750 ng of cRNA per sample was hybridized to Human HT-12 Expression BeadChips (Illumina) using the Whole-Genome Expression Direct Hybridization Kit (Illumina). The chips were scanned on an Illumina Beadstation 500. The original data were corrected by background subtraction and invariant normalization. Before calculating the fold change, a value of 20 was added to all means to remove negative signals, p-values for the difference between the mean signals were calculated in GenomeStudio using the t-test and adjusted according to multiple hypothesis testing using the Benjamini-Hochberg method in combination with the traditional model Illumina False Positive Rate (FDR).

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Claims (11)

1. Method for differentiating PDX1-positive pancreatic progenitors into NKX6.1-positive pancreatic progenitors using a culture medium containing a) KGF, b) SANT1 and c) retinoic acid (RA), wherein the culture medium does not contain a protein kinase activator C (PKC). 2. The method of claim 1, wherein the PKC activator is phorbol 12,13-dibutyrate (PdBu) or TBP. 3. The method according to p. 1, characterized in that the PKC activator is PdBu. 4. The method according to p. 1, characterized in that the PCK activator is TRB. 5. A method for differentiating PDX1-positive pancreatic progenitors into NKX6.1-positive pancreatic progenitors using a culture medium containing a) KGF, b) SANT1 and c) retinoic acid, the culture medium not containing an inhibitor of the BMP signaling pathway. 6. The method according to claim 5, characterized in that the inhibitor of the BMP signaling pathway is noggin, chordin, gremlin and follistatin, as well as LDN 193189. 7. The method according to claim 5, characterized in that the inhibitor of the BMP signaling pathway is noggin. 8. The method according to claim 5, characterized in that the inhibitor of the BMP signaling pathway is chordine. 9. The method according to claim 5, characterized in that the inhibitor of the BMP signaling pathway is gremlin. 10. The method according to claim 5, characterized in that the inhibitor of the BMP signaling pathway is follistatin. 11. The method according to claim 5, characterized in that the inhibitor of the BMP signaling pathway is LDN 193189.

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