JP7550830B2 - Cellular compositions derived from dedifferentiated reprogrammed cells - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. Patent Application No. 13/761,078, filed February 6, 2013.
Title CELL COMPOSITIONS DERIVED FROM DEDIFFE
This application claims priority to U.S. Patent Application No. 12/765,714, entitled "REPROGRAMMED CELLS," filed April 22, 2010.
LL COMPOSITIONS DERIVED FROM DEDIFFERENT
119(e) of U.S. Provisional Patent Application No. 61/171,759, filed April 22, 2009, entitled CELL COMPOSITIONS DERIVED REPROGRAMMED CELLS.
FROM DEDIFFERENTIATED REPROGRAMMED CELLS
No. 6,399,433, filed on Dec. 13, 2003, the disclosures of which are incorporated herein by reference in their entireties.

SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format. The Sequence Listing is a sequence listing of size 8.
The information in the electronic format of the Sequence Listing is provided in a 92 Kb file and is hereby incorporated by reference in its entirety.

The present invention relates generally to the isolation, maintenance and use of cell cultures. More specifically, the present invention relates to cell compositions derived from induced pluripotent stem cells.

An important application of pluripotent cells is their use in cell therapy. Pluripotent stem cells include, but are not limited to, human embryonic stem (hES) cells, human embryonic germ (hEG) cells. There are still other types of pluripotent cells, for example, dedifferentiated mouse and human stem cells, i.e., differentiated somatic adult cells are dedifferentiated to become pluripotent-like stem cells. These dedifferentiated cells, which are induced to establish cells with pluripotency and growth capacity similar to ES cells, are called "induced pluripotent stem (iPS) cells,""embryonic stem cell-like cells," and "ES-like cells."
or their equivalents. Such cells are potentially viable alternative pluripotent cells. Therapeutic application of iPS cells requires demonstrating that these cells are stable and exhibit a suitable safety profile in preclinical studies to treat diabetes and other diseases. Reprogramming differentiated human somatic cells to a pluripotent state allows for patient- and disease-specific stem cells. Takahashi, K. et al. Cell, 1-12, 2
See, for example, 2007 and Ju, J. et al. Science 2007.
and Ju et al. introduced four genes into adult and fetal/neonatal fibroblasts, respectively, to generate iPS cells: Oct4, Sox2, Klf4, and c-myc, Ta
Kahashi et al.; Oct4, Sox2, Nanog and Lin28, Ju et al. In all cases, iPS cells had some characteristics of hES cells, including hES cell morphology, marker expression, prolonged proliferation, normal karyotype and pluripotency.

Although iPS cells can provide cell therapy-based regenerative medicine without associated ethical controversies, the differentiation properties of iPS cells, such as in vitro differentiation potential and differentiation efficiency, are still unclear, and no directed differentiation method for iPS cells has been demonstrated. Therefore, there is a need to determine and demonstrate the detailed differentiation properties and directed differentiation efficiency of iPS cells.

Embodiments described herein provide cell compositions derived from pluripotent cells, e.g., dedifferentiated reprogrammed cells, such as induced pluripotent stem (iPS) cells.

One embodiment provides a composition comprising human cells and a method of making an in vitro cell culture comprising human cells, wherein at least about 15% of the human cells are definitive endoderm cells, and the embryonic somatic cells are derived from the dedifferentiated genetically reprogrammed cells.
Definitive endoderm cells are multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

Another embodiment provides a composition comprising human cells and a method of making an in vitro cell culture comprising human cells, wherein at least about 15% of the human cells are pancreatic-duodenal homeobox factor 1 (PDX1) positive foregut endoderm cells, and the PDX1 positive foregut endoderm cells are derived from dedifferentiated genetically reprogrammed cells.
1 positive foregut endoderm cells are co-positive for PDX1, SOX9, PROX1 and HNF6.

Further embodiments provide compositions comprising human cells and methods of making in vitro cell cultures comprising human cells, wherein at least about 15% of the human cells are pancreatic-duodenal homeobox factor 1 (PDX1) positive pancreatic progenitor cells, and the PDX1 positive pancreatic progenitor cells are derived from the dedifferentiated genetically reprogrammed cells.
NKX6.1 positive pancreatic progenitor cells are co-positive for PDX1 and NKX6.1.

Yet another embodiment provides compositions comprising human cells and in vitro methods comprising human cells.
The present invention provides a method for producing a cell culture, wherein at least about 15% of the human cells are Neurogene.
nin3 (NGN3) positive endocrine precursor cells, NGN3 endocrine precursor cells are derived from dedifferentiated genetically reprogrammed cells. In one aspect, NGN3 positive endocrine precursor cells are co-positive for NGN3, PAX4 and NKX2.2.
Further embodiments of the cell culture compositions described herein include in vitro human pancreatic endoderm cell cultures comprising differentiated cells derived from the dedifferentiated genetically reprogrammed cells and an ERBB receptor tyrosine kinase activating agent.
Additional embodiments described herein relate to methods of producing insulin, hi some such embodiments, the methods include: (a) at least a foregut endoderm cell culture derived from dedifferentiated genetically reprogrammed cells and/or at least PDX
ERBB receptor tyrosine kinase activating agents were administered in
(b) contacting in vitro, thereby generating a pancreatic endoderm cell population comprising endocrine cells and a non-endocrine cell subpopulation; and (b) transplanting and maturing the pancreatic endoderm cell population of step (a) or the cell subpopulation of step (a) in vivo, thereby obtaining insulin-secreting cells, wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.
Yet other embodiments described herein include a method of producing insulin, comprising:
(a) contacting in vitro the dedifferentiated genetically reprogrammed cells with a first culture medium comprising an agent that activates a TGFβ receptor family member; (b) culturing in vitro the cells of step (a) in a second culture medium lacking the agent that activates a TGFβ receptor family member, thereby generating at least foregut endoderm cells and/or at least PDX1-negative foregut endoderm cells; and (c) culturing in vitro the cells of step (a) in a second culture medium lacking the agent that activates a TGFβ receptor family member, thereby generating at least foregut endoderm cells and/or at least PDX1-negative foregut endoderm cells.
) contacting the cell of step (b) with an ERBB receptor tyrosine kinase activating agent;
thereby generating a cell population comprising endocrine cells and a non-endocrine cell subpopulation; and (d) in vivo culture of the cell population of step (c) or the cell subpopulation of step (c).
and (b) transplanting and maturing the cells in a culture medium containing at least PDX1-negative foregut endoderm cells and/or at least PDX1-positive pancreatic endoderm cells with an ERBB receptor tyrosine kinase activating agent, thereby generating a cell population capable of maturing in vivo into glucose-responsive insulin-secreting cells.
Still other embodiments described herein relate to contacting a population comprising at least foregut endoderm cells, at least PDX1-negative foregut endoderm cells and/or at least PDX1-positive pancreatic endoderm cells with an ERBB receptor tyrosine kinase activating agent and a rho kinase inhibitor, thereby generating a cell population capable of maturing in vivo into glucose-responsive insulin-secreting cells.
As used herein, the phrases "at least foregut endoderm cells,""at least PDX
"PDX1-negative foregut endoderm cells" and "at least PDX1-positive pancreatic endoderm cells" mean that some or a portion of the cells of the cell population have differentiated from iPSCs to foregut endoderm cells or higher, from iPSCs to PDX1-negative foregut endoderm cells or higher, and/or from iPSCs to PDX1-positive pancreatic endoderm cells or higher, during their differentiation into pancreatic islet cells.
As used herein, when referring to cells of a cell population, the terms "some" and/or "part" refer to cells of a cell population that are at least 5%, at least 10%, at least 15%,
%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 6
By this definition, it is meant that the cells contain 0%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or greater than at least 95% of cells of a specified cell type.

1 is a photographic image of an aggregated suspension culture of dedifferentiated reprogrammed cells, or cells also referred to herein as iPS cells.

2A-2C are bar graphs showing relative gene expression levels of OCT4 (FIG. 2A), BRACHYURY (FIG. 2B), CER1 (FIG. 2C), GSC (FIG. 2D), FOXA2 (FIG. 2E), FOXA1 (FIG. 2F), HNF6 (FIG. 2G), PDX1 (FIG. 2H), PTF1A (FIG. 2I), NKX6.1 (FIG. 2J), NGN3 (FIG. 2K), and INS (FIG. 2L). Expression levels are normalized to the mean expression levels of housekeeping genes, cyclophilin G, and TATA binding protein (TBP) expression. Graphs represent fold upregulation from the lowest data point in the data set.

3A-3D are immunocytochemistry (ICC) photomicrographs of human iPS cell cultures from stage 4 differentiation using antibodies specific for (3A) PDX-1; (3B) NKX6.1; (3C) PTF1A; and (3D) Dapi.

Photographs of immunocytochemistry (ICC) of iPS cell cultures from stage 4 differentiation using ligands specific for (FIG. 4A) glucagon, (FIG. 4B) insulin, (FIG. 4C) somatostatin, and (FIG. 4D) Dapi. FIG. 5 shows an array location map and array key provided with the Proteome Profiler™ Human Phospho-RTK Antibody Array from R&D Systems. The location map in FIG. 5A shows the coordinates or locations of the RTK antibodies. The RTK family and antibody identity or name are listed in the key in FIG. 5B. Thus, a positive signal observed in the developed film can be identified by overlaying a transparency as in FIG. 5A and identifying the signal by referencing the coordinates on the overlay (FIG. 5A) with the name of the RTK in FIG. 5B. Figure 1 shows RTK array analysis of iPS cell-derived pancreatic endoderm cells (PEC) under four different conditions (Figures A, B, C and D, as described in Example 5). Tyrosine phosphorylation of specific RTKs is observed by identification of high to low intensity signals. IGF 1R/IR and ERBB (EGFR) family members are identified or boxed. Graphs showing concentrations of human C-peptide and insulin in serum of transplanted mice from experiments E2314, E2356 and E2380 (FIG. 7A), E2347 (FIG. 7B) and E2354 (FIG. 7C) as shown in Table 9. Mice transplanted with PECs were analyzed at the indicated post-implantation time points for serum levels of human C-peptide at fasting and 30 and 60 minutes after intraperitoneal glucose administration. In FIG. 7C, PECs were encapsulated with a cell encapsulation device (Encaptra® EN20 or EN20, ViaCyte, San Diego, CA), and in some cases the device had microperforations (pEN20, ViaCyte, San Diego, CA). Such devices are described in U.S. Pat. No. 8,278,106, the disclosure of which is incorporated herein by reference in its entirety. 8A-8B are graphs showing the results of blood glucose analysis of STZ-treated mice from experiment #2347. FIG. 8A shows blood glucose for each of the 13 mice (baseline with and without Heregulin), and FIG. 8B shows the combined average measurements for each treatment (baseline with and without Heregulin). For 13 mice that received iPEC grafts up to 14 days prior to treatment with STZ (day 0), random non-fasting blood glucose level measurements are shown for the same mice after STZ treatment and after explantation of the graft. STZ-treated animals received STZ approximately 26 weeks after implantation of the graft (day 0). 28 weeks after graft implantation, iPEC grafts were explanted (removed) approximately 2 weeks after the start of STZ treatment. Non-fasting blood glucose measurements were collected over time for each animal.

The present invention can be readily understood by reference to the following detailed description of the preferred embodiments and examples of the invention contained herein. However, the present compounds, compositions,
Before the disclosure and description of the present invention and methods, it should be understood that the present invention is not limited to a specific cell type, a specific feeder cell layer, specific conditions, or a specific method, etc., which may vary as such. Numerous modifications and variations will be apparent to those skilled in the art. It should also be understood that the terms used herein are merely for the purpose of describing specific embodiments and are not limiting.

DEFINITIONS The numerical ranges expressed herein are understood to be inclusive of the recited endpoints and to recite every integer between the endpoints of the stated numerical range.

Unless otherwise specified, the terms used herein are understood according to conventional usage by those skilled in the art.Also, for the purposes of this specification and the appended claims, unless otherwise specified, all amounts of ingredients, percentages or proportions of materials, numbers expressing reaction conditions, and other numerical values used in this specification and the appended claims should be understood to be modified in all cases by the term "about".Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that can vary depending on the desired properties to be obtained by the present invention.
At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

The practice of the embodiments described herein will employ, unless otherwise specified, conventional techniques of cell biology, molecular biology, genetics, chemistry, microbiology, recombinant DNA, and immunology.

In this specification and the appended claims, the singular forms "a,""an," and "th" are used interchangeably.
It should be understood that "e" includes the plural unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes one or more of such different cells, and reference to "a method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified or substituted for the methods described herein.

The term "cell" as used herein refers to an individual cell, a cell line, or a culture derived from such a cell. A "culture" refers to a composition containing isolated cells of the same or different type.

As used herein, the phrase "totipotent stem cells" refers to cells that have the potential to differentiate into all cells that make up an organism, such as cells resulting from the fusion of an egg cell and a sperm cell. Cells resulting from the first few divisions of a fertilized egg may also be totipotent. These cells can differentiate into embryonic and extraembryonic cell lineages. Pluripotent stem cells, such as ES cells, can give rise to any fetal or adult cell type. However, they cannot develop into a fetus or adult animal on their own, as they lack the ability to develop into extraembryonic tissues. Extraembryonic tissues are derived, in part, from the extraembryonic endoderm and are called distal endoderm.
doderm (Reichert's membrane) and visceral endoderm
m) (which form part of the yolk sac). Both distal and proximal endoderm support the development of the embryo, but do not themselves form embryonic structures. Other extraembryonic tissues also exist, including extraembryonic mesoderm and extraembryonic ectoderm.

In some embodiments, "pluripotent cells" are used as starting material for differentiation into endoderm lineages, and more particularly, pancreatic endoderm-type cells. As used herein, "pluripotent" refers to
or "pluripotent cells" or equivalents refer to cells that can be expanded in cell culture and differentiated into various lineage-restricted cell populations that exhibit multipotent properties;
For example, both pluripotent ES cells and induced pluripotent stem (iPS) cells can give rise to each of the three embryonic cell lineages, but pluripotent cells may not be able to generate an entire organism, i.e., pluripotent cells are not totipotent.

In certain embodiments, the pluripotent cells used as starting material are hES cells, hE
As used herein, "embryonic" refers to the various developmental stages of an organism that begins with a single zygote and ends with a multicellular structure that no longer contains pluripotent or totipotent cells other than the gamete cells that developed. The term "embryonic" refers to embryos derived by gamete fusion as well as embryos derived by somatic cell nuclear transfer. In yet another embodiment, the pluripotent cells are not derived or directly derived from an embryo, e.g.
iPS cells are derived from non-pluripotent cells, such as multipotent cells or terminally differentiated cells.

Human pluripotent stem cells can also be defined or characterized by the presence of several transcription factors and cell surface proteins, such as the transcription factors Oct-4, Nanog, and Sox-2, that form a core regulatory complex that ensures the repression of genes that lead to differentiation and the maintenance of pluripotency, as well as cell surface antigens, such as the glycolipids SSEA3, SSEA4 and the keratan sulfate antigens, Tra-1-60 and Tra-1-81.

As used herein, "induced pluripotent stem cells" or "iPS cells" or "iPS cells"
The phrase "SC" refers to non-pluripotent cells, generally adult somatic cells, or, for example, fibroblasts,
It refers to a type of pluripotent stem cell artificially prepared from terminally differentiated cells such as hematopoietic cells, muscle cells, neurons, and epidermal cells by inserting specific genes or gene products called reprogramming factors, the disclosure of which is fully incorporated herein by reference.
Takahashi et al., Cell, 131:861-872 (2007); Wernig
et al., Nature, 448:318-324 (2007); Park et al., Nature,
451:141-146 (2008). Induced pluripotent stem cells are substantially similar to naturally occurring human pluripotent stem cells, such as hES cells, in many respects, including expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and developmental potential and differentiability. Human iPS cells provide a source of pluripotent stem cells without the use of embryos.

A variety of methods can be used to generate iPS cells, which are described in more detail herein below. However, all methods involve the use of Sox-2, Oct-4, Na
nog and optionally an expression cassette encoding Lin-28, or Sox-2, O
an expression cassette encoding ct-4, Klf4 and optionally c-myc, or So
Specific reprogramming factors are used, such as expression cassettes encoding Oct-2, Oct-4 and optionally Esrrb. The nucleic acids encoding these reprogramming factors may be in the same expression cassette, different expression cassettes, the same reprogramming vector, or different reprogramming vectors. Oct-3/4 and specific members of the Sox gene family (Sox-1, Sox-2, Sox-3 and Sox-15) are
It is a key transcription factor involved in the induction process, the absence of which renders induction impossible. Oct-3/4 (Pou5f1) is a member of the family of octameric ("Oct") transcription factors and plays a key role in the maintenance of pluripotency. For example, the absence of Oct-3/4 in cells that are normally Oct-3/4+, such as blastomeres and embryonic stem cells, leads to spontaneous trophoblast differentiation, whereas the presence of Oct-3/4 results in the pluripotency and differentiation potential of embryonic stem cells. Furthermore, other genes in the "Oct" family, such as Oct1 and Oct6, do not induce pluripotency, and therefore this pluripotency induction process can be attributed to Oct-3/4. Another family of genes related to the maintenance of pluripotency similar to Oct-3/4 is the Sox family.
The Sox family is a family of Sox kinases. However, the Sox family is not restricted to pluripotent cell types, but is also associated with multipotent and unipotent stem cells. The Sox family has also been found to play a role in the induction process. In the initial study by Takahashi et al., 2006, supra, Sox2 was used. Since then, Sox1, Sox3, Sox15 and Sox18 genes have also generated iPS cells. The Klf family of genes (Klf-1, Klf2, Klf3, Klf4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16, Klf17, Klf18, Klf19, Klf20, Klf21, Klf22, Klf23, Klf24, Klf25, Klf26, Klf27, Klf28, Klf29, Klf30, Klf31, Klf32, Klf33, Klf34, Klf35, Klf36, Klf37, Klf38, Klf40, Klf41, Klf42, Klf43, Klf44, Klf45, Klf46, Klf47, Klf48, Klf50, Klf51, Klf52, Klf53, Klf54, Klf55, Klf56, Klf57, Klf58, Klf69, Klf69, Klf69, Klf70, Klf71, Klf72, Klf73, Klf74, Klf75, Klf76, Klf77, Klf78, Klf79, Klf80, Klf81, Klf82, Klf83, Klf84, Klf85, Klf96, Klf97, Klf98, Klf9
Klf4, Klf5, and Klf6 were initially identified by Yamanaka et al., 2006, supra, as a factor in the generation of mouse iPS cells.
Human iPS cells from .ka were used to explore the cell therapy application of hIPS cells.
However, Yu et al. (2007) noted above that Klf4 is not required and, indeed, human iPSCs
They reported that they were unable to generate cells that were specifically engineered to express Klf1, Klf2, and Klf5.
Other members of the Klf family are capable of generating iPS cells, including: Finally, the Myc family (C-myc, L-myc, and N-myc), proto-oncogenes that have been linked to cancer; c-myc has been implicated in the generation of mouse and human iPS cells, but Yu et al. (2007) supra reported that c-myc was not required for the generation of human iPS cells.

As used herein, "multipotency" or "multipotent cells" or equivalents refer to a cell type that can give rise to a limited number of other specific cell types, i.e., multipotent cells are committed to one or more embryonic cell fates and thus, in contrast to pluripotent cells, are unable to give rise to each of the three embryonic cell lineages, as well as extraembryonic cells.
Multipotent somatic cells are more differentiated than pluripotent cells, but are not terminally differentiated. Thus, pluripotent cells have a higher developmental potential than multipotent cells. Developmental potential determining factors that can be used to reprogram somatic cells or generate iPS cells include, but are not limited to, Oct-4, Sox2, FoxD3, UTF1, Stella
, Rexl, ZNF206, Soxl5, Mybl2, Lin28, Nanog, DPP
A2, ESG1, Otx2 or a combination thereof.
As used herein, an "ERBB receptor tyrosine kinase activating agent" includes, but is not limited to, at least 16 different EGF family ligands that bind to the ERBB receptor: EGF (epidermal growth factor), AG or AREG (amphiregulin), and TGF-alpha (transforming growth factor-alpha), Btc (betacellulin), HBEGF (heparin-binding EGF), and Ereg (epiregulin).
ERBB receptors include neuregulins (or heregulins), such as neuregulin-1, -2, -3 and -4 (or heregulin-1, -2, -3 and -4. However, the present invention contemplates any ligand that can bind to any one or combination of the four ERBB receptors and induce the formation of homo- and heterodimeric receptor complexes that lead to activation and subsequent phosphorylation of the intrinsic kinase domain. See also Table 11.
Some embodiments of the methods for producing insulin described herein can include treating an animal for diabetes or controlling glucose concentrations in the blood of an animal by providing the animal with pancreatic endoderm cells that can mature in vivo into insulin-producing cells that secrete insulin in response to glucose stimulation.

One embodiment described herein includes a population of pluripotent or progenitor cells that are capable of selectively, and in some embodiments selectively and reversibly, developing into different cell lineages when cultured under appropriate conditions. As used herein, the term "population" refers to a cell culture of multiple cells having the same identifying characteristic. The term "cell lineage" refers to all stages of development of a cell type from the earliest precursor cells to fully mature cells (i.e., specialized cells). A "progenitor cell" or "precursor cell" may be any cell within a cell differentiation pathway that can differentiate into a more mature cell. As such, a progenitor cell may be a pluripotent cell, a partially differentiated multipotent cell, or a reversibly differentiated cell. The term "progenitor cell population" refers to a group of cells that can develop into a more mature or differentiated cell type. Progenitor cell populations may include pluripotent cells, cells restricted to a stem cell lineage (i.e., cells that cannot develop into all of the ectodermal lineages or cells that can only develop into cells of, for example, a neuronal lineage), and cells that are reversibly restricted to a stem cell lineage. Thus, the term "progenitor cell" or "precursor cell" can be "pluripotent cell" or "multipotent cell".

As used herein, the terms "reprogramming,""reprogrammed," or equivalent terms, refer to a process that endows a cell with a measurably greater ability to form at least one new cell-type progeny in culture or in vivo than it would have under the same conditions without reprogramming. In certain embodiments described herein, somatic cells are "reprogrammed" into pluripotent cells.
In certain aspects, somatic cells are reprogrammed when, after sufficient proliferation, in vivo or in vitro cell culture, a measurable proportion of cells display phenotypic characteristics of the new pluripotent cell type. Without reprogramming, such somatic cells would not generate progeny that display phenotypic characteristics of the new pluripotent cell type. If somatic cells can generate progeny that display phenotypic characteristics of the new pluripotent cell type without reprogramming, then the proportion of progeny from these somatic cells that display phenotypic characteristics of the new pluripotent cell type is measurably higher than before reprogramming.

As used herein, the phrase "differentiation programming" refers to differentiation of cells in culture or in vivo.
In the present specification, differentiation reprogramming refers to a process of changing a cell to form at least one new cell type progeny in a new differentiation state than it would have under the same conditions without differentiation reprogramming. This process includes differentiation, dedifferentiation and transdifferentiation. Thus, as used herein, the term "differentiation" refers to a process in which a less specialized cell becomes a more specialized cell type. In contrast, the term "dedifferentiation" refers to a cellular process in which a partially or terminally differentiated cell reverts to an earlier developmental stage, e.g., a cell with pluripotency or multipotency. In further contrast, the term "transdifferentiation" refers to a process in which one differentiated cell type is converted to another differentiated cell type.

As used herein, the terms "develop from pluripotent cells", "differentiate from pluripotent cells", "mature from pluripotent cells" or "generate from pluripotent cells", "derived from pluripotent cells", "differentiate from pluripotent cells" and equivalent expressions are intended to mean any of the foregoing terms and equivalent expressions as described, for example, in International Patent Application No. PCT/US2007, the disclosure of which is incorporated herein by reference in its entirety.
/015536, entitled "METHODS OF PRODUCING PANCREA
In the case of endocrine cells that mature from in vivo transplanted PDX1 pancreatic endoderm cells, as described in "TIC HORMONES," in vitro or in vivo
All such terms refer to the generation of differentiated cell types from pluripotent cells in
It refers to the progression of a cell from a stage with the potential to differentiate into at least two different cell lineages to becoming a specialized and terminally differentiated cell. Such terms may be used interchangeably for the purposes of this application. In the embodiments described herein, culture conditions are contemplated that render such differentiation reversible, thus allowing selective regaining of pluripotency or the ability to differentiate into at least two or more cell lineages.

The term "feeder cells" refers to cell cultures that grow in vitro, secrete at least one factor into the cell culture, and can be used to support the growth of other cells of interest in the culture. As used herein, the term "feeder cell layer" can be used interchangeably with the term "feeder cells." Feeder cells may comprise a monolayer in which the surface of a culture dish is covered with a complete layer of feeder cells before overlapping and amplifying, or may comprise clusters of cells. In a preferred embodiment, the feeder cells comprise an adherent monolayer.

As used herein, the terms "cluster,""aggregate," or "aggregate" may be used interchangeably and generally refer to a group of cells that have not been dissociated into single cells. Clusters can be dissociated into smaller clusters. This dissociation is generally manual in nature (e.g., using a Pasteur pipette), although other means of dissociation are contemplated. Aggregated suspension pluripotent or multipotent cell cultures are essentially those described in International Publication No. PCT/US2007/0
62755, Title COMPOSITIONS AND METHODS FOR CULTURE
TURING DIFFERENTIAL CELLS and PCT/US2008/0
82356, Title STEM CELL AGGREGATE SUSPENSION C
OMPOSITIONS AND METHODS OF DIFFERENTIATI
ON THEREOF, which are hereby incorporated by reference in their entireties.

Similarly, embodiments in which pluripotent cell cultures or pluripotent aggregate suspension cultures are grown in defined conditions without the use of feeder cells are "feeder-free." Feeder-free culture methods increase the scalability and reproducibility of pluripotent cell cultures and reduce the risk of contamination with infectious agents, for example, from feeder cells or other animal-sourced culture components. Feeder-free methods are described in U.S. Patent No. 6,800,480 to Bodnar et al. (Geron Corporation, Met.
However,
In contrast to the '480 patent, the embodiments described herein, whether pluripotent, multipotent or differentiated cell cultures, are feeder-free and do not further contain endogenous or exogenous extracellular matrix; that is, the cultures described herein are not only feeder-free, but also extracellular matrix-free. For example, in '480, the extracellular matrix is prepared by culturing fibroblasts, lysing the fibroblasts in situ, and then washing what remains after lysis. Alternatively ...
In issue 0,480, collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan,
The extracellular matrix may also be prepared from isolated matrix components or combinations of components selected from biglycan, thrombospondin, vitronectin, and decorin. In the embodiments described herein, a feeder layer or fibroblast layer is grown;
There is no need to generate an extracellular matrix by lysing cells to generate the extracellular matrix, nor is there a need to first coat a tissue culture vessel with an extracellular matrix component or combination of extracellular matrix components selected from collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin. Thus, the aggregate suspension cultures described herein for pluripotent, multipotent, and differentiated cells do not require a feeder layer, lysed feeder cells, or fibroblasts to generate an extracellular matrix coating that is an exogenously added extracellular matrix or matrix component, as described in International Application PCT/US2008/080516, entitled METHODS AND METHOD FOR TRANSFORMATION OF HIGH VOLUME ATTACHED BY ...
AND COMPOSITIONS FOR FEEDER-FREE PLURIP
OTENT STEM CELL MEDIA CONTAINING HUMAN S
As described in ERUM, the use of soluble human serum components overcomes the need for feeder cells or feeder monolayers, as well as the need for endogenous extracellular matrix derived from feeder cells or fibroblasts or exogenously added extracellular matrix components.

In a preferred embodiment, the culture method is free of animal-sourced products. In another preferred embodiment, the culture method is xeno-free. In a further preferred embodiment, the culture method described herein meets or exceeds one or more conditions or requirements for commercial manufacturing of human cell therapeutics.

The population of pluripotent cells can be further cultured in the presence of certain supplemental growth factors to obtain populations of cells that are or will develop into different cell lineages, or can be selectively reversed to allow development into different cell lineages.
The term "supplemental growth factor" is used in its broadest context and refers to a substance that is effective to promote the growth of pluripotent cells, to maintain cell survival, to stimulate cell differentiation, and/or to stimulate the reversal of cell differentiation. Additionally, supplemental growth factors may be substances secreted by feeder cells into their medium. Such substances include, but are not limited to, cytokines, chemokines, small molecules, neutralizing antibodies, and proteins. Growth factors may also include intercellular signaling polypeptides that control the development and maintenance of cells and tissue morphology and function. In a preferred embodiment, the supplemental growth factor is a stem cell factor (SCF) or a stimulatory factor (SCF) that is capable of regulating the differentiation and function of cells.
), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), a combination of interleukin-6 (IL-6) and soluble interleukin-6 receptor (IL-6R), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), tumor necrosis factor (TNF), and granulocyte macrophage colony stimulating factor (GM-CSF).

In certain processes for generating the cells described herein, growth factors are removed from the cell culture or cell population after addition. For example, activin A, activin B, GD
Growth factors, such as F-8, or GDF-11, can be added and removed within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days after their addition. In some embodiments, differentiation factors are not removed from the cell culture.

The efficiency of the differentiation process can be adjusted by modifying certain parameters, including, but not limited to, cell growth conditions, growth factor concentrations, and timing of culture steps, so that the differentiation procedures described herein can result in a differentiation efficiency of about 5%, about 10%, about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%, or about 150%, or about 250%, or about 300%, or about 400%, or about 500%, or about 600%, or about 1000%, or about 1500%, or about 2500, or about 3000, or about 4000, or about 5000, or about 6000, or about 10000, or about 15000, or about 25000
%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55
%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95
%, or greater than about 95% of the cells differentiated from pluripotent cells, including induced pluripotent cells, such as definitive endoderm, foregut endoderm, PDX1-positive foregut endoderm, PDX1-positive pancreatic endoderm or PDX1/NKX6.1 co-positive pancreatic endoderm, endocrine precursor or NGN3
/NKX2.2 co-positive endocrine precursors, and hormone-secreting endocrine cells or INS, GC
The process can result in conversion to G, GHRL, SST, PP single positive endocrine cells. In processes using isolation of preprimitive streak or mesendoderm cells, a substantially pure population of preprimitive streak or mesendoderm cells can be recovered.

Various cell compositions derived from pluripotent stem cells are described herein. One embodiment includes iPS cells and cells derived therefrom. Still other processes and compositions related to but different from the embodiments described herein are described in U.S. Provisional Patent Application No. 60/532,004, filed Dec. 23, 2003, entitled DEFINITIVE ENDODER
M; U.S. Provisional Patent Application No. 60/566,293, filed April 27, 2004, entitled P.D.
X1 EXPRESSING ENDODERM; U.S. Provisional Patent Application No. 60/586,566, filed July 9, 2004, entitled CHEMOKINE CELL SURFACE
RECEPTOR FOR THE ISOLATION OF DEFINITIV
E ENDODERM; U.S. Provisional Patent Application No. 60/587, filed July 14, 2004;
No. 942, entitled CHEMOKINE CELL SURFACE RECEPTOR F
OR THE ISOLATION OF DEFINITIVE ENDODERM;
U.S. Patent Application Serial No. 11/021,618, filed December 23, 2004, entitled DEFI
No. 11/115,868, filed April 26, 2005, entitled PDX1 EXPRESSING ENDODERM;
No. 11/165,305, filed June 23, 2005, entitled METHOD
S FOR IDENTIFYING FACTORS FOR DIFFERENTI
No. 60/730,917, filed Oct. 27, 2005, entitled PDX1-EXPRESSING ENDODERM;
ORSAL AND VENTRAL FOREGUT ENDODERM; U.S. Provisional Patent Application No. 60/736,598, filed Nov. 14, 2005, entitled MARKERS
OF DEFINITIVE ENDODERM; U.S. Provisional Patent Application No. 60/778,649, filed March 2, 2006, entitled INSULIN-PRODUCING CELL
US Provisional Patent Application No. 60/833,633, filed on July 26, 2006, entitled INSULIN-PRODUCING
US Provisional Patent Application No. 60/852,878, filed October 18, 2006, entitled ENRICHMENT CELLS AND METHOD OF PRODUCTION;
OF ENDOCRINE PRECURSOR CELLS, IMMATURE PA
NCREATIC ISLET CELLS AND MATURE PANCREAT
IC ISLET CELLS USING NCAM; U.S. Patent Application No. 11/588,693, filed October 27, 2006, entitled PDX1-EXPRESSING DOCUMENT
RSAL AND VENTRAL FOREGUT ENDODERM; March 2007
No. 11/681,687, filed on the 2nd of this month, entitled ENDOCRINE PR
ECURSOR CELLS, PANCREATIC HORMONE-EXPRESS
ING CELLS AND METHODS OF PRODUCTION;2007
No. 11/773,944, filed on July 5, 2002, entitled METHODS OF
PRODUCING PANCREATIC HORMONES; September 13, 2007
No. 60/972,174, filed on 2006, entitled METHODS OF TREES
No. 11/860,494, filed Sep. 24, 2007, entitled METHODS FOR INCREASING DELIVERY DEVICES AND SYSTEMS FOR USE IN DIABETES;
FINITIVE ENDODERM PRODUCTION; U.S. Patent Application No. 60/977,349, filed October 3, 2007, entitled CELL SURFACE MATERIAL
KERS OF HUMAN EMBRYONIC STEM CELLS AND C
ANCER STEM CELLS; and U.S. patent application Ser. No. 12/099,759, filed Apr. 8, 2008, entitled METHODS OF PRODUCING PANCR
EATIC HORMONES; and U.S. patent application Ser. No. 1, filed Apr. 21, 2008.
No. 2/107,020, entitled "METHODS FOR PURIFYING ENDOD"
ERM AND PANCREATIC ENDODERM CELLS DERIVE
D FORM HUMAN EMBRYONIC STEM CELLS, the disclosures of which are incorporated herein by reference in their entireties.

General methods for generating endoderm-lineage cells derived from hES cells are described in the related US patent applications mentioned above, as well as in D'Amour et al., 2005 Nat Biotechnol.
ol. 23:1534-41, and D'Amour et al., 2006 Nat Biol.
Chnol. 24(11):1392-401, the disclosures of which are incorporated herein by reference in their entireties, D'Amour et al. describe a five stage differentiation protocol: stage 1 (resulting in mostly definitive endoderm production), stage 2 (resulting in mostly PDX1-negative foregut endoderm production), stage 3 (resulting in mostly PDX1-positive foregut endoderm production), stage 4 (resulting in mostly pancreatic endoderm or pancreatic endocrine precursor production), and stage 5 (resulting in mostly hormone-expressing endocrine cell production).

The term "trophectoderm" refers to HAND1, Eomes, MASH2, ESXL1, H
CG, KRT18, PSG3, SFXN5, DLX3, PSX1, ETS2, and ER
BB genes in a non-trophectoderm cell or cell population.
It refers to multipotent cells with relatively high expression levels compared to SG3, SFXN5, DLX3, PSX1, ETS2, and ERBB.

"Extraembryonic endoderm" refers to the SOX7 gene, SOX17 gene, THBD gene, SPAR
The expression level of a marker selected from the group consisting of SOX7, SOX17, THB, C, DAB1, or AFP in a non-extraembryonic endoderm cell or cell population.
D, refers to multipotent cells with relatively high expression levels compared to SPARC, DAB1, or AFP.

The term "preprimitive streak cells" refers to cells in which the expression levels of the FGF8 marker gene and/or the NODAL marker gene are BRACHURY low, FGF4 low, SNAI1 low, SOX1 low,
7 low, FOXA2 low, SOX7 low and SOX1 low refer to relatively more multipotent cells compared to FOXA2 low, SOX7 low and SOX1 low.

The term "mesendoderm cells" refers to the cells that express the brachyury marker gene, FGF4, SN
The expression levels of the AI1, MIXL1 and/or WNT3 marker genes are
Low, CXCR4 low, FOXA2 low, SOX7 low and SOX1 low refer to relatively highly multipotent cells compared to CXCR4 low, FOXA2 low, SOX7 low and SOX1 low.

The term "definitive endoderm (DE)" refers to multipotent endoderm lineage cells that can differentiate into cells of the gut or gut-derived organs. According to certain embodiments, the definitive endoderm cells are mammalian cells, and in preferred embodiments, the definitive endoderm cells are human cells. In some embodiments of the invention, the definitive endoderm cells express certain markers or fail to significantly express certain markers. In some embodiments, the S
OX17, CXCR4, MIXL1, GATA4, HNF3β, GSC, FGF17, V
One or more markers selected from WF, CALCR, FOXQ1, CMKOR1 and CRIP1 are expressed in definitive endoderm cells.
, alpha-fetoprotein (AFP), thrombomodulin (TM), SPARC,
One or more markers selected from SOX7 and HNF4alpha are not expressed or are not significantly expressed in definitive endoderm cells. Definitive endoderm cell populations and methods for producing same are described in U.S. Patent Application Serial No. 11/021,618, entitled DEFINITIV
E ENDODERM, filed Dec. 23, 2004, which is incorporated herein in its entirety.

Yet other embodiments relate to cell cultures referred to as "PDX1-negative foregut endoderm cells" or "foregut endoderm cells," or equivalents thereof. In some embodiments, the foregut endoderm cells are selected from the group consisting of SOX17, HNF1 beta (HNF1B), HNF4 alpha (HNF4A) and F
The PDX1-negative foregut endoderm cell population and methods for producing the same are also described in U.S. patent application Ser. No. 11/588,693, filed Oct. 27, 2006, entitled PDX1-express
ing dorsal and ventral foregut endoderm, which is incorporated herein by reference in its entirety.

Other embodiments described herein relate to cell cultures of "PDX1-positive dorsally biased foregut endoderm cells" (dorsal PDX1-positive foregut endoderm cells) or simply "PDX1-positive endoderm." In some embodiments, the PDX1-positive endoderm cells express one or more markers selected from Table 1 and/or one or more markers selected from Table 2, which are also described in related U.S. patent application Ser. No. 11/2006, filed Oct. 27, 2006.
No. 588,693, entitled PDX1 EXPRESSING DOSAL AND VEN
No. 11/115,868, filed April 26, 2005, entitled PDX1-expressing endo
derm, which are incorporated herein by reference in their entireties.

PDX1-positive foregut endoderm cells, such as those produced by the methods described herein, are precursors that can be used to generate fully differentiated pancreatic hormone-secreting or endocrine cells, such as insulin-producing β-cells.
PDX1-positive foregut endoderm cells are generated by differentiating definitive endoderm cells that do not substantially express PDX1 (PDX1-negative definitive endoderm cells; also referred to herein as definitive endoderm) to form PDX1-positive foregut endoderm cells.

As used herein, "pancreatic endoderm,""pancreaticepithelium,"
pancreatic epithelium
"Pancreatic progenitors," and their equivalents, such as "PDX-1 positive pancreatic endoderm or pancreatic endoderm cells ("PECs") (all of which may be abbreviated as "PE"), are all precursor or progenitor pancreatic cells. As described herein, PECs are a population of progenitor cells following stage 4 differentiation (about days 12-14) and are divided into at least two major distinct populations: i) those that express NKX6.1 but not C
pancreatic progenitor cells that do not express HGA (or CHGA-negative, CHGA-); and ii) C
Without being bound by theory, the polyhormonal endocrine cells expressing NKX6.1 but not CHGA (CHGA positive, CHGA+)
It is hypothesized that the cell population that does not express A represents the more active and therapeutic component of PEC.
On the other hand, it is hypothesized that the population of CHGA-positive polyhormonal endocrine cells further differentiates and matures into glucagon-expressing pancreatic islet cells in vivo. Kelly et al. (2011) Cell-surface matrix metalloproteinases (CMM), the disclosures of both of which are fully incorporated herein by reference.
arkers for the isolation of pancreatic c
ell types derived from human embryonics
tem cells, Nat Biotechnol. 29(8):750-756, 2
Published online July 31, 2011 and Schulz et al. (2012), A. Scala
ble System for Production of Functional
Pancreatic Progenitors from Human Embryo
See nic Stem Cells, PLosOne 7(5):1-17, e37004.
Furthermore, pancreatic endoderm cells are sometimes used to refer generally to at least stage 3 and stage 4 type cells, rather than to PECs as described immediately above. The usage and meaning will be clear from the context. Thus, pancreatic endoderm derived from pluripotent stem cells, and at least hES and hIPS cells, is distinct from other endodermal lineage cell types, as are pancreatic endoderm cells.
DX1, NKX6.1, PTF1A, CPA1, cMYC, NGN3, PAX4, ARX
and NKX2.2 markers, the expression of which is differentially or at a high level, is a characteristic of pancreatic endocrine cells, e.g., CHGA, INS, GCG, GHR
L, SST, MAFA, PCSK1 and GLUT1 are not substantially expressed. Furthermore, some "endocrine precursor cells" expressing NGN3 can differentiate into other non-pancreatic structures (e.g., duodenum). In one embodiment, the NGN3-expressing endocrine precursors described herein differentiate into mature pancreatic lineage cells, e.g., pancreatic endocrine cells. Pancreatic endoderm or endocrine precursor cell populations and methods thereof are disclosed in U.S. Patent Application Serial No. 11/7
No. 73,944, entitled "Methods of Producing Pancreativity"
c hormones, filed July 5, 2007, and U.S. patent application Ser. No. 12/107,
No. 020, entitled METHODS FOR PURIFYING ENDODERM AND
D PANCREATIC ENDODERM CELLS DERIVED FORM
No. 6,313,939, filed Apr. 21, 2008, the disclosures of which are hereby incorporated by reference in their entireties.

As used herein, "endocrine precursor cells" refer to cells that express neurogenin 3 (NEUROG
3) and can be further differentiated into cells of the endocrine lineage, such as, but not limited to, pancreatic islet hormone-expressing cells. Endocrine progenitor cells cannot differentiate into as many different cell, tissue and/or organ types as less specifically differentiated definitive endoderm lineage cells, such as PDX1-positive pancreatic endoderm cells.

As used herein, "pancreatic islet hormone-expressing cells", "pancreatic endocrine cells" or equivalents refer to cells derived in vitro from pluripotent cells, which may be polyhormonal or monohormonal. Thus, endocrine cells can express one or more pancreatic hormones, with at least some of the functions of human pancreatic islet cells. Pancreatic islet hormone-expressing cells may be mature or immature. Immature pancreatic islet hormone-expressing cells can be distinguished from mature pancreatic islet hormone-expressing cells based on differential expression of certain markers or based on their functional capabilities, such as glucose responsiveness.

Many stem cell medium cultures or growth environments are contemplated in the embodiments described herein, including defined medium, conditioned medium, feeder-free medium, serum-free medium, and the like. As used herein, the term "growth environment" or "environment" or equivalents is the environment in which undifferentiated or differentiated stem cells (e.g., primate embryonic stem cells) are grown in vitro. Characteristics of the environment include the medium in which the cells are cultured and, if present, a support structure (e.g., a substrate on a solid surface, etc.). Methods for culturing or maintaining pluripotent cells and/or for differentiating pluripotent cells are described in PCT/US2007/062755, entitled "Citation and Methods of Differentiating Pluripotent Cells."
OMPOSITIONS AND METHODS USEFUL FOR CULTU
RING DIFFERENTIABLE CELLS, filed February 23, 2007; U.S. Patent Application Serial No. 11/993,399, entitled EMBRYONIC STEM CELL
CULTURE COMPOSITIONS AND METHODS OF USE
THEREOF, filed December 20, 2007; and U.S. patent application Ser. No. 11/875,0
No. 57, entitled "Methods and compositions for feed"
r-free pluripotent stem cell media conta
and injecting human serum, filed Oct. 19, 2007, the disclosures of which are incorporated herein by reference in their entireties.

The terms "essentially" or "substantially" refer to the fact that a component or cell is present in minimal or low amounts in any cell aggregate suspension type, such as the cell aggregates in the suspensions described herein, is "essentially or substantially homogeneous,""essentially or substantially homocellular," or alternatively, "essentially hES cells,""essentially or substantially definitive endoderm cells,""essentially or substantially foregut endoderm cells,""essentially or substantially PDX cells,""essentially or substantially homogeneous," or "essentially or substantially homogeneous."
"PDX1-negative foregut endoderm cells,""essentially or substantially PDX1-positive prepancreatic endoderm cells,"
"essentially or substantially PDX1-positive pancreatic endoderm or progenitor cells", "essentially or substantially PDX1-positive pancreatic endoderm tip cells", "essentially or substantially pancreatic endocrine precursor cells",
It means that the pancreatic endocrine tissue is composed essentially or substantially of pancreatic endocrine cells.

With respect to cells in a cell culture or cell population, the term "substantially free of" means
The cell types identified as not being included in the cell culture or cell population represent less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 10% of the total number of cells present in the cell culture or cell population.
By "absolutely non-limiting," it is meant being present in an amount of less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1%.

The cell cultures can be grown in media containing reduced serum or substantially no serum or no serum. Under certain culture conditions, the serum concentration is about 0.
For example, in some differentiation processes, the serum concentration in the medium can range from less than about 0.05% (v/v), about 0.1% (v/v), or about 10% (v/v).
), less than about 0.2% (v/v), less than about 0.3% (v/v), about 0.4% (v/v)
less than about 0.5% (v/v), less than about 0.6% (v/v), less than about 0.7% (v/v), less than about 0.8% (v/v), less than about 0.9% (v/v), less than about 1% (v/v), less than about 2% (v/v), less than about 3% (v/v), less than about 4% (v/v), less than about 5% (v/v)
The concentration of B27 supplement may range from about 0.1% (v/v) to about 20% (v/v). In some processes, the preprimitive streak cells are grown without serum or without serum replacement. In still other processes, the preprimitive streak cells are grown in the presence of B27. In such processes, the concentration of B27 supplement may range from about 0.1% (v/v) to about 20% (v/v).

In yet another process, immature pancreatic islet hormone-expressing cells are grown in the presence of B27. In such processes, the concentration of B27 supplement is from about 0.1% (v/v) to about 2.5% (v/v).
In certain processes, the concentration of B27 in the medium may range from about 0.1% (v/v), about 0.0% (v/v), or may be greater than about 20% (v/v).
．． 2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v
), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9%
(v/v), approximately 1% (v/v), approximately 2% (v/v), approximately 3% (v/v), approximately 4% (v/v)
), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v), about 9%
% (v/v), about 10% (v/v), about 15% (v/v), or about 20% (v/v). Alternatively, the concentration of B27 supplement added can be measured as a multiple of the strength of a commercially available B27 stock solution. For example, B27 is available from Invitrogen (Carl
B27 is available as a 50x stock solution from BioStation, Inc., Sanford, Calif. Adding a sufficient amount of this stock solution to a sufficient volume of growth medium will produce a medium supplemented with the desired amount of B27. For example, adding 10 ml of 50x B27 stock solution to 90 ml of growth medium will produce a growth medium supplemented with 5x B27.
The supplement concentrations are approximately 0.1x, approximately 0.2x, approximately 0.3x, approximately 0.4x, and approximately 0.5x.
, about 0.6x, about 0.7x, about 0.8x, about 0.9x, about 1x, about 1.1x, about 1.2x
, about 1.3x, about 1.4x, about 1.5x, about 1.6x, about 1.7x, about 1.8x, about 1.
9x, about 2x, about 2.5x, about 3x, about 3.5x, about 4x, about 4.5x, about 5x, about 6x
, about 7x, about 8x, about 9x, about 10x, about 11x, about 12x, about 13x, about 14x, about 1
It may be 5x, about 16x, about 17x, about 18x, about 19x, about 20x and greater than about 20x.

As used herein, "exogenously added" refers to, for example, growth factors, differentiation factors, etc.
Compound, in reference to a culture or conditioned medium, refers to a growth factor that is added to the culture or medium to supplement any compounds or growth factors that may already be present in the culture or medium. For example, in some embodiments, the cell culture and/or cell population does not contain an exogenously added retinoid.

As used herein, "retinoid" refers to retinol, retinal or retinoic acid, as well as derivatives of any of these compounds. In a preferred embodiment, the retinoid is retinoic acid.

"FGF family growth factor", "fibroblast growth factor" or "member of the fibroblast growth factor family" refers to FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF19, FGF19, FGF110, FGF111, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19 ...
F6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF1
3, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FG
In some embodiments, "FGF family growth factor" refers to an FGF selected from the group consisting of FGF20, FGF21, FGF22, and FGF23.
Or, "a member of the fibroblast growth factor family" means any growth factor that has homology to and/or similar function to a known member of the fibroblast growth factor family.

As used herein, "expression" refers to the production of a material or substance as well as the level or amount of production of a material or substance. Thus, determining the expression of a particular marker refers to detecting the relative or absolute amount of the marker expressed or simply detecting whether the marker is present or absent.

As used herein, "marker" refers to any molecule that can be observed or detected. For example, markers include, but are not limited to, nucleic acids such as transcripts of particular genes, polypeptide products of genes, non-gene product polypeptides, glycoproteins, carbohydrates, glycolipids, lipids, lipoproteins, or small molecules (e.g., molecules with a molecular weight of 10,000 or less).
0 amu molecules).

For the majority of markers described herein, the official Human Genome Or
HUGO Gene No.
Such symbols, developed by the Menculation Committee, provide unique abbreviations for each named human gene and gene product, which those of skill in the art can readily recognize and associate with the corresponding unique human gene and/or protein sequences.

According to the HUGO nomenclature, the following gene symbols are defined as follows: GHRL-
Ghrelin; IAPP-islet amyloid polypeptide; INS-insulin; GCG-glucagon; ISL1-ISL1 transcription factor; PAX6-paired box gene 6; PAX4-
Paired box gene 4; NEUROG3-neurogenin 3 (NGN3); NKX2
-2-NKX2 transcription factor associated locus 2 (NKX2.2); NKX6-1-NKX6 transcription factor associated locus 1 (NKX6.1); IPF1-insulin promoter factor 1 (IPF1)
DX1);ONECUT1-Onecut domain, family member 1 (HNF6);
HLXB9 - homeobox B9 (HB9); TCF2 - transcription factor 2, liver (HNF1b
); FOXA1-Forkhead box A1; HGF-Hepatocyte growth factor; IGF1-Insulin-like growth factor 1; POU5F1-POU domain, class 5, transcription factor 1 (OCT
4); NANOG-Nanog homeobox; SOX2-SRY (sex determining region Y)-box 2; CDH1-cadherin 1, type 1, E-cadherin (ECAD); T-brac
hyury homolog (BRACH); FGF4-fibroblast growth factor 4; WNT3-wingless MMTV integration site family, member 3; SOX17-SRY (sex determining region Y)
-box 17; GSC-goosecoid; CER1-(cerberus 1, cysteine knot superfamily, homolog (CER); CXCR4-chemokine (C-X-C motif) receptor 4; FGF17-fibroblast growth factor 17; FOXA2-forkhead box A2; SOX7-SRY (sex determining region Y)-box 7; SOX1-SRY (sex determining region Y)-box 1; AFP-alpha-fetoprotein; SPARC-secreted protein, acidic, cysteine-rich (osteonectin); and THBD-thrombomodulin (TM), NCAM-neuronal cell adhesion molecule; SYP-synaptophysin; ZIC1-
Zic family member 1; NEF3-neurofilament 3 (NFM); SST-somatostatin; MAFA-v-maf musculoaponeurotic fibrosarcoma oncogene homolog A; MAFB-v-
maf-musculoaponeurotic fibrosarcoma oncogene homolog B; SYP-synaptophysin; CHGA-chromogranin A (parathyroid secretory protein 1).

Full gene names corresponding to the non-HUGO gene symbols, as well as other abbreviations that may be used herein, are provided below: SS-somatostatin (SOM); PP-pancreatic polypeptide; C-peptide-connecting peptide; Ex4-exendin 4; NIC-nicotinamide and DAPT-N-[N-(3,5-difluorophenacetyl)-L-alanyl]-
S-Phenylglycine t-butyl ester; RA-Retinoic acid; RPMI-Roswe
ll Park Memorial Institute medium; CMRL-Connau
ght Medical Research Labs medium; FBS-fetal bovine serum; N
BP 10-NCAM binding protein 10; PTF1a-pancreatic specific transcription factor 1a.

The progression from pluripotent cells to various multipotent and/or differentiated cells can be monitored by determining the relative expression of a gene, or gene marker, a characteristic of a particular cell, compared to the expression of a second or control gene, e.g., a housekeeping gene. In some processes, the expression of a particular marker is determined by detecting whether the marker is present or absent. Alternatively, the expression of a particular marker can be determined by measuring the level at which the marker is present in the cells of a cell culture or cell population. In such processes, the measurement of the expression of the marker can be qualitative or quantitative. One way to quantify the expression of a marker produced by a marker gene is by using quantitative PCR (Q-PCR). Methods for performing Q-PCR are well known in the art. Other methods known in the art can also be used to quantify marker gene expression. For example, the expression product of a marker gene can be detected by using an antibody specific for the marker gene product of interest.

In some processes, high expression of the following genes indicates certain populations of cells: SOX17, SOX7, AFP or THBD indicate extraembryonic endoderm, NODAL and/or FGF8 indicate the anterior primitive streak, brachyury, F
GF4, SNAI1 and/or WNT3 indicate mesendoderm, while CER, GSC,
CXCR4, SOX17 and FOXA2 indicate definitive endoderm cells; SOX17,
FOXA2, FOXA1, HNF1B and HNF4A mediate the development of the foregut endoderm (or PDX
1-negative endoderm), and PDX1, HNF6, SOX9 and PROX1 are responsible for PDX
1 positive endoderm, PDX1, NKX6.1, PTFA1, CPA and cMYC indicate pancreatic epithelium (PE or pancreatic precursor), NGN3, PAX4, ARX and N
KX2.2 represents the endocrine precursor cells, and INS, GCG, GHRL, SST and P
P indicates a variety of endocrine cells, with high MAFA gene expression relative to MAFB gene expression indicating insulin-secreting endocrine cells and high expression of MAFB relative to MAFA gene expression indicating glucagon-secreting endocrine cells.

The terms fibroblast growth factor 7 (FGF7) and keratinocyte growth factor (KGF) are
Synonymous.

Methods for generating induced pluripotent stem (iPS) cells The embodiments described herein can be used to generate any one type of iPS cell or any one iPS cell.
There is also no limitation on the method of generating PS cells. Various methods exist, so the embodiments are not limiting or depend on the level of efficiency of generating iPS cells. The embodiments described herein apply to differentiation of iPS cells into endoderm lineage cells and uses thereof.

Adenovirus, plasmid, or Cre-loxP3 or piggy BAC
Induced pluripotent stem cells (iPSCs) using ablation of reprogramming factors using transposition
Viral, non-viral and non-integrating viral methods for generating
tfeld, M. et al., Science 322, 945-949 (2008);
a, K. et al., Science 322, 949-953 (2008); Kaji, K. et al., Nature 458, 771-775 (2009); Soldner, F. et al., Ce
ll 136, 964-977 (2009); and Woltjen, K. et al., Natl.
re 458, 766-770 (2009), which are also fully incorporated by reference herein.
No. 0100003757 (published Jan. 7, 2010) and PCT/U.S. Pat. No. 6,233,363 to Shi et al.
See also S2009/037429. However, the reprogramming efficiency of these methods is low (<0.003%) and vector sequences may remain despite excision, limiting their therapeutic application. For example, viral integration and overexpression of the above transcription factors into the host genome has been linked to tumorigenesis, and residual transgene expression is a potential distinguishing feature between ES cells and iPS cells. Solder, F. et al., Cell 136, incorporated herein by reference in its entirety.
:964-977 (2009); Foster et al., Oncogene 24:1491-
1500 (2005); and Hochedlinger, K. et al., Cell 121:
Please refer to 465-477 (2005).

In another embodiment of the present invention, the method for generating iPSCs includes an episomal vector derived from Epstein-Barr virus.
u, J. et al., Science 324, 797-801 (2009) and Mack, A.
See U.S. Patent Application Publication No. 20100003757 to Gregg et al., published Jan. 7, 2010. These methods require three separate plasmids carrying combinations of seven factors, including the oncogene SV40.

In another embodiment of the present invention, the method for generating iPSCs includes iPSCs based on proteins from mouse cells and human fetal and neonatal cells. Zhou, H. et al., Cell Stem Cell 4, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
81-384 (2009); and Kim, D. et al., Cell Stem Cell 4
, 472-476 (2009). These methodologies are achieved using chemical treatments (e.g., valproic acid in the case of Zhou et al., 2009, supra) or multiple treatments (Kim et al., 2009, supra).

In another embodiment of the present invention, minicircle vectors or plasmids, which are supercoiled DNA molecules lacking a bacterial origin of replication and an antibiotic resistance gene, can be used. Chen, Z.-Y. et al., Mol. T, incorporated herein by reference in its entirety.
her. 8, 495-500 (2003); Chen, Z. -Y. et al., Hum. Ge
ne Ther. 16, 126-131 (2005); and Jia, F. et al., Natl.
re Methods Advance Publication Online 7
See February 2010. These methodologies result in iPSCs with higher transfection efficiency and longer term ectopic expression due to less activation of endogenous silencing mechanisms.

In yet another embodiment of the present invention, iPS cells can be generated from human patients with various diseases, including diabetes, ALS, spinal muscular dystrophy, and Parkinson's patients. Maehr et al., PNAS USA 10, incorporated herein by reference in its entirety.
6(37):15768-73(2009); Dimos et al., Science, 321:
1218-21 (2008); Ebert et al., Nature 457:277-80 (2
009); Park et al., Cell 134:877-886 (2008); and Sol
See, e.g., Nat. Dner et al., Cell 136:964-977. At least one advantage of generating hIPS cells from patients with a particular disease is that the derived cells contain the genotype and cellular responses of the human disease. See also Table 3, which lists at least some of the existing human iPS cell lines. This information is provided in the literature and in, e.g., Nat.
National Institutes of Health (NIH) Stem Cell
Registry, the Human Embryonic Stem Cell
Registry and University of Massachusetts M
educational School, Worcester, Massachusetts, US
The data are taken from publicly available databases, including the International Stem Cell Registry at http://www.ietf.org/TR/A. These databases are updated regularly as cell lines become available and are registered.
In some embodiments of the compositions and methods described herein, other cytochrome P450 antibodies may be used, including, but not limited to, CyT49, CyT212, CyT203, CyT25, (available at least at the time of the filing of this application from ViaCyte Inc., 3550 General Atpmics Court, San Jose, CA).
(commercially available from San Diego, CA 92121), BGO1, BG02
and MEL1, and human pluripotent stem cells such as hESCs, including iPSC-482c.
7 and iPSC-603 (Cellular Dynamics International
onal, Inc., Madison, Wisconsin), and iPSC-G4
(hereinafter referred to as "G4") and iPSC-B7 (hereinafter referred to as "B7") (Shinyah Yaman
aka, Center for iPS Cell Research, Kyoto U
The use of a variety of differentiable primate pluripotent stem cells is contemplated, including induced pluripotent stem (iPS) cells such as primate pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) such as G4 and B7, and studies using these cells are described in detail herein. Certain of these human pluripotent stem cells are available from the National Institute of Integrative Medicine (NII).
It is registered with international registration agencies such as the Institutes of Health (NIH) and is
H Human Stem Cell Registry (e.g.,
The accession number for yT49 is 0041.) Other available cell lines are available from stemcells.
Further cell lines, such as BG01 and BG01v, can be found on the World Wide Web at nih.gov/research/registry.
isconsin International Stem Cell (WISC)Ba
WiCell (registered trademark) (catalog name: BG01) and ATC, which are affiliates of .nk.
The cell lines described herein are sold and distributed to third parties by WiCell® (catalog number SCRC-2002). Other cell lines described herein may not be registered or distributed by a biological repository such as WiCell® or ATCC, but such cell lines are publicly available directly or indirectly from the principal investigator, laboratory and/or institution. Requests for public release of cell lines and reagents are routine for those skilled in the art, for example, in the life sciences. Generally, transfer of these cells or materials is via a standard material transfer agreement between the cell line or material owner and the recipient. These types of material transfers occur frequently, especially in the research environment in the life sciences. Indeed, Applicant has released CyT49 (2006), CyT203 (2005), Cyt21 (2006), Cyt10 (2007), Cyt12 (2009), Cyt16 (2010), Cyt18 (2011), Cyt19 (2012), Cyt20 (2013), Cyt21 (2014), Cyt22 (2015), Cyt23 (2016), Cyt24 (2017), Cyt25 (2018), Cyt26 (2019), Cyt27 (2010), Cyt28 (2011), Cyt29 (2012), Cyt30 (2013), Cyt31 (2014), Cyt32 (2015), Cyt33 (2016), Cyt34 (2017), Cyt35 (2013), Cyt36 (2014), Cyt37 (2015), Cyt38 (2016), Cyt39 (2017), Cyt39 (2018), Cyt39 (2019), Cyt39 (2013), Cyt39 (2014), Cyt39 (2015), Cyt39 (2016), Cyt39 (2017
2 (2009), CyT25 (2002), BG01 (2001), BG02 (2001
Since the cells were derived and characterized, including BG03 (2001) and BGOlv (2004), we have routinely received transfers of cells through such agreements with commercial and non-commercial industrial partners and collaborators. The years in parentheses next to each cell line in the above list indicate:
Indicates the year that the cell lines or materials became publicly available and immortalized (e.g., a cell bank was created), and therefore, from the time that these cell lines were established, no further destruction of embryos has been performed or required to produce the compositions and practice the methods described herein.
In August 2006, Klimanskaya et al. demonstrated that hESCs can be derived from a single blastomere, thus leaving the embryo intact and not causing its destruction. Using micromanipulation techniques, biopsies were taken from each embryo, resulting in nineteen (19) ES cell-like outgrowths and two (2) stable hESC lines. These hESC lines could be maintained in an undifferentiated state for more than six (6) months, and showed normal karyotypes, as well as Oct-4, SSEA-3 and IL-1 expression.
These hESCs showed expression of pluripotency markers including SSEA-4, TRA-1-60, TRA-1-81, Nanog and alkaline phosphatase.
Differentiate and form derivatives of all three (3) embryonic germ layers in vitro, and in vivo.
These methods of generating new stem cell lines without the destruction of the embryo address the ethical concerns of using human embryos. See Klimanskaya et al. (2006) Nature, vol. 14, no. 1, 2006, the disclosure of which is incorporated herein by reference in its entirety.
e 444:481-5, Epub 23 Aug. 2006.
Anskaya et al. co-cultured the derived hESC line with other hESCs.
In 2003, Chung Y. et al. were able to obtain hES cell lines again from a single blastomere without co-culture with hESCs. See Chung Y. et al., Cell Stem Cell 2008, 2(2), pp. 113-117, which is fully incorporated herein by reference. Thus, the compositions and methods described herein, particularly such compositions and methods as they relate to induced pluripotent stem cells or genetically dedifferentiated pluripotent stem cells, do not require the destruction of human embryos.
Tables 3 and 4 are non-exhaustive lists of certain iPSCs and hESCs, respectively, that are available worldwide for research and/or commercial purposes and are suitable for use in the methods and compositions of the invention. The information in Tables 3 and 4 is taken from the literature and from, for example, Natl.
National Institutes of Health (NIH)Human Stem
Cell Registry, Human Embryonic Stem Cell
Registry and University of Massachusetts
Medical School, Worcester, Massachusetts, U.
The data are taken from publicly available databases, including the International Stem Cell Registry located in San Gorgonio, CA. These databases are updated regularly as cell lines become available and are registered.
Human iPSCs as described herein (at least iPSC-603 and iPSC-482)
-c7) is Cellular Dynamics International, Inc.
(Madison, Wisconsin, USA).

Another advantage is that such hIPS cells are an immunologically compatible autologous cell population, allowing patient-specific cells to study disease origin and progression.
Thus, it is possible to understand the underlying causes of disease, which may provide insights that guide the development of preventative and therapeutic treatments for disease.

Pluripotent human embryonic stem (hES) cells Some embodiments are directed to methods for deriving definitive endoderm cells and ultimately any endoderm lineage-derived cell type, including but not limited to foregut endoderm, pancreatic endoderm, endocrine precursor cells and/or pancreatic islet hormone-expressing cells, using human embryonic stem (hES) cells as starting material. These hES cells may originate from the morula, embryonic inner cell mass or from the gonadal ridges of an embryo. Human embryonic stem cells can be maintained in a pluripotent state without substantial differentiation in culture using methods known in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,453,357, 5,463, 5,521, 5,532, 5,542, 5,551, and 5,621, respectively.
No. 670,372, No. 5,690,926, No. 5,843,780, No. 6,2
Nos. 6,251,671 and 6,000,806, the disclosures of which are incorporated herein by reference in their entireties.

In some processes, pluripotent stem cells, such as hES cells, are maintained on a feeder layer. In such processes, any feeder layer that allows the pluripotent cells to be maintained in a pluripotent state can be used.
One commonly used feeder layer for cultivation of pluripotent cells is a layer of mouse fibroblasts. More recently, human fibroblast feeder layers have been developed for use in the cultivation of pluripotent cells (US Patent Application Publication No. 2002/0072117, the disclosure of which is incorporated herein by reference in its entirety). Alternative processes allow for the maintenance of pluripotent cells without the use of feeder layers. A method for maintaining pluripotent cells under feeder-free conditions is described in US Patent Application Publication No. 2003/0,175,956, the disclosure of which is incorporated herein by reference in its entirety.

The pluripotent cells described herein can be maintained in culture with or without serum, with or without extracellular matrix, with or without FGF. Some pluripotent cell maintenance procedures use serum replacements. These and other methods for culturing and differentiating pluripotent or multipotent cells are described in PCT/US200, respectively.
No. 7/062755, filed February 23, 2007, entitled COMPOSITIONS AND
METHODS FOR CULTURING DIFFERENTIAL CELL
S and PCT/US2008/080516, filed October 20, 2008, entitled ME
THODS AND COMPOSITIONS FOR FEEDER-FREE P
LURIPOTENT STEM CELL MEDIA CONTAINING HU
MAN SERUM, which are incorporated herein by reference in their entirety.

The invention described herein is useful with all hES cell lines, and at least those listed in Table 4. This information is available from the literature and from, for example, National Institute of Standards and Technology.
Institutes of Health (NIH) Stem Cell Registry
, Human Embryonic Stem Cell Registry and
University of Massachusetts Medical School
International, Inc., Worcester, Massachusetts, USA
The data are taken from publicly available databases, including the International Stem Cell Registry, which are updated periodically as cell lines become available and are registered. As of the filing date of this application, the International Stem Cell Registry is available at:
254 iPSC lines listed in the TEM Cell Registry, and 121
There was one hESC line: Table 4 below is not an inclusive list of hESCs and iPSCs, but is an example of potentially available pluripotent stem cells.

Pluripotent dedifferentiated somatic cells Recently, tests using certain nuclear reprogramming factors have made it possible to derive pluripotent stem cells or pluripotent-like stem cells from a patient's own somatic cells. These cells are also called induced pluripotent stem (iPS) cells.
Various iPS cell lines provided by Kyoto University have been described. However, other iPS cell lines have been described, for example James Thomson et al., A1.
No. 20090047263, International Publication WO2005/8059, all of which are incorporated herein by reference in their entirety.
8. U.S. Patent Application Publication No. 20080233610 and International Publication WO2008/118
82. Thus, as used herein, "induced pluripotent stem (iPS)
By "pluripotent stem cells" is meant cells that have similar properties to other pluripotent stem cells, such as hES cells, hEG cells, pPS (primate pluripotent stem) cells, parthenogenetic cells, and the like.

Nuclear programming factors are described in U.S. Patent Application Publication No. 20090047263 and International Publication No. WO 2009/023116.
US2005/80598, US20080233610 and International Publication WO2008/11882, and are used to induce reprogramming of differentiated cells without the use of eggs, embryos, or ES cells. The method for preparing iPS cells derived from somatic cells by using nuclear reprogramming factors similar to those used and described in the present invention is not particularly limited. In a preferred embodiment, the nuclear reprogramming factors are contacted with somatic cells in an environment where somatic cells and induced pluripotent stem cells can grow. The advantage of certain embodiments described herein is that induced pluripotent stem cells can be prepared by contacting nuclear reprogramming factors with somatic cells in the absence of eggs, embryos, or embryonic stem (ES) cells. By using nuclear reprogramming factors, the nuclei of somatic cells can be reprogrammed to obtain iPS cells or "ES-like cells".

Pluripotent stem cells as described herein, whether hES cells or iPS 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/ECAT5, E-cadherin;.beta.III-tubulin;.alpha.-smooth muscle actin (alpha.-SMA)
fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (N
at1); (ES cell-associated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2
;ECAT3;ECAT6;ECAT7;ECAT8;ECAT9;ECAT10;EC
AT15-1; ECAT15-2; Fthl17; Sall4; blastocyte transcription factor (
Utf1); Rex1; p53; G3PDH; telomerase such as TERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box-containing protein 15 (Fbx15); Nanog/ECAT4; Oct3/4; Sox2; Klf4
;c-Myc;Esrrb;TDGF1;GABRB3;Zfp42, FoxD3;GD
F3; CYP25A1; developmental pluripotency-associated 2
otency-associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1)
DPPA3/Stella; DPPA4. The present invention is not limited to the markers listed herein, and may include cell surface markers, antigens, and ESTs, RNA (including microRNA and antisense RNA).
It is understood that the term encompasses markers such as DNA (including genes and cDNA) and other gene products, including portions thereof.

In one embodiment, the iPS cell lines used herein contain the following nuclear reprogramming factor genes: Oct family genes, Klf family genes, and Sox family genes.
Family genes. In one iPS cell line, the following three genes: Oct3/4
Three types of genes are provided: Oct family genes, Klf family genes, and Myc family genes, e.g., O
Other iPS cell line gene products of each of ct3/4, Klf4 and c-Myc were used, and it is therefore understood that nuclear reprogramming factors may or may not include Myc family genes.

Nuclear reprogramming factors described herein and known in the art can be used to generate iPS cells from differentiated adult somatic cells and are not limited to the type of somatic cell that is reprogrammed, i.e., any kind of somatic cell can be reprogrammed or dedifferentiated. Since somatic cell reprogramming does not require an egg and/or embryo, iPS cells can be mammalian cells, thus providing the opportunity to generate patient or disease specific pluripotent stem cells.

Aggregate Suspensions of Pluripotent Stem Cells In contrast to previously known tissue engineering methods based on seeding individual cells onto polymer scaffolds, matrices and/or gels, the embodiments described herein may use cell aggregate suspensions formed from pluripotent stem cells, single cell suspensions or differentiated single cell suspensions derived therefrom. Methods for processing and/or producing stem cell aggregate suspensions and differentiation of the cells thereof are described in International Applications PCT/US2007/062,755 and PCT/US2008/082,356, which are incorporated herein by reference in their entireties.

In one embodiment, a method is provided for producing pluripotent stem cell aggregate suspension from a single cell suspension of pluripotent stem cell culture, such as hES or iPS cell culture.Pluripotent stem cells can be initially cultured on fibroblast feeders or can be feeder-free.Methods for isolating hES cells and culturing them on human feeder cells are described in U.S. Patent No. 7,432,104, which is incorporated herein by reference in its entirety.

As used herein, the term "single cell suspension" or equivalents refers to a pluripotent, multipotent or terminally differentiated single cell suspension, or a single cell suspension derived by any mechanical or chemical means from pluripotent or multipotent cells.
Several methods are available for dissociating cell clusters from primary tissues, cells in attached cultures, and aggregates to form single cell suspensions, e.g., physical forces (cell scrapers,
There are several methods for dissociation, including mechanical dissociation such as trituration through a narrow bore pipette, fine needle aspiration, disaggregation by vortexing and forced filtration through a fine nylon or stainless steel mesh, enzymatic dissociation (e.g., trypsin, collagenase, Accutase™, etc.), or a combination of both. Additionally, methods and culture media conditions that can support dissociation of pluripotent, multipotent or differentiated cells into single cells are useful for expansion, cell sorting, and defined seeding for multi-well plate assays, and also allow automation of culture sequences and clonal expansion.

Other embodiments provide methods of generating pluripotent stem cell aggregate suspensions directly in a differentiation medium, such as a differentiation medium containing an agent, preferably a TGFβ family member capable of activating a receptor of the TGFβ family, such as activin A, activin B, GDF-8, GDF-11 and Nodal. Growth factors for the generation of endoderm are described in International Application No. PCT/US2008/065,686, which is incorporated herein by reference in its entirety. The method of generating cell aggregate suspensions in a differentiation medium can be distinguished from other methods, also described herein, that provide for the generation of cell aggregate suspension cultures in a pluripotent stem cell medium, such as StemPro® hES SFM (Invitrogen), which is based on the heregulin-based DC-HAIF medium described in PCT/US2007/062,755.

Embodiments described herein relate to a method for generating pluripotent cell aggregates in suspension by culturing pluripotent cells from a pluripotent adherent culture in adherent growth culture conditions that allow for proliferation in an undifferentiated state, dissociating the adherent pluripotent cell culture into a single cell suspension culture, and contacting the single cell suspension culture with first differentiation culture conditions that allow for the formation of hES-derived cell aggregates in suspension by agitating the single cell suspension culture from the single cell suspension culture for a period of time until pluripotent-derived cell aggregates in suspension are formed, thereby generating pluripotent-derived cell aggregates in suspension. In a preferred embodiment, agitation of the single cell suspension culture is performed by rotating at about 80 rpm to 160 rpm. In certain other embodiments described herein, a rho-kinase inhibitor is used to facilitate aggregation, growth, proliferation, expansion and/or cell clusters of pluripotent stem cells.

Embodiments described herein include a method for culturing hES cells in adherent growth culture conditions that allow for expansion in an undifferentiated state; contacting the undifferentiated hES cells with first differentiation culture conditions suitable for differentiation of the hES cells to result in adherent pluripotent-derived cells; dissociating the adherent hES-derived cells into a single cell suspension culture; contacting the single cell suspension culture with second differentiation culture conditions that allow for the formation of suspended hES-derived cell aggregates by agitating the single cell suspension culture until such time that the single cell suspension culture forms suspended pluripotent-derived cell aggregates;
The present invention also relates to a method of producing pluripotent-derived cell aggregates in suspension from a pluripotent-derived single cell suspension, thereby producing pluripotent-derived cell aggregates in suspension. In a preferred embodiment, the agitation of the single cell suspension culture is performed by rotation at about 80 rpm to 160 rpm.

In a preferred embodiment, manufacturing-scale suspension cultures utilize continuous perfusion of medium as a method to maintain cell viability while maximizing cell density. In this context, medium exchange contributes fluid shear to the culture that affects adherent cells and suspension aggregates differently. Fixed, adherent cells are subjected to fluid shear stress as medium flows tangentially over the cell surface. In contrast, suspension aggregates experience much weaker shear stress across the aggregate surface as the aggregates are free to roll in response to the application of shear forces. It is anticipated that prolonged shear stress is detrimental to adherent pluripotent cells, and that the suspension aggregate format is preferred for optimal survival and function. Thus,
Based on the need for an efficient manufacturing process for the generation of pluripotent stem cells and/or multipotent progenitor cells derived from pluripotent stem cells, and the above observed mechanisms regarding shear rate and shear stress, the embodiments described herein provide, for the first time, a manufacturing method for the generation of pluripotent stem cells and/or multipotent progenitor cells derived from pluripotent stem cells in a suspension format, in particular a cell aggregate suspension format.

In yet another embodiment, the hES cell aggregate suspension is cultured in a medium that is substantially free of serum.
Additionally, they were cultured in the absence of exogenously added fibroblast growth factor (FGF), which is similar to culturing hE cells in medium that is serum-free but contains exogenously added growth factors such as FGF.
U.S. Patent No. 7,005,200 to Thomson, J., which requires culturing S cells.
In some embodiments, the iPS cell aggregate suspension is cultured in a medium that is substantially free of serum and/or further contains exogenously added fibroblast growth factor (FGF).
The cells are cultured in the absence of GF.

In contrast to cell aggregates produced by known methods that may vary in both size and shape, the cell aggregates and methods described herein have a narrow distribution of size and shape, i.e., the cell aggregates are substantially uniform in size and/or shape. Size uniformity of cell aggregates is important for differentiation performance and culture uniformity. Applying basic mass transport analysis to aggregates, assuming equal permeability, oxygen and nutrients are expected to diffuse slower into the center of larger aggregates compared to diffusion into smaller aggregates. Because differentiation of aggregated ES cells or iPS cells into pancreatic lineage cells depends on the temporal application of specific growth factors, cultures with a mixture of aggregates of different diameters may be desynchronized compared to uniform (size and shape) cultures of cell aggregates. This mixture of cell aggregates may result in heterogeneity, resulting in poor differentiation performance and ultimately incompatibility with manufacturing, scale-up and production. Thus, as used herein, the phrase "substantially uniform" or "substantially uniform in size and shape" or equivalents refer to the extent of uniformity of the aggregates, which is about 20% or less. In another embodiment, the degree of spread of aggregate uniformity is less than about 15%, 10% or 5%.

The exact number of cells per aggregate is not critical, but one of skill in the art will appreciate that the size of each aggregate (and therefore the number of cells per aggregate) is limited by the maximum diffusion of oxygen and nutrients to the central cell, and that this number may also vary depending on the cell type and the nutrient requirements of that cell type. Cell aggregates are generally formed by culturing a minimum number of cells (e.g.,
The cell aggregates may contain as few as 100 or 200 cells (2 or 3 cells) or may contain hundreds or thousands of cells per aggregate. Generally, the cell aggregates contain hundreds to thousands of cells per aggregate. For purposes of the present invention, cell aggregates are generally from about 50 microns to about 600 microns in size, although depending on the cell type, the size may be below or above this range. In one embodiment, the cell aggregates are from about 50 microns to about 250 microns in size, or about 70 microns to about 100 microns in size.
They are between 5 and 200 microns in size, and preferably between about 100 and 150 microns in size.

Yet another method describes the generation of embryoid bodies (EBs). As used herein, the terms "embryoid bodies", "aggregate bodies" or equivalents refer to aggregates of differentiated and undifferentiated cells that arise when ES cells are overgrown in monolayer cultures, or maintained in suspension cultures in undefined media, or differentiated into multiple germ layer tissues by non-directed protocols. That is, EBs are not formed from single cell suspensions of pluripotent stem cells as described herein, nor are EBs formed from adherent cultures of pluripotent-derived multipotent cells. These features alone clearly distinguish the present invention from embryoid bodies.

In contrast to embryoid bodies, which are mixtures of differentiated and undifferentiated cells, generally consisting of cells derived from several germ layers and which undergo random differentiation, the cell aggregates described herein are essentially or substantially homogeneous cells and exist as aggregates of pluripotent, multipotent, bipotent, or unipotent types of cells, e.g., embryonic cells, definitive endoderm, foregut endoderm, PDX1-positive pancreatic endoderm, pancreatic endocrine cells, etc.

The embodiments described herein address the above problems by providing a cost-effective manufacturing process or method capable of repeatedly producing cell aggregates that are substantially uniform in size and shape using a process that can be easily adapted for large-scale manufacturing. In one particular embodiment, the differentiable cells are grown in suspension culture using the cell medium of the present invention. In another particular embodiment, the differentiable cells can be maintained and grown in suspension, i.e., they remain undifferentiated or are prevented from further differentiation. The term "growing" in the context of cell culture is used as it is used in the art and refers to cell proliferation and an increase in cell number, preferably an increase in viable cell number. In a specific embodiment, by culturing for about one day, i.e., more than about 24 hours,
In a more specific embodiment, the cells are grown in culture suspension.
The cells are grown in suspension culture by culturing for at least 2, 3, 4, 5, 6, 7, 8 days, or for at least 2, 3, 4, 5, 6, 7, 8 weeks.

Yet another embodiment of the invention is a differentiated pluripotent stem cell culture, e.g.,
The present invention provides a method for producing a cell aggregate suspension formed from cells generated from the pluripotent cells described herein, including cells from stages 1, 2, 3, 4 and 5 described in our 2005 and 2006. Thus, the methods for producing cell aggregates described herein are not limited to any one pluripotent or multipotent cell or cell stage,
Rather, the method of use and the need for cell type optimization will determine which method is preferred.

The methods described herein for producing aggregated suspension cultures of pluripotent cells, e.g., hES or iPS cells, and other sources of pluripotent cells, e.g., cells derived from embryonic or parthenogenetic organism cells, are substantially similar to those described in the patent application Ser. No. 11/200,333, filed Feb. 23, 2007, entitled "Composite Cell Line," which is incorporated herein by reference in its entirety.
tions and methods for culturing different
and PCT/US2007/062,755, entitled "Methods and compositions," filed on October 20, 2008.
for feeder-free pluripotent stem cell me
PCT/US2008/
080,516, which are incorporated herein by reference in their entireties.

The methods described herein include, for example, those to Bodnar et al.
No. 6,800,480, assigned to the same assignee as U.S. Patent No. 6,800,480, issued to the same assignee as U.S. Patent No. 6,800,480, assigned ...
r-free pluripotent stem cell media conta
ining human serum, U.S. Patent Application PCT/US2008/080
The cells can be cultured in the same manner as they are cultured using soluble human serum as described in, et al., J. Clin. Pharmacol., 2003, 516.

The methods described herein are directed to Thomson, J., and Wiscon
No exogenously added fibroblast growth factor (FGF) is required, supplied from a source other than simply a fibroblast feeder layer, as described in U.S. Pat. No. 7,005,252, assigned to the Sin Alumni Research Foundation (WARF), which is incorporated by reference in its entirety herein.

Pluripotent and Differentiated Cell Compositions The cell compositions produced by the methods described herein include pluripotent stem cells, preprimitive streak, mesendoderm, definitive endoderm, foregut endoderm, PDX1 positive foregut endoderm, PDX1 positive pancreatic endoderm or PDX1/NKX6.1 co-positive pancreatic endoderm, endocrine precursors or NGN3/NKX2.2
Co-positive endocrine precursors and hormone-secreting endocrine cells or INS, GCG, GHRL,
SST, cell cultures comprising PP single-positive endocrine cells, wherein at least about 5-90% of the cells in the culture are generated preprimitive streak, mesendoderm, definitive endoderm, foregut endoderm, PDX1-positive foregut endoderm, PDX1-positive pancreatic endoderm or PDX1/NKX6.1 co-positive pancreatic endoderm;
endocrine precursors or NGN3/NKX2.2 co-positive endocrine precursors, and hormone-secreting endocrine cells or INS, GCG, GHRL, SST, PP single positive endocrine cells.

Some embodiments described herein relate to compositions such as cell populations and cell cultures that contain both pluripotent cells, such as stem cells and iPS cells, and multipotent cells, such as preprimitive streak, mesendoderm, or definitive endoderm, as well as more differentiated, but potentially multipotent, cells, such as PDX1-positive foregut endoderm, PDX1-positive pancreatic endoderm, or PDX1/NKX6.1 co-positive pancreatic endoderm, endocrine precursors, or NGN3/NKX2.2 co-positive endocrine precursors, and hormone-secreting endocrine cells, or INS, GCG, GHRL, SST, PP single positive endocrine cells. For example, the methods described herein can be used to generate compositions that contain a mixture of pluripotent stem cells and other multipotent or differentiated cells. In some embodiments, compositions are generated that contain at least about 5 multipotent or differentiated cells for every about 95 pluripotent cells. In other embodiments, compositions are generated that contain at least about 95 multipotent or differentiated cells for every about 5 pluripotent cells. Additionally, compositions containing other ratios of multipotent or differentiated cells to pluripotent cells are contemplated.
a composition comprising at least about 1 multipotent cell or differentiated cell for every 00,000 pluripotent cells; a composition comprising at least about 1 multipotent cell or differentiated cell for every 100,000 pluripotent cells; a composition comprising at least about 1 multipotent cell or differentiated cell for every 10,000 pluripotent cells; a composition comprising at least about 1 multipotent cell or differentiated cell for every 1000 pluripotent cells; a composition comprising at least about 1 multipotent cell or differentiated cell for every 500 pluripotent cells; Compositions comprising differentiated cells, compositions comprising at least about 1 multipotent or differentiated cell for about every 100 pluripotent cells, compositions comprising at least about 1 multipotent or differentiated cell for about every 10 pluripotent cells, compositions comprising at least about 1 multipotent or differentiated cell for about every 5 pluripotent cells, and up to about 1 pluripotent cell, and compositions comprising at least about 1,000,000 multipotent or differentiated cells for about every 1 pluripotent cell are contemplated.

Some embodiments described herein relate to cell cultures or cell populations comprising at least about 5% multipotent or differentiated cells to at least about 99% multipotent or differentiated cells. In some embodiments, the cell cultures or cell populations comprise mammalian cells. In preferred embodiments, the cell cultures or cell populations comprise human cells. For example,
Certain embodiments provide a cell culture comprising human cells, comprising at least about 5% of the human cells.
The present invention relates to cell cultures in which from about 99% to at least about 99% are multipotent or differentiated cells.
Other embodiments relate to cell cultures comprising human cells, wherein at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more than 99% of the human cells are multipotent or differentiated cells. In embodiments where the cell culture or cell population comprises human feeder cells, the above percentages are calculated without consideration of the human feeder cells in the cell culture.

Pluripotent, multipotent or differentiated cells can differentiate or further differentiate into preprimitive streak, mesoendoderm, definitive endoderm cells, and cells, tissues and/or organs derived therefrom. Mesoendoderm cells can differentiate into mesoderm and/or definitive endoderm cells, and cells, tissues and/or organs derived from any of these lineages. In some embodiments, preprimitive streak cells are converted through a mesoendoderm intermediate into terminally differentiated cells of either the mesoderm or definitive endoderm lineages. For example, such a process can provide the basis for efficient generation of human endoderm-derived tissues, such as the pancreas, liver, lung, stomach, intestine, thyroid, parathyroid gland, thymus, pharynx, gallbladder and urinary bladder. Importantly, the generation of preprimitive streak and/or mesoendoderm cells is an early step in the differentiation of pluripotent stem cells into functional insulin-producing beta cells. As another example,
Differentiation of preprimitive streak cells and/or mesoendodermal cells can provide the basis for the efficient generation of human mesodermally derived tissues, such as blood cells, cardiovascular tissue, skeletal tissue and other structural and connective tissues.

The compositions and methods described herein have several useful features. For example, cell cultures and cell populations including pluripotent cells, such as preprimitive streak cells and/or mesendoderm cells, and methods for generating such cell cultures and cell populations are useful for modeling early human development. In addition, the compositions and methods described herein may also serve as therapeutic interventions for pathologies such as diabetes mellitus. For example, preprimitive streak cells and/or mesendoderm cells serve as a source for only a limited number of tissues, and therefore can be used in the generation of pure tissue or cell types. In some processes for generating preprimitive streak cells, the pluripotent cells used as starting material are pluripotent stem cells, such as hESCs.
ES cells, hEG cells or iPS cells.

Trophectoderm Cells The methods described herein can be used to generate compositions comprising trophectoderm cells, substantially free of other cell types. In some embodiments described herein, the trophectoderm cell populations or cell cultures generated by the methods described herein contain HAND1, E
omes, MASH2, ESXL1, HCG, KRT18, PSG3, SFXN5, DL
Expression of a marker selected from the group consisting of HAND1, Eomes, MASH2, X3, PSX1, ETS2, and ERBB genes in a non-trophectoderm cell or cell population.
, ESXL1, HCG, KRT18, PSG3, SFXN5, DLX3, PSX1, ET
S2, and ERBB expression levels are substantially higher.

Preprimitive Streak Cells The methods described herein can be used to generate compositions comprising preprimitive streak cells, substantially free of other cell types. In some embodiments described herein, the preprimitive streak cell populations or cell cultures generated by the methods described herein express the FGF8 marker gene and/or the NODAL marker gene as BRACHURY low, FGF4 low, SNAI low,
1 low, SOX17 low, FOXA2 low, SOX7 low and SOX1 low.

The methods described herein can be used to generate compositions comprising extraembryonic cells, substantially free of other cell types. Primitive endoderm cells, proximal endoderm cells and distal endoderm cells are extraembryonic cells. Primitive endoderm cells give rise to proximal endoderm cells and distal endoderm cells.
Proximal endoderm cells are endoderm cells that form part of the yolk sac. Proximal endoderm cells function in both nutrient uptake and transport. Distal endoderm cells are in close proximity to an extraembryonic tissue known as Reichert's membrane. One of the roles of distal endoderm cells is the production of components of the basement membrane. Taken together, proximal and distal endoderm cells form what is often referred to as extraembryonic endoderm. As the name suggests, extraembryonic endoderm cells do not give rise to embryonic structures that form during development. In contrast, the definitive endoderm cells and other endoderm lineage or pancreatic lineage cells described herein are embryonic or derived from embryonic cells, and give rise to tissues derived from the gut tube that forms during embryonic development. In some embodiments described herein, the extraembryonic cell populations or cell cultures generated by the methods described herein exhibit expression of a marker selected from the group consisting of SOX7 gene, SOX17 gene, THBD gene, SPARC gene, DAB1 gene, FTNF4alpha gene, or AFP gene, at least one of which is not expressed in other cell types or cell populations, such as SOX7, SOX17, THBD, SPARC, DAB1, or AFP, which is not expressed in definitive endoderm.
is substantially higher compared to the expression level of

The methods described herein can be used to generate compositions comprising mesendoderm cells that are substantially free of other cell types. In some embodiments described herein, the mesendoderm cell populations or cell cultures generated by the methods described herein are characterized by the expression of FGF4, SNAI1,
, MIXL1 and/or WNT3 marker genes is substantially higher compared to SOX17 low, CXCR4 low, FOXA2 low, SOX7 low and SOX1 low.

Screening method In some embodiments, the screening method is used to obtain a certain cell population, such as pluripotent cells, multipotent cells and/or differentiated cells, such as human pluripotent stem cells, induced pluripotent stem cells, preprimitive streak cells, mesendoderm cells, definitive endoderm cells, foregut endoderm or PDX1-negative foregut endoderm cells, PDX1-positive foregut endoderm or PDX1-positive pancreatic endoderm cells or pancreatic progenitor cells, endocrine precursor cells, and/or endocrine cells. A candidate differentiation factor is then provided to the cell population. At a first time point, before or at about the same time as providing the candidate differentiation factor, the expression of the marker is determined. Alternatively, the expression of the marker can be determined after providing the candidate differentiation factor. At a second time point, after the first time point and after the step of providing the candidate differentiation factor to the cell population, the expression of the same marker is determined again. Whether the candidate differentiation factor can promote the differentiation of pancreatic precursor cells is determined by comparing the expression of the marker at the first time point with the expression of the marker at the second time point. If expression of the marker at the second time point is increased or decreased relative to expression of the marker at the first time point, then the candidate differentiation factor is capable of promoting differentiation of pancreatic progenitor cells.

In some embodiments of the screening methods described herein, human definitive endoderm, PDX
The present invention utilizes cell populations or cell cultures that contain PDX-1-negative foregut endoderm, PDX-1-positive foregut endoderm, PDX-1-positive pancreatic endoderm, or pancreatic precursor or endocrine progenitor cells. For example, the cell population can be a substantially purified population of PDX-1-positive pancreatic endoderm or pancreatic progenitor cells. For example, the cell population can be an enriched population of human pancreatic progenitor cells, where at least about 50%-97% of the human cells in the cell population are human pancreatic progenitor cells, with the remainder being made up of endocrine precursor or endocrine cells and other cell types.

In some embodiments of the screening methods described herein, the cell population is a candidate (test)
The cells are contacted with or otherwise provided with a candidate (test) differentiation factor. Candidate differentiation factors may include any molecule that may have the potential to promote differentiation of any of the above cells, e.g., human pancreatic progenitor cells. In some embodiments described herein, the candidate differentiation factor is
The candidate differentiation factors include molecules known to be differentiation factors for one or more types of cells. In alternative embodiments, the candidate differentiation factors include molecules not known to promote cell differentiation. In a preferred embodiment, the candidate differentiation factors include molecules not known to promote differentiation of human pancreatic progenitor cells.

In some embodiments of the screening methods described herein, the candidate differentiation factor comprises a small molecule. In preferred embodiments, the small molecule is a molecule having a molecular weight of about 10,000 amu or less.

In other embodiments described herein, the candidate differentiation factors include large molecules, such as polypeptides. The polypeptides may be any polypeptide, including, but not limited to, glycoproteins, lipoproteins, extracellular matrix proteins, cytokines, chemokines, peptide hormones, interleukins, or growth factors. Preferred polypeptides include growth factors.

In some embodiments of the screening methods described herein, the candidate differentiation factor is amphiregulin, B lymphocyte stimulatory substance, IL-16, thymopoietin, TRAIL/A.
po-2, pre-B cell colony-stimulating factor, endothelial differentiation-associated factor 1 (EDF1), endothelial monocyte-activating polypeptide II, macrophage migration inhibitory factor (MIF), natural killer cell-enhancing factor (NKEFA), bone morphogenetic protein 2, bone morphogenetic protein 8 (bone morphogenetic protein 2), bone morphogenetic protein 6, bone morphogenetic protein 7, connective tissue growth factor (CTGF), CG
I-149 protein (neuroendocrine differentiation factor), cytokine A3 (macrophage inflammatory protein 1-alpha), glioblastoma cell differentiation-related protein (GBDR1), hepatoma-derived growth factor, neuromedin U-25 precursor, vascular endothelial growth factor (VEGF),
Vascular endothelial growth factor B (VEGF-B), T cell-specific RANTES precursor, thymic dendritic cell-derived factor 1, transferrin, interleukin-1 (IL1), interleukin-2 (IL2), interleukin-3 (IL3), interleukin-4 (IL4)
, interleukin-5 (IL5), interleukin-6 (IL6), interleukin-7 (IL7), interleukin-8 (IL8), interleukin-9 (IL9)
, interleukin-10 (IL10), interleukin-11 (IL11), interleukin-12 (IL12), interleukin-13 (IL13), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin, thrombopoietin,
Vitamin D3, epidermal growth factor (EGF), brain-derived neurotrophic factor, leukemia inhibitory factor, thyroid hormone, basic fibroblast growth factor (bFGF), aFGF, FGF-4, FGF-6
, FGF-7/keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF),
Platelet-derived growth factor-BB, beta nerve growth factor, activin A, transforming growth factor beta 1 (TGF beta 1), interferon-alpha, interferon-beta, interferon-gamma, tumor necrosis factor-alpha, tumor necrosis factor-beta, burst promoting activity (BPA), erythroid promoting activity (EPA), PGE2, insulin growth factor-1 (IGF-1), IGF-II, neurotrophin growth factor (NGF), neurotrophin-
3, neurotrophin 4/5, ciliary neurotrophic factor, glial-derived nexin, dexamethasone, β-mercaptoethanol, retinoic acid, butylated hydroxyanisole, 5-azacytidine, amphotericin B, ascorbic acid, ascorbate, isobutylxanthine, indomethacin, β-glycerol phosphate, nicotinamide, DMSO, thiazolidinedione, TWS119, oxytocin, vasopressin, melanocyte-stimulating hormone, adrenocorticotropic hormone, lipotropin, thyroid-stimulating hormone, growth hormone, prolactin, luteinizing hormone, human chorionic gonadotropin, follicle-stimulating hormone, corticotropin-releasing factor, gonadotropin-releasing factor, prolactin-releasing factor, prolactin-inhibiting factor, growth hormone-releasing factor, somatostatin thyrotropin-releasing factor, calcitonin gene-related peptide, parathyroid hormone, glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, gastrin, secretin, cholecystokinin, motilin, vasoactive intestinal peptide, substance P, pancreatic polypeptide, peptide tyrosine, neuropeptide tyrosine, insulin, glucagon, placental lactogen, relaxin, angiotensin II, calctriol, atrial natriuretic peptide, and melatonin, thyroxine, triiodothyronine, calcitonin, estradiol, estrone, progesterone, testosterone, cortisol, corticosterone, aldosterone, epinephrine, norepinephrine, androgen.
ostiene), calcitriol, collagen, dexamethasone, β-mercaptoethanol, retinoic acid, butylated hydroxyanisole, 5-azacytidine, amphotericin B, ascorbic acid, ascorbate, isobutylxanthine, indomethacin,
The composition includes one or more growth factors selected from the group consisting of β-glycerol phosphate, nicotinamide, DMSO, a thiazolidinedione, and TWS119.

In some embodiments of the screening methods described herein, the candidate differentiation factor is
The candidate differentiation factor is provided to the cell population at one or more concentrations. In some embodiments, the candidate differentiation factor is provided to the cell population such that the concentration of the candidate differentiation factor in the medium surrounding the cells is about 0.1 ng/ml to 10 mg/ml. In some embodiments, the concentration of the candidate differentiation factor in the medium surrounding the cells is about 1 ng/ml to about 1 mg/ml. In other embodiments, the concentration of the candidate differentiation factor in the medium surrounding the cells is about 10 ng/ml to about 100 μg/ml. In yet other embodiments, the concentration of the candidate differentiation factor in the medium surrounding the cells is about 100 ng/ml to about 10 μg/ml. In a preferred embodiment, the concentration of the candidate differentiation factor in the medium surrounding the cells is about 5 ng/ml to about 1000 μg/ml.

In some embodiments, the steps of the screening methods described herein include determining the expression of at least one marker at a first time point and at a second time point. In some of these embodiments, the first time point may be before or at about the same time as providing the cell population with the candidate differentiation factor. Alternatively, in some embodiments, the first time point is after providing the cell population with the candidate differentiation factor. In some embodiments, the expression of multiple markers is determined at the first time point.

In addition to determining the expression of at least one marker at a first time point, some embodiments of the screening methods described herein contemplate determining the expression of at least one marker at a second time point that is subsequent to the first time point and subsequent to providing the cell population with the candidate differentiation factor. In such embodiments, the expression of the same marker is determined at both the first and second time points. In some embodiments, the expression of multiple markers is determined at both the first and second time points. In such embodiments, the expression of the same multiple markers is determined at both the first and second time points. In some embodiments, the expression of markers is determined at multiple time points, each subsequent to the first time point and each subsequent to providing the cell population with the candidate differentiation factor. In certain embodiments, the expression of markers is determined by Q-PCR. In other embodiments, the expression of markers is determined by immunocytochemistry.

In certain embodiments of the screening methods described herein, the markers whose expression is determined at the first and second time points are markers associated with the differentiation of pancreatic progenitor cells into cells that are precursors of the terminally differentiated cells that make up pancreatic islet tissue, including immature pancreatic islet hormone-expressing cells.

In some embodiments of the screening methods described herein, a sufficient amount of time can elapse between providing the cell population with the candidate differentiation factor and determining expression of the marker at a second time point. The sufficient amount of time between providing the cell population with the candidate differentiation factor and determining expression of the marker at a second time point can be as short as about 1 hour to as long as about 10 days. In some embodiments, the expression of at least one marker is determined multiple times after providing the cell population with the candidate differentiation factor. In some embodiments, the sufficient amount of time can be at least about 1 hour, at least about 6 hours, at least about 12 hours,
At least about 16 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11
days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, or at least about 8 weeks.

In some embodiments of the methods described herein, the method further determines whether the expression of the marker at the second time point is increased or decreased compared to the expression of the marker at the first time point. An increase or decrease in the expression of at least one marker indicates that the candidate differentiation factor can promote the differentiation of endocrine precursor cells. Similarly, when the expression of multiple markers is determined, the method further determines whether the expression of the multiple markers at the second time point is increased or decreased compared to the expression of the multiple markers at the first time point.
The increase or decrease in expression of a marker can be determined by measuring or otherwise assessing the amount, level or activity of the marker in the cell population at the first and second time points. Such determinations can be comparative to other markers, such as housekeeping gene expression, or can be absolute. In certain embodiments where expression of a marker at the second time point is increased compared to the first time point, the amount of increase can be at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 10 ...500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, at least about 200-fold, at least about 200-fold, at least about 300
fold or at least about 100-fold more. In some embodiments, the amount of increase is less than 2-fold. In embodiments where expression of the marker at the second time point is decreased compared to the first time point, the amount of decrease is at least about 2-fold, at least about 5-fold, at least about 10-fold
, at least about 20 times smaller, at least about 30 times smaller, at least about 40 times smaller, at least about 50 times smaller, at least about 60 times smaller, at least about 70 times smaller, at least about 8 times smaller
0 times lower, at least about 90 times lower, at least about 100 times lower, or at least about 10 times lower
In some embodiments, the amount of reduction is no more than a factor of 2.

Monitoring the generation of multipotent or differentiated cells The progression of pluripotent cells to multipotent cells, and from multipotent cells to more multipotent or differentiated cells, such as pancreatic precursors or hormone endocrine cells, can be monitored by determining the expression of markers characteristic of certain cells, including genetic and phenotypic markers, such as the expression of islet hormones in endocrine cells and the processing of proinsulin to insulin and C-peptide.In some processes, the expression of a certain marker is determined by detecting whether the marker is present or absent.Alternatively, the expression of a certain marker can be determined by measuring the level of the marker present in the cells of a cell culture or cell population.For example, in certain processes, the expression of markers characteristic of immature pancreatic islet hormone-expressing cells, as well as pluripotent cells, definitive endoderm, foregut endoderm, PDX1-positive foregut endoderm, endocrine precursors, extraembryonic endoderm,
The lack of significant expression of markers characteristic of mesoderm, ectoderm, mature pancreatic islet hormone-expressing cells and/or other cell types is determined.

As described in connection with monitoring the generation of other less differentiated cell types of the definitive endoderm lineage, qualitative or semi-quantitative techniques such as blot transfer and immunocytochemistry can be used to measure expression of the markers. Alternatively, expression of the markers can be precisely quantified by using techniques such as Q-PCR. Furthermore, at the polypeptide level, it will be appreciated that many of the markers of pancreatic islet hormone-expressing cells are secreted proteins. Thus, techniques for measuring extracellular marker content such as ELISA can be utilized.

For example, in one embodiment, PDX1 is a marker gene associated with PDX1-positive foregut endoderm. Thus, in some embodiments of the invention, expression of PDX1 is determined. In other embodiments, expression of other markers expressed in PDX1-positive foregut endoderm is also determined, such as, but not limited to, SOX17, HNF6, SOX9 and PROX1. Because PDX1 can also be expressed by certain other cell types (i.e., visceral endoderm and certain neuroectoderm), some embodiments of the invention relate to demonstrating the absence or substantial absence of marker gene expression associated with visceral endoderm and/or neuroectoderm. For example, in some embodiments, expression of markers associated with visceral endoderm and/or neuroectoderm is determined, such as, but not limited to, SOX7, AFP, SOX1, ZIC1 and/or NFM.
The expression of markers expressed in visceral endoderm and/or neural cells, including:

In some embodiments, the PDX1-positive foregut endoderm cell cultures produced by the methods described herein are substantially free of cells expressing SOX7, AFP, SOX1, ZIC1 or NFM marker genes. In certain embodiments, the PDX1-positive foregut endoderm cell cultures produced by the processes described herein are substantially free of proximal endoderm, distal endoderm and/or neural cells.

The developmental progress of pluripotent cells described herein (e.g., cells generated as a result of stages or steps 1-5 described in D'Amour et al. 2006, supra) can be monitored by determining the expression of markers characteristic of each hES- or iPS-derived cell type along the developmental pathway. In some processes, the identification and characterization of hES- or iPS-derived cell types is by expression of a particular marker or by differential expression levels and patterns of multiple markers. That is, the presence or absence, high or low expression of one or more markers characterizes and identifies the cell type. Furthermore, a particular marker can have a transient expression, whereby the marker is highly expressed during one stage of development and weakly expressed at another stage of development. The expression of a particular marker can be determined by measuring the level of that marker present in the cells of the cell culture or cell population compared to a standardized or normalized control marker. In such processes, the measurement of marker expression can be qualitative or quantitative. One method of quantifying the expression of a marker generated by a marker gene is quantitative PCA.
By use of Q-PCR. Methods for performing Q-PCR are well known in the art.

In some embodiments of the invention, the presence, absence and/or expression level of a marker is determined by quantitative PCR (Q-PCR). For example, certain genetic markers, such as SOX17, CXCR4, OCT4, AFP, TM, SPARC, SOX7, CDX2,
MIXL1, GATA4, HNF3β, HNF4α, GSC, FGF17, VWF
The amount of transcripts produced by , CALCR, FOXQ1, CMKOR1, CRIP1 and other markers described herein is determined by quantitative Q-PCR.

In other embodiments, immunohistochemistry is used to detect proteins expressed by the above genes. In yet other embodiments, Q-PCR can be used in conjunction with immunohistochemistry or flow cytometry techniques to effectively and accurately characterize and identify cell types and determine both the amount and relative proportion of such markers in the cell type of interest. In one embodiment, Q-PCR can quantify the level of RNA expression in cell cultures containing a mixed population of cells. However, Q-PCR cannot provide or qualify whether markers or proteins of interest are co-expressed in the same cell. In another embodiment, Q-PCR is used in conjunction with flow cytometry methods to characterize and identify cell types. Thus, by using the methods described herein in combination with, for example, those described above, complete characterization and identification of various cell types, including endoderm lineage cells, can be achieved and demonstrated.

For example, in a preferred embodiment, the pancreatic progenitor or pancreatic endoderm or PDX-1 positive pancreatic endoderm is at least PDZ-1 positive as demonstrated by Q-PCR and/or ICC.
PDX1 positive expressing cells express PDX1, Nkx6.1, PTF1A, CPA and/or cMYC, but such cells must co-express at least PDX1 and Nkx6.1 as demonstrated by ICC and not express other markers, including SOX17, CXCR4, or CER, to be identified as PDX1 positive expressing cells.
To adequately identify mature hormone-secreting pancreatic cells in vo, for example, ICC demonstrates that in insulin-secreting cells, C-peptide (the product of proper processing of proinsulin in mature and functional β-cells) and insulin are co-expressed.

In addition, other methods known in the art can be used to quantify marker gene expression. For example, expression of marker gene products can be detected by using antibodies specific to the marker gene products of interest (e.g., Western blot, flow cytometry analysis, etc.). In certain processes, expression of marker genes specific to hES-derived cells as well as the lack of significant expression of marker genes specific to hES-derived cells. Further methods for characterizing and identifying hES-derived cell types are described in the related applications listed above, which are fully incorporated herein by reference.

Suitable amplification probe/primer combinations for use in the amplification assay are as follows: Insulin (INS) (GenBank NM_000207): Primer AA
GAGGCCATCAAGCAGATCA (SEQ ID NO: 1); CAGGAGGCGCATC
CACA (SEQ ID NO:2); Nkx6.1 (NM_006168): primer CTGGC
CTGTACCCCTCATCA (SEQ ID NO: 3); CTTCCCGTCTTTGTCCA
ACAA (SEQ ID NO: 4); Pdx1 (NM_000209): primer AAGTCTA
CCAAAGCTCACGCG (SEQ ID NO: 5); GTAGGCGCCGCCCTGC (SEQ ID NO: 6); Ngn3 (NM_020999): primer GCTCATCGCTCTCT
ATTCTTTTGC (SEQ ID NO: 7); GGTTGAGGCGTCATCCTTTCT (
SEQ ID NO: 8); FOXA2 (HNF3B) (NM_021784): primer GGGA
GCGGTGAAGATGGA (SEQ ID NO: 9); TCATGTTGCTCACGGAGG
AGTA (SEQ ID NO: 10); Glucagon (GCG) (NM_002054): primers AAGCATTTACTTTGTGGCTGGATTT (SEQ ID NO: 11); TGATCTG
GATTTCTCCTCTGTGTCT (SEQ ID NO: 12); HNF6 (NM_03071
2): Primer CGCTCCGCTTAGCAGCAT (SEQ ID NO: 13); GTGTT
GCCTCTATCCTTCCCAT (SEQ ID NO: 14); HNF4 alpha (NM_00
0457): Primer GAAGAAGGAAGCCGTCCAGA (SEQ ID NO: 15);
GACCTTCGAGTGCTGATCCG (SEQ ID NO: 16); Sox17 (NM_02
2454): Primer GGCGCAGCAGAATCCAGA (SEQ ID NO: 17);
NNNNNNNNNNNNNNN NNNNN (SEQ ID NO: 18); HLxB9 (NM_005
515): Primer CACCGCGGGCATGATC (SEQ ID NO: 19); ACTTC
CCCAGGAGGTTCGA (SEQ ID NO: 20); Nkx2.2 (NM_002509)
Primer GGCCTTCAGTATCCCTGCA (SEQ ID NO: 21); GGGAC
TTGGAGCTTGAGTCCT (SEQ ID NO: 22); PTF1a (NM_178161
): Primer GAAGGTCATCATCTGCCATCG (SEQ ID NO: 23); GGC
CATAATCAGGGTCGCT (SEQ ID NO: 24); SST (NM_001048):
Primer CCCCAGACTCCGTCAGTTTC (SEQ ID NO:25); TCCGTC
TGGTTGGGTTCAG (SEQ ID NO: 26); PAX6 (NM_000280): primer CCAGAAAGGATGCCTCATAAAGG (SEQ ID NO: 27); TCTGC
GCGCCCCTAGTTA (SEQ ID NO: 28); Oct4 primer: TGGGCTCG
AGAAGGATGTG (SEQ ID NO: 29); GCATAGTCGCTGCTTGATCG
(SEQ ID NO: 30); MIXL1 primer CCGAGTCCAGGATCCAGGTA (
SEQ ID NO:31) CTCTGACGCCGAGACTTGG (SEQ ID NO:32); GATA4
Primer CCTCTTGCAATGCGAAAG (SEQ ID NO: 33) CGGGAGGA
AGGCTCTCACT (SEQ ID NO: 34); GSC primer GAGGAGAAAGTG
GAGGTCTGGTT (SEQ ID NO: 35) CTCTGATGAGGGACCGCTTCTG
(SEQ ID NO:36); CER primers ACAGTGCCCTTCAGCCAGACT (SEQ ID NO:37) ACAACTACTTTTTCACAGCCTTCGT (SEQ ID NO:38);
AFP primer GAGAAACCCACTGGAGATGAACA (SEQ ID NO: 39) C
TCATGGCAAAGTTCTTCCAGAA (SEQ ID NO: 40); SOX1 primer ATGCACCGCTACGACATGG (SEQ ID NO: 41) CTCATGTAGCCCT
GCGAGTTG (SEQ ID NO: 42); ZIC1 primer CTGGCTGTGGCAAG
GTCTTC (SEQ ID NO: 43) CAGCCCTCAAACTCGCACTT (SEQ ID NO: 4
4); NFM primer ATCGAGGAGCGCCACAAC (SEQ ID NO: 45) TGC
TGGATGGTGTCCTGGT (SEQ ID NO: 46). The other primers were FGF17(
Hs00182599_m1), VWF (Hs00169795_m1), CMKOR1
(Hs00604567_m1), CRIP1 (Hs00832816_g1), FOX
Q1 (Hs00536425_s1), CALCR (Hs00156229_m1) and CHGA (Hs00154441_m1), all available through ABI Taqman.

Summary of In Vivo Generation of PDX1-Positive Pancreatic Endoderm (Stages 1-4) and Insulin Production Methods for the generation of specific endoderm lineages and pancreatic endoderm lineage cells are provided herein and are described in related applications, such as U.S. Patent Application No. 11/773,944, filed July 5, 2007, entitled METHODS OF PRODUCING PANCREATIC HORMOGENESIS.
NES, which is incorporated herein by reference in its entirety.
No. 681,687, entitled ENDOCRINE PRECURSOR CELLS, PAN
CREATIC HORMONE-EXPRESSING CELLS AND MET
This is a continuation-in-part of HODS OF PRODUCTION, which is incorporated by reference herein in its entirety.

Briefly, the directed differentiation method of the present invention for pluripotent stem cells, e.g., hES cells and iPS cells, can be described in at least four or five stages. Stage 1 is the generation of definitive endoderm from pluripotent stem cells, which takes about 2-5 days, preferably 2 or 3 days. The pluripotent stem cells are cultured in RPMI, a TGF superfamily member growth factor, e.g., activin A, activin B, GDF-8 or GDF-11 (100 ng/ml).
l), a Wnt family member or a Wnt pathway activator, such as Wnt3a (
5 ng/ml), or a rho kinase or ROCK inhibitor, such as Y-27632
(10 μM) to enhance growth, survival and proliferation as well as promote cell-cell adhesion. After about 24 hours, the medium is changed to serum, e.g., 0.2% FBS, and T
GFβ superfamily member growth factors, such as activin A, activin B, GD
F-8 or GDF-11 (100 ng/mL), or rho-kinase or R
The medium was replaced with RPMI containing an OCK inhibitor, and then incubated for another 24 hours (day 1) to 48 hours.
Alternatively, after about 24 hours in medium containing Activin/Wnt3a, the cells are cultured for the next 24 hours in medium containing Activin alone (i.e., the medium does not contain Wnt3a). Importantly, the generation of definitive endoderm requires cell culture conditions with low serum content, and therefore low insulin or insulin-like growth factor content. McLean et al. (2007) Stem Cell Immunol. 2009, 143:1311-1323, which is incorporated herein in its entirety.
See McLean et al., in Stage 1 hE
It has also been shown that contacting S cells with insulin at a concentration of as little as 0.2 μg/mL can be detrimental to the generation of definitive endoderm. Others skilled in the art have modified stage 1 differentiation of pluripotent cells to definitive endoderm substantially as described herein and in D'Amour et al. (2005). For example, see at least Agarwal et al., Efficient
Differentiation of Functional Hepatocytes
s from Human Embryonic Stem Cells, Stem C
ells (2008) 26:1117-1127; Borowiak et al., Small M
olecules Efficiently Direct Endodermal D
Differentiation of Mouse and Human Embryon
c Stem Cells, (2009) Cell Stem Cell 4:348-
358; and Brunner et al., Distinct DNA Methylation
patterns
man embryonic stem cells and developing
human fatal liver, (2009) Genome Res. 19:1
Proper differentiation, specification, characterization and identification of definitive endoderm is necessary to derive other endoderm lineage cells. Definitive endoderm cells at this stage are derived from SO
Co-expression of HNF3β (FOXA2) and HNF4α, HNF
F6, PDX1, SOX6, PROX1, PTF1A, CPA, cMYC, NKX6.1
, NGN3, PAX3, ARX, NKX2.2, INS, GSC, GHRL, SST, or PP are not appreciably expressed.

Stage 2 is the process of taking definitive endoderm cell cultures from stage 1 and culturing them in suspension at 1:
Low serum levels such as 0.2% FBS in 1000 dilution, 25 ng KGF (or F
Foregut endoderm or PDX1-negative foregut endoderm is generated by incubating the cells with RPMI containing RPMI-100001 (RPMI 100001) or RPMI containing ROCK inhibitor for 24 hours (days 2 to 3) to enhance growth, survival, proliferation, and promote cell-cell adhesion. After 24 hours (days 3 to 4),
The medium was replaced with the same medium without TGFβ inhibitors but instead still containing ROCK inhibitors to enhance cell growth, survival and proliferation, and incubated for another 24 hours (days 4-5).
Stage 2 cell cultures are then left for 48 hours (day 6) to 48 hours (day 7). A critical step for proper specification of foregut endoderm is the removal of TGFβ family growth factors. Therefore, a TGFβ inhibitor such as 2.5 μM TGFβ inhibitor number 4 or 5 μM SB431542, a specific inhibitor of the TGFβ type I receptor, activin receptor-like kinase (ALK), can be added to stage 2 cell cultures. Stage 2 generated foregut endoderm or PDX1-negative foregut endoderm cells co-express SOX17, HNF1β and HNF4alpha and are consistent with definitive endoderm,
At least SOX17 and HNF3β (FOXA2), which are hallmarks of PDX1-positive pancreatic endoderm or pancreatic progenitor or endocrine precursor cells and single or multiple hormone type cells, are also expressed in the human pancreatic endoderm and pancreatic progenitor cells.
6.1, NGN3, PAX3, ARX, NKX2.2, INS, GSC, GHRL, SS
Neither T nor PP are appreciably co-expressed.

At stage 3 (days 5-8), foregut endoderm cell cultures were obtained from stage 2 and cultured at 1%
B27, 0.25 μM KAAD cyclopamine, 0.2 μM retinoid such as retinoic acid (RA) or 3 nM retinoic acid analogue such as TTNPB, and 50 ng/m
PDX1-positive foregut endoderm cells are generated in L. noggin with DMEM or RPMI over a period of about 24 hours (day 7) to 48 hours (day 8). Specifically, Applicants have been using DMEM-high glucose since approximately 2003, and all patent and non-patent disclosures to that time have used DMEM-high glucose even without any reference to "DMEM-high glucose" or the like. This is in part due to the fact that manufacturers such as Gibco use DMEM-high glucose, e.g.
MEM (Cat. No. 11960) and Knockout DMEM (Cat. No. 108
29), DMEM has not been so named. It should be noted that as of the filing date of this application, Gibco offers additional DMEM products, but has not traditionally labeled certain of their high glucose-containing DMEM products, such as Knockout DMEM (Cat No. 10829-018). Thus, in each instance where DMEM is mentioned, it can be presumed that DMEM containing high glucose is meant, which would have been obvious to others conducting research and development in this field. Furthermore, the use of ROCK inhibitors or rho inhibitors has not been shown to be effective in treating certain conditions, such as chronic kidney disease.
Kinase inhibitors, such as Y-27632, can be used to enhance growth, survival, proliferation and promote cell-cell adhesion. PDX1-positive foregut cells generated at stage 3 co-express PDX1 and HNF6, as well as SOX9 and PROX, and are considered to be definitive endoderm cells or foregut endoderm cells (PDX1) as described above in stages 1 and 2.
Markers indicative of PDX1-negative foregut endoderm cells or PDX1-positive foregut endoderm cells are not appreciably co-expressed.

In stage 4 (days 8 to 14), the medium was taken from stage 3 and diluted with 1% vol.
/vol B27 supplement, plus 50 ng/mL KGF and 50 ng/mL
The medium is replaced with DMEM containing 100 ng/mL EGF, and sometimes additionally 50 ng/mL Noggin. Again, Y-27 is added to enhance growth, survival, proliferation, and promote cell-cell adhesion.
ROCK inhibitors such as 632 can be used. PDX generated from stage 4
1-positive pancreatic endoderm cells express at least PDX1 and Nkx6.1, as well as PTF1A
and do not express significant amounts of markers indicative of definitive endoderm or foregut endoderm (PDX1-negative foregut endoderm) cells as described for stages 1, 2 and 3 above.

Alternatively, cells from stage 4 can be further differentiated at stage 5 in DMEM containing medium in 1% v/v B27 supplement for about 1-6 days (days 15-20) to generate endocrine precursor or progenitor type cells and/or mono- and polyhormonal pancreatic endocrine type cells from stage 4 cells. Endocrine precursor cells generated from stage 5 co-express at least NGN3 and PAX4 and Nkx2.2 and are characterized as definitive endoderm or foregut endoderm (
These cells do not appreciably express markers indicative of PDX1-negative foregut endoderm) or PDX1-positive pancreatic endoderm or progenitor cells.

The PDX1-positive pancreatic endoderm generated at stage 4 is loaded into a macroencapsulation device, fully contained, and implanted into a patient, and the PDX1-positive pancreatic endoderm cells are allowed to mature in vivo into pancreatic hormone-secreting cells, e.g., insulin-secreting cells. Encapsulation of PDX1-positive pancreatic endoderm cells and in vivo production of insulin is described in U.S. patent application Ser. No. 12/6
No. 18,659 (the '659 application"), entitled "ENCAPSULATION OF PAN
CREATIC LINEAGE CELLS DERIVED FROM HUMAN
The '659 application is a continuation of provisional patent application Ser. No. 61/114,857, entitled "E PLURIPOTENT STEM CELLS," filed Nov. 13, 2009.
NCAPSULATION OF PANCREATIC PROGENITORS D
No. 61/121,084, entitled "ENCAPSULATION OF HYDROXY-INDUCED HYDROGEN SURFACE FORMATIONS FOR HYDROGEN-INDUCED HYDROGEN SURFACE CELLS" filed Nov. 14, 2008; and U.S. Provisional Patent Application No. 61/121,084, entitled "ENCAPSULATION OF HYDROGEN-INDUCED HYDROGEN SURFACE FORMATIONS FOR HYDROGEN-INDUCED HYDROGEN SURFACE CELLS" filed Nov. 14, 2008;
This application claims priority to "PANCREATIC ENDODERM CELLS," filed December 9, 2008, the disclosures of each of which are incorporated herein by reference in their entirety.

The methods, compositions and devices described herein presently represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art that are encompassed within the spirit of the invention and are defined by the scope of the disclosure. Thus, it will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

For example, activin A, a member of the TGFβ superfamily of growth factors or signaling proteins, is used to generate definitive endoderm from pluripotent cells, such as hES cells and iPS cells, as described in International Patent Application No. PCT/US2008/065686, entitled "GROWTH FACTORS," which is incorporated herein by reference in its entirety.
FOR PRODUCTION OF DEFINITIVE ENDODERM”, 2
Other TGFβ superfamily members, such as those described in the Jun. 3, 2008 application, eg, GDF-8 and GDF-11, can be used to generate definitive endoderm.

Retinoic acid (RA) is used to differentiate stage 2 PDX1-negative foregut endoderm cells into stage 3 PDX1-positive foregut cells.
Other retinoids or retinoic acid analogs such as 7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (or TTNPB) and similar analogs (eg, 4-HBTTNPB) can be used.

Noggin is a protein that inactivates members of the TGFβ superfamily signaling proteins, such as bone morphogenetic protein-4 (BMP4). However, other BMP4 inhibitors, such as Chordin and Twisted Gastrulation (Tsg), or anti-BMP neutralizing antibodies, can also prevent the binding of BMP to its cell surface receptor, thereby effectively inhibiting BMP signaling. Alternatively, the human Noggin gene has been cloned and sequenced. See U.S. Patent No. 6,075,007, which is incorporated herein by reference. Analysis of the Noggin sequence shows a carboxy-terminal region with homology to Kunitz-type protease inhibitors, indicating that potentially other Kunitz-type protease inhibitors may have similar effects on inhibiting BMPs.

Finally, the macroencapsulation devices described herein and in US Application No. 12/618,659, fully incorporated herein by reference, are again merely exemplary and do not limit the scope of the present invention. In particular, variations in device design, such as device size, multiple chambers or subcompartments or multiple ports within the device, as well as device loading and extraction mechanisms, are all encompassed within the spirit of the present invention. Thus, it will be apparent to those skilled in the art that various substitutions and modifications to the differentiation methods described herein, as well as various substitutions and modifications to the encapsulation devices, can be made to the invention disclosed herein without departing from the scope and spirit of the present invention.

Generation and Composition of Definitive Endoderm (Stage 1)
In some processes, differentiation to definitive endoderm is achieved by providing the pluripotent cell culture with a growth factor of the TGFβ superfamily in an amount sufficient to promote differentiation to definitive endoderm. Growth factors of the TGFβ superfamily that are beneficial for the generation of definitive endoderm are selected from Nodal/activin, GDF-8, -9, -10, -11, etc., or the BMP subgroup. In some preferred differentiation processes, the growth factor is Nodal.
, activin A, activin B, GDF-8, GDF-11 and BMP4. In addition, the growth factors Wnt3a, other Wnt family members, and Wnt3b are also included.
nt pathway activators are beneficial for the generation of definitive endoderm cells. In certain differentiation processes, combinations of any of the above growth factors can be used. This and other methods are described in detail in U.S. Patent No. 7,510,876, which is incorporated herein by reference in its entirety.

For some processes for differentiating pluripotent cells into definitive endoderm cells, the growth factors are provided to the cells such that the growth factors are present in the culture at a concentration sufficient to promote differentiation of at least a portion of the pluripotent cells into definitive endoderm cells. In some processes, the growth factors are provided at a concentration of at least about 5 ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, at least about 20 ...
0 ng/mL, at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, at least about 100 ng/mL, at least about 200 ng/mL, at least about 300 ng/mL, at least about 400 ng/mL, at least about 500 ng
/mL, at least about 1000 ng/mL, at least about 2000 ng/mL, at least about 3000 ng/mL, at least about 4000 ng/mL, at least about 5000 ng/mL
/mL or greater than about 5000 ng/mL.

In certain processes for differentiation of pluripotent cells into definitive endoderm cells, the aforementioned growth factors are removed from the cell culture after they are added.For example, they can be removed within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days after adding the growth factors.In a preferred process, the growth factors are removed about 4 days after adding them.

In some embodiments of the processes described herein, the definitive endoderm cells are enriched, isolated and/or purified prior to further differentiation. In such embodiments, the definitive endoderm cells can be enriched, isolated and/or purified using any known method. In a preferred embodiment, the definitive endoderm cells are enriched, isolated and/or purified using any known method, as described in U.S. patent application Ser. No. 11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, and entitled "Definitive Endoderm Cells," ...
No. 60/736,598, filed Nov. 14, 2005, entitled MARKER
The enriched, isolated and/or purified proteins may be enriched, isolated and/or purified using one or more of the methods described in S OF DEFINITIVE ENDODERM, the disclosure of which is incorporated herein by reference in its entirety.

In preferred embodiments, the definitive endoderm cells produced and described herein have relatively high levels of CER, GSC, CXCR4, SOX17 and FOXA2 gene expression when normalized to and compared to the levels of control genes, such as housekeeping genes.

Generation and Composition of PDX1-Negative Foregut Endoderm (Stage 2)
Definitive endoderm cells can be specified towards pancreatic differentiation by further differentiation of these cells to generate PDX1-negative foregut endoderm cells. In some of the differentiation processes described herein, cell cultures and enriched or purified cell populations comprising definitive endoderm cells can be used for further differentiation into cell cultures and/or enriched cell populations comprising PDX1-negative foregut endoderm cells.

Generally, definitive endoderm cells are differentiated into PDX1-negative foregut endoderm cells by reducing, eliminating or removing TGFβ superfamily growth factor signaling in a cell culture or cell population of SOX17-positive definitive endoderm cells. In some embodiments, reducing or eliminating TGFβ superfamily growth factor signaling is mediated by diluting or removing exogenously added TGFβ superfamily growth factor, e.g., activin A, from the definitive endoderm cell culture or cell population. In other embodiments, TGFβ superfamily growth factor signaling is reduced or eliminated by providing the definitive endoderm cells with a compound that blocks TGFβ superfamily growth factor signaling, e.g., follistatin and/or noggin. In some embodiments, TGFβ superfamily growth factor signaling is reduced or eliminated about 1 day, about 2 days, about 3 days, or about 4 days after differentiation of human pluripotent cells into definitive endoderm cells.
It can be reduced or eliminated for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days or for more than about 10 days.

In some embodiments, differentiation of definitive endoderm cells to foregut endoderm cells is enhanced by providing an FGF-family growth factor and/or a Hedgehog pathway inhibitor to the definitive endoderm cell culture or cell population. In such embodiments, the FGF-family growth factor and/or Hedgehog pathway inhibitor is administered about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days after reducing or eliminating TGFβ superfamily growth factor signaling in the definitive endoderm cell culture.
about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more than about 10 days later. In preferred embodiments, the FGF family growth factor and/or Hedgehog pathway inhibitor is provided at about the same time as the reduction or elimination of TGFβ superfamily growth factor signaling in the definitive endoderm cell culture.

In a preferred embodiment, the FGF family growth factor provided to the definitive endoderm cell culture or cell population is FGF10 and/or FGF7. However, it is understood that other FGF family growth factors or FGF family growth factor analogs or mimetics can be provided in place of or in addition to FGF10 and/or FGF7. For example, an FGF family growth factor selected from the group consisting of FGF1, FGF2, FGF3, and up to FGF23 can be provided. In such embodiments, the FGF family growth factor and/or FGF family growth factor analogs or mimetics are provided at a concentration of at least about 10 ng/ml, at least about 25 ng/ml, at least about 50 ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at least about 200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, at least about 500 ng/ml, at least about 600 ng/ml, at least about 750 ng/ml, at least about 800 ng/ml, at least about 900 ng/ml, at least about 1000 ng/ml, at least about 250 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, at least about 5 ...500 ng/ml, at least about 1000 ng/ml, at least about 250 ng/ml, at least about 300 ng/ml, at least about 1000 ng/ml,
The antibody may be provided to the cells of the cell culture such that the antibody is present at a concentration of at least about 100 ng/ml, at least about 500 ng/ml, or at least about 1000 ng/ml.

In another preferred embodiment, the hedgehog inhibitor is KAAD-cyclopamine.
However, it is understood that other hedgehog inhibitors can be used, including, but not limited to, KAAD-cyclopamine analogs, jervine, jervine analogs, hedgehog pathway blocking antibodies, and any other inhibitors of hedgehog pathway function known to those of skill in the art. When used alone or in combination with an FGF family growth factor, the hedgehog inhibitor can be provided at a concentration of at least about 0.01 μM to 50 μM.

In a preferred process for generating a population of PDX1-negative foregut endoderm cells from definitive endoderm cells, TGFβ superfamily growth factor signaling is reduced or removed for about 2 days from the differentiation of a substantial portion of human pluripotent cells to definitive endoderm (e.g., after the 3, 4 or 5 day differentiation protocol described in the Examples below). Around the same time, cell cultures or cell populations of definitive endoderm cells are treated with noggin and KAAD-cyclopamine, e.g., 2 mM RA.
or 50 ng/ml Noggin and 0 ng/ml in DMEM medium in the presence of 3 nM TTNPB.
. Supply 25 μM KAAD-cyclopamine.

In some embodiments, PDX1-negative foregut endoderm cells can be further differentiated into PDX1-positive foregut endoderm cells by contacting or otherwise providing the cells with medium containing a retinoid, such as retinoic acid (RA). In some embodiments, the retinoid is provided to the cells of the cell culture such that it is present at a concentration of at least about 1 nM to 50 μM. In such embodiments, the retinoid is provided to the cells about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more than about 10 days after reducing or eliminating TGFβ superfamily growth factor signaling in the definitive endoderm cell culture. In preferred embodiments, the retinoid is provided to the cells about 2-3 days after reducing or eliminating TGFβ superfamily growth factor signaling at a concentration of about 0.05
From about 1 μM RA to about 2 μM RA is delivered to PDX1-negative foregut endoderm cell cultures.

In some of the differentiation processes described herein, the differentiation factors are added and then removed from the cell culture. For example, the differentiation factors are added about 1 day, about 2 days, about 3 days, or about 4 days after addition.
The tumor may be removed within about 1 day, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

In preferred embodiments, the PDX1-negative foregut endoderm cells generated and described herein have relatively high levels of SOX17, FOXA1, HNF1B and HNF4A gene expression when normalized to the levels of a control gene, such as a housekeeping gene. In particular, HNF4A gene expression levels do not increase substantially until removal of TGFβ superfamily growth factor signaling (e.g., activin) from, for example, the definitive endoderm cultures.

Generation and composition of PDX1-positive foregut endoderm (Stage 3)
PDX1-negative foregut endoderm cells can be further specified toward pancreatic differentiation by further differentiation of these cells to generate PDX1-positive foregut endoderm cells or PDX1-positive pancreatic endoderm or their equivalents. As part of the differentiation processes described herein, cell cultures and enriched or purified cell populations comprising definitive endoderm cells are enriched for PDX1.
The cells can be used for further differentiation into cell cultures and/or enriched cell populations containing positive foregut endoderm cells.

Typically, PDX1-negative foregut endoderm cells are differentiated into PDX1-positive foregut endoderm cells by providing a retinoid, such as retinoic acid (RA), to a cell culture comprising SOX17-positive foregut endoderm cells. In part of the differentiation process, the PDX1-negative foregut endoderm cells in culture are also provided with a member of the fibroblast growth factor family prior to or at about the same time as the addition of RA. A preferred fibroblast growth factor is FGF-7. In another preferred process, the fibroblast growth factor includes any fibroblast growth factor or ligand that stimulates or otherwise interacts with fibroblast growth factor 2 receptor IIIb (FGFR2(IIIb)). In a further preferred process, the FGF family growth factor is used in combination with a hedgehog pathway inhibitor. A preferred hedgehog pathway inhibitor is KAAD
In a particularly preferred differentiation process, cell cultures comprising PDX1-negative definitive endoderm cells in the presence of RA are provided with FGF-10 and/or KAAD-cyclopamine. In certain processes, BMP4 may be included with FGF10 and/or KAAD-cyclopamine in the presence of RA. In some processes, the retinoid is a member of the ΤGFβ superfamily of growth factors and/or Connaught.
Medical Research Labs medium (CRML medium) (In vitro
gen, Carlsbad, Calif.).

For some of the embodiments of the differentiation process described herein, retinoids and/or combinations of the aforementioned differentiation factors may be used to promote differentiation of PDX1-positive foregut endoderm cells by inhibiting the differentiation of PDX1-positive foregut endoderm cells.
The antibody or antibody fragments are provided to the cells such that they are present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the cell culture or cell population.

In other processes, the FGF-10 is at least about 1 ng/ml, at least about 2 ng/ml
/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 25
The fibroblast growth factor or ligand that stimulates or otherwise interacts with fibroblast growth factor 2 receptor IIIb (FGFR2(IIIb)) is provided to cells in cell culture, either alone or in combination with a Hedgehog pathway inhibitor, so as to be present at a concentration of up to 50 μM.

In a preferred process for generating a population of PDX1-positive foregut endoderm cells from PDX1-negative foregut endoderm cells, a cell culture or enriched cell population of PDX1-negative foregut endoderm cells is treated with FGF-7, KAAD-cyclopamine and retinoic acid (RA), e.g., 2μ
25 ng/ml FGF-7 and 0.2 μM KA in CMRL medium in the presence of 1 μM RA
AD-cyclopamine is provided.

In some processes described herein, TGFβ signaling factors, such as activin A and/or activin B, GDF-8, GDF-11, etc., are provided to the cell culture together with a retinoid and/or fibroblast growth factor and a hedgehog inhibitor. For example, in such processes, activin A and/or activin B and/or GDF-8 and/or GDF-11 are provided at least about 5 ng/ml, at least about 10 ng/ml, at least about 25 ng/ml, at least about 50 ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at least about 200 ng/ml,
, at least about 300 ng/ml, at least about 400 ng/ml, at least about 500
The antibody is provided to the cell culture at a concentration of about 1000 ng/ml or at least about 1000 ng/ml.

In some processes, the differentiation factors and/or CRML medium are provided to the PDX1-negative foregut endoderm cells about 1 day, 2 days, 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more than about 10 days after initiation of differentiation from pluripotent cells. In preferred processes, the differentiation factors and/or CRML medium are provided to the PDX1-negative foregut endoderm cells about 3 days, or 4 days, or 5 days after initiation of differentiation from pluripotent stem cells.

In certain processes described herein, the differentiation factors are added and then removed from the cell culture. For example, the differentiation factors are added about 1 day, about 2 days, about 3 days, or about 4 days after the addition.
The tumor may be removed within about 1 day, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

These and other methods are described in U.S. Provisional Patent Application No. 60/7, filed October 27, 2005.
No. 30,917, entitled PDX1-EXPRESSING DORSAL AND VENUE
PDX1-positive foregut endoderm cells are described in detail in TRAL FOREGUT ENDODERM, and PDX1-positive foregut endoderm cells can be found in U.S. patent application Ser. No. 11/115,868, filed Apr. 26, 2005, entitled PDX1 EXPRESSING ENDODERM, the disclosure of which is incorporated herein by reference in its entirety.

In a preferred embodiment, when normalized and compared to the levels of a control gene, such as a housekeeping gene, the PDX1 positive foregut endoderm cells generated and described herein exhibit:
have relatively high levels of PDX1, NKX6.1, PTF1A, CPA and cMYC gene expression.

Generation and Composition of PDX1-Positive Pancreatic Endoderm (Stage 4)
The generation and composition of PDX1-positive pancreatic endoderm or "pancreatic epithelium" or more generally pancreatic precursors are described in U.S. patent application Ser. No. 12/167,227, filed Jul. 2, 2008, entitled "PDX1-Positive Pancreatic Endoderm and Pancreatic Epithelium," and more generally, in U.S. Pat. No. 6,313,363, entitled "PDX1-Positive Pancreatic Endoderm and Pancreatic Epithelium and Pancreatic Progenitors."
The methods and methods of producing PANCREATIC HORMONES are described in detail in U.S. Patent Application No. 12/167,227, which was filed on July 5, 2007, and is incorporated herein by reference in its entirety.
CREATIC HORMONES, which is a continuation of U.S. patent application Ser. No. 11/681,687, filed Mar. 2, 2007, entitled ENDOCRINE PRECURSO
R CELLS, PANCREATIC HORMONE-EXPRESSING CE
This application is a continuation-in-part of LLS AND METHODS OF PRODUCTION.
The disclosure of each of these applications is incorporated herein by reference in its entirety.

Example 22 of the 12/167,227 application details various modifications of the cell culture conditions described herein for stages 1-4. Generally, stage 3 PDX1-positive foregut endoderm type cells are cultured in medium containing DMEM in 1% v/v B27 supplement plus Noggin or another BMP4 specific inhibitor for about 1-3 days, or about 1-4 days, or about 1-5 days, or about 1-6 days. At the end of stage 4, PDX1-positive pancreatic endoderm is generated, the cells of which at least co-express PDX1 and Nkx6.1 and PTF1A. The cell cultures do not appreciably express cells indicative of stage 5 single or multiple hormonal endocrine cells, or stage 1 definitive endoderm cells, or stage 2 PDX1-negative foregut endoderm cells, or stage 3 PDX1-positive foregut endoderm cells.

Generation and composition of endocrine precursor cells (Stage 5)
Some embodiments described herein relate to methods of generating endocrine precursor cells starting from pluripotent cells. As described above, endocrine precursor cells can be generated by first differentiating pluripotent cells to generate definitive endoderm cells, and then further differentiating the definitive endoderm cells to generate PDX1-positive foregut endoderm cells. In such embodiments, PDX1 is
Positive foregut endoderm cells further differentiate into multipotent endocrine progenitor cells, which are capable of differentiating into human pancreatic islet hormone-expressing cells.

In one embodiment, PDX1-positive foregut endoderm cells are differentiated into endocrine precursor cells by continuing incubation of the PDX1-positive foregut endoderm cells in the presence of a retinoid, such as retinoic acid, for a time sufficient to generate endocrine precursor cells. In some embodiments, the time sufficient to generate endocrine precursor cells is from about 1 hour to about 10 days following expression of the PDX1 marker in a portion of the cells in the cell culture. In some embodiments, the retinoid is added to the cell culture for about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 16 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 12 hours, about 16 hours, about 1 day, about 2 days ...3 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 12 hours, about 16 hours, about 1 day, about 2 days, about 1 day, about 3 days, about 5 days, about 1 day, about 1 day, about 2 days, about 1 day, about 3 days, about 5 days, about 1 day, about 1 day
The effect is maintained for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more than about 10 days.

In some processes described herein, the concentration of retinoid used to differentiate PDX1-positive foregut endoderm cells in a cell culture or cell population into endocrine precursor cells is
From about 1 nM to about 100 μM.

In some preferred embodiments, the provision of a gamma secretase inhibitor to a cell culture or cell population comprising human PDX1-positive foregut endoderm cells mediates the differentiation of the PDX1-positive foregut endoderm cells into pancreatic endocrine progenitor cells. In one preferred embodiment, the gamma secretase inhibitor is N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S
-phenylglycine t-butyl ester (DAPT).

In other embodiments, the gamma secretase inhibitor is provided at the beginning of the differentiation process, for example at the pluripotent stage, and remains in the cell culture throughout differentiation into pancreatic islet hormone-expressing cells. In yet other embodiments, the gamma secretase inhibitor is added after the initiation of differentiation, but prior to differentiation into the PDX1-positive foregut endoderm stage. In a preferred embodiment,
The gamma secretase inhibitor is provided to the cell culture or cell population at the same time as the provision of a differentiation factor that promotes conversion of definitive endoderm to PDX1-positive endoderm. In other preferred embodiments, the gamma secretase inhibitor is provided to the cell culture or cell population after a substantial portion of the cells in the cell culture or cell population have differentiated into PDX1-positive foregut endoderm cells.

For some embodiments relating to differentiation of PDX1-positive foregut endoderm cells into endocrine precursor cells, the gamma secretase inhibitor is provided to the cells so that it is present in the cell culture or cell population at a concentration sufficient to promote the differentiation of at least some of the PDX1-positive cells into endocrine precursor cells.In some embodiments, the gamma secretase inhibitor is present in the cell culture or cell population at a concentration of about 0.01 μM to about 1000 μM.In preferred embodiments, the gamma secretase inhibitor is present in the cell culture or cell population at a concentration of about 0.1 μM to about 100 μM.

In certain embodiments of the processes for generating endocrine precursor cells described herein,
The gamma secretase inhibitor is provided after one or more previously provided differentiation factors are removed from the cell culture. For example, the previously provided one or more differentiation factors can be removed about 1 to 10 days, or about 10 days or more, prior to the addition of the gamma secretase inhibitor. In other embodiments, the gamma secretase inhibitor is provided after one or more previously provided or removed from the cell culture.
The cell culture or cell population is provided with one or more differentiation factors provided contemporaneously with the gamma secretase inhibitor. In a preferred embodiment, the differentiation factors provided prior to or contemporaneously with the gamma secretase inhibitor include FGF-10, KAAD-cyclopamine, activin A, activin B, GDF-8, GDF-11, BMP4 and/or FGF-10.
or RA.

In some embodiments of the invention described herein, exendin 4 is provided to the differentiating cell culture or cell population at about the same time as the gamma selenase inhibitor. In certain embodiments, exendin 4 is provided to the cell culture or cell population at a concentration of at least about 0.1
It is provided to be present at concentrations from ng/mL to 1000 ng/mL.

In a preferred process for the generation of endocrine precursor cells from PDX1-positive foregut endoderm cells, cell cultures or cell populations of PDX1-positive foregut endoderm cells are provided with 3 μM DAPT and 40 ng/ml exendin 4. In a particularly preferred embodiment, the cells are differentiated in CMRL. In another particularly preferred process, for the generation of endocrine precursor cells from PDX1-positive foregut endoderm cells, cell cultures or cell populations of PDX1-positive foregut endoderm cells are provided with 3 μM DAPT and 40 ng/ml exendin 4 in the presence of 2 μM RA.

It will be appreciated that expression of the NGN3, NKX2.2 and/or PAX4 markers is induced in endocrine progenitor cells across a variety of different levels depending on the state of differentiation. Thus, in some embodiments described herein, expression of the NGN3, NKX2.2 and/or PAX4 markers in endocrine progenitor cells or cell populations is induced across a variety of different levels depending on the state of differentiation. As such, in some embodiments described herein, expression of the NGN3, NKX2.2 and/or PAX4 markers in endocrine progenitor cells or cell populations is induced across a variety of different levels depending on the state of differentiation. As such, in some embodiments described herein, expression of the NGN3, NKX2.2 and/or PAX4 markers in endocrine progenitor cells or cell populations is induced across a variety of different levels depending on the state of differentiation.
In other embodiments, the expression of the NGN3, NKX2.2 and/or PAX4 markers in the endocrine progenitor cells or cell population is at least about 4-fold, at least about 6-fold to at least about 10,000-fold greater than the expression of the NGN3, NKX2.2 and/or PAX4 markers in the non-endocrine progenitor cells or cell populations, e.g., pluripotent stem cells, definitive endoderm cells, PDX1 positive foregut endoderm cells, immature pancreatic islet hormone expressing cells, mature pancreatic islet hormone expressing cells, extraembryonic endoderm cells, mesoderm cells and/or ectoderm cells. In some embodiments, expression of NGN3, NKX2.2 and/or PAX4 markers in endocrine progenitor cells or cell populations indicates expression of NGN3, NKX2.2 and/or PAX4 markers in non-endocrine progenitor cells or cell populations, such as pluripotent cell-like iPS cells and hES cells, definitive endoderm cells, PDX1 positive foregut endoderm cells, immature pancreatic islet hormone expressing cells, mature pancreatic islet hormone expressing cells, extraembryonic endoderm cells, mesoderm cells and/or ectoderm cells.
3, much higher than the expression of NKX2.2 and/or PAX4 markers.

Another embodiment of the invention is a composition, such as a cell culture or cell population, comprising human cells, such as human endocrine precursor cells, wherein at least about 2% of the human cells have expression of the NGN3 markers AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, or the like.
In another embodiment, the expression of the NGN3 marker is greater than the expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, INS, GCG, SST, GHRL, and/or PAX6 markers in at least about 5%-98% of the human cells.
In some embodiments, the expression of NGN3 in the cell culture or cell population is greater than the expression of SYP, CHGA, INS, GCG, SST, GHRL, and/or PAX6 markers.
The expression of AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, and CHGA
The percentage of human cells with greater than expression of the INS, GCG, SST, GHRL, and/or PAX6 markers is calculated without taking into account feeder cells.

Some embodiments of the invention provide a composition, such as a cell culture or cell population, comprising human endocrine precursor cells, wherein expression of NKX2.2 and/or PAX4 is at least about 2% to at least about 98% of the human cells, and expression of AFP, SOX7, SOX1, ZIC1,
It will be appreciated that the present invention relates to compositions in which the expression of NKX2.2 and/or PAX4 is greater than the expression of AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL, and/or PAX6 markers in at least about 5% of human cells to 98% of human cells.
In some embodiments, the expression of NKX, SYP, CHGA, INS, GCG, SST, GHRL, and/or PAX6 markers is greater than that of NKX, SYP, CHGA, INS, GCG, SST, GHRL, and/or PAX6 markers in the cell culture or cell population.
2.2 and/or PAX4 expression is AFP, SOX7, SOX1, ZIC1, NFM
, MAFA, SYP, CHGA, INS, GCG, SST, GHRL, and/or P
The percentage of human cells with greater expression of the AX6 marker is calculated without taking into account the feeder cells.

Additional embodiments of the invention relate to compositions, e.g., cell cultures or cell populations, comprising mammalian cells differentiated in vitro from definitive endoderm, e.g., human cells differentiated in vitro from definitive endoderm, wherein expression of the NGN3, NKX2.2 and/or PAX4 markers is at least about 2% of the cells differentiated in vitro from definitive endoderm.
FP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS,
In another embodiment, at least about 5% of cells differentiated in vitro from definitive endoderm have greater than or equal to the expression of GCG, SST, GHRL and/or PAX6 markers.
In 98% of cells differentiated from definitive endoderm in vitro, expression of NGN3, NKX2.2 and/or PAX4 markers was significantly higher than that of AFP, SOX7, SOX1, ZIC1, NFM,
MAFA, SYP, CHGA, INS, GCG, SST, GHRL and/or PAX
Greater than expression of 6 markers.

The processes described herein can be used to generate compositions comprising endocrine precursor cells that are substantially free of other cell types. In some embodiments of the invention, the endocrine precursor cell populations or cell cultures generated by the methods described herein are characterized by the expression of AFP, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX16, SOX17, SOX18, SOX19, SOX20, SOX21, SOX22, SOX30, SOX31, SOX40, SOX41, SOX50, SOX51, SOX60, SOX71, SOX82, SOX93, SOX104, SOX116, SOX126, SOX138, SOX140, SOX156, SOX160, SOX182, SOX194, SOX196, SOX198, SOX1
In some embodiments, the endocrine precursor cell population of the cell culture generated by the methods described herein is substantially free of cells that significantly express the markers AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, C
Substantially free of cells that significantly express HGA, INS, GCG, SST, GHRL, and/or PAX6 markers.

In one embodiment of the present invention, the description of endocrine precursor cells based on the expression of markers is
3. High NKX2.2, high PAX4, low AFP, low SOX7, low SOX1, low ZIC1, low NFM, low MAFA; low SYP; low CHGA; low INS, low GCG, low SST, low GHR
L and/or low PAX6.

[0023] The embodiments described herein relate to a method for generating immature pancreatic islet hormone-expressing cells starting from pluripotent cells. As described above, immature pancreatic islet hormone-expressing cells can be generated by first differentiating pluripotent cells to generate definitive endoderm cells, differentiating the definitive endoderm cells to generate foregut endoderm cells, differentiating the foregut endoderm to generate PDX1-positive foregut endoderm cells, and then differentiating the PDX1-positive foregut endoderm cells.
These can be generated by further differentiating positive foregut endoderm cells to generate endocrine precursor cells, in some embodiments, the process is continued by further differentiating the endocrine precursor cells into immature pancreatic islet hormone-expressing cells.

In some embodiments of the invention, the cells are substantially inhibited from expressing NGN3 and
Differentiation of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells is progressed by continuing incubation of the culture of endocrine precursor cells with the gamma secretase inhibitor for a time sufficient to initiate expression of X6 and such that the cells have the ability to express at least one pancreatic islet cell hormone. In some embodiments, the gamma secretase inhibitor is administered for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about
It is removed after about 9 days, about 10 days, or about 10 or more days. In one preferred embodiment, the gamma secretase inhibitor is N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT).

Certain processes for the generation of immature pancreatic islet hormone-expressing cells disclosed herein are mediated by the provision of one or more factors selected from the group consisting of nicotinamide, exendin 4, hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF1) to a cell culture or cell population comprising human endocrine precursor cells. In some embodiments, all four of the above factors are provided together. In some embodiments, one or more of the above factors are provided to the cell culture prior to differentiation of endocrine precursor cells and remain in the cell culture during differentiation of at least a portion of the cells in the cell culture into endocrine precursor cells. In other embodiments, one or more of the above factors are provided to the cell culture at or about the time of differentiation of a substantial portion of the cells into endocrine precursor cells and remain in the cell culture until at least a substantial portion of the cells have differentiated into immature pancreatic islet hormone-expressing cells. In some embodiments of the invention, one or more of the above factors are provided at the beginning of the differentiation process, for example at the pluripotent cell stage, and remain in the cell culture throughout differentiation into immature pancreatic islet hormone-expressing cells.

In some processes for generating immature pancreatic islet hormone-expressing cells disclosed herein, nicotinamide, nicotinamide adenine dinucleotide (NAD) or nicotinic acid is provided to cells so that it is present in the cell culture or cell population at a concentration sufficient to promote the differentiation of at least a portion of endocrine precursor cells into immature pancreatic islet hormone-expressing cells.In some embodiments, nicotinamide is present in the cell culture or cell population at a concentration of at least about 0.1 mM, at least about 0.5 mM to 1000 mM.

In another process for the generation of immature pancreatic islet hormone-expressing cells disclosed herein,
Exendin 4 is provided to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, Exendin 4 is present in the cell culture or cell population at a concentration of at least about 1 ng/ml, at least about 5 ng/ml to 1000 ng/ml.

In yet other processes for the production of immature pancreatic islet hormone-expressing cells disclosed herein, HGF is supplied to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, HGF is provided at a concentration of at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 40 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml, at least about 25 ...15 ng/ml, at least about 25
The antibody is present in the cell culture or cell population at a concentration of from 1000 ng/ml to 1000 ng/ml.

In yet other processes for the production of immature pancreatic islet hormone-expressing cells disclosed herein, IGF1 is provided to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, IGF1 is provided at a concentration of at least about 1 ng/ml to 1000 ng/ml.
The antibody is present in the cell culture or cell population at a concentration of 1000 ml.

In certain embodiments of the processes for generating immature pancreatic islet hormone-expressing cells described herein, one or more of nicotinamide, exendin 4, HGF, and IGF1 are provided after the previously provided one or more differentiation factors are removed from the cell culture. In other embodiments, nicotinamide, exendin 4, HGF, and IGF1 are provided after the previously provided one or more differentiation factors are removed from the cell culture.
One or more of nicotinamide, exendin 4, HGF, and IGF1 are provided to a cell culture or cell population that includes one or more differentiation factors provided prior to or at about the same time as one or more of nicotinamide, exendin 4, HGF, and IGF1. In a preferred embodiment, the differentiation factors provided prior to or at about the same time as one or more of nicotinamide, exendin 4, HGF, and IGF1 include, but are not limited to, DAPT, FGF-10, KAAD-cyclopamine, activin A, activin B, activin C, activin D, activin E, activin F, activin F, activin G, activin H, activin I ...
, activin B, BMP4 and/or RA.

Another embodiment of the invention is a composition, such as a cell culture or cell population, comprising human cells, such as human immature pancreatic islet hormone-expressing cells, wherein at least about 2% of the human cells are MAb-expressing.
FB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX1
, HB9, GHRL, IAPP, INS, GCG, SST, PP, and/or C-peptide marker expression is NGN3, MAFA, MOX1, CER, POU5F1, AFP
In other embodiments, the expression of MAFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, P in at least about 5% of human cells, in at least about 10% to 95% of human cells, or in at least about 98% of human cells is greater than expression of MAFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, P
DX1, HB9, GHRL, IAPP, INS, GCG, SST, PP, and/or C-peptide marker expression is higher than NGN3, MAFA, MOX1, CER, POU5F1,
greater than expression of AFP, SOX7, SOX1, ZIC1 and/or NFM markers.

In some embodiments, MAFB, SYP, CHGA, NKX2.2, ISL1, PAX
6, NEUROD, PDX1, HB9, GHRL, IAPP, INS GCG, SST,
PP and/or C-peptide expression is associated with NGN3, MAFA, MOX1, CER, and POU
If the expression of 5F1, AFP, SOX7, SOX1, ZIC1 and/or NFM markers is greater than or equal to the percentage of human cells in the cell culture or population, then the percentage of human cells in the cell culture or population is calculated regardless of the feeder cells.

In certain embodiments of the invention, the cell cultures and/or cell populations comprising immature pancreatic islet hormone-expressing cells also comprise medium comprising one or more secreted hormones selected from ghrelin, insulin, somatostatin and/or glucagon. In other embodiments, the medium comprises C-peptide. In preferred embodiments, the concentration of the one or more secreted hormones or C-peptide in the medium ranges from at least about 1 pmol of ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of intracellular DNA to at least about 1000 picomoles (pmol) of ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of intracellular DNA.

Methods for Producing Insulin In Vivo In some embodiments, the in vitro derived pancreatic progenitor cells or PDX described above are
The -1 positive pancreatic endoderm cells or their equivalent are then transplanted into a mammalian subject. These methods are described in International Application No. PCT/US2007/015536, entitled "METHODS OF TRANSPLANTATION
RODUCING PANCREATIC HORMONES, which is incorporated herein by reference in its entirety. In a preferred embodiment, the mammalian subject is a human subject. Particularly preferred subjects are those identified as having a condition that limits the subject's ability to produce sufficient levels of insulin in response to physiologically high blood glucose concentrations. The range of blood glucose levels that constitute physiologically high blood glucose levels for any particular mammalian species can be readily determined by one of skill in the art. Any sustained blood glucose level that results in a recognized disease or condition is considered a physiologically high blood glucose level.

Further embodiments of the present invention relate to the production of in vitro pluripotent stem cells or their progeny, such as PDX-1 positive foregut endoderm cells, PDX-1 positive pancreatic endoderm or pancreatic progenitor cells, N
The present invention relates to in vivo insulin-secreting cells derived from multipotent cells, such as endocrine precursors, such as GN3-positive endocrine precursors, or functionally differentiated hormone-secreting cells, such as insulin-secreting cells, glucagon-secreting cells, somatostatin-secreting cells, ghrelin-secreting cells, or pancreatic polypeptide-secreting cells. Any of the above terminally differentiated or multipotent cells can be transplanted into a host, or mammal, and matured into a physiologically functional hormone-secreting cell, such as a cell that secretes insulin in response to the host blood glucose level. In preferred embodiments, the cells do not form teratomas in vivo, and if so formed, they remain localized in the area of transplantation and can be easily excised or removed. In particularly preferred embodiments, the cells are capable of differentiation during the differentiation process in vitro, or when transplanted in vivo into a mammal, or when transplanted in vivo into a mammal.
Upon maturation and development into functional islets in vivo, they do not contain any karyotypic abnormalities.

Additionally, embodiments described herein relate to engineered or genetically modified pluripotent cells, multipotent cells or differentiated cells derived based on the description provided herein from pluripotent cells, such as human iPS cells, where the iPS cells are derived from hE
Having demonstrated similar physiological functions and gene marker expression profiles as S cells and hES-derived cells, iPS cells are predicted to have similar physiological properties in vivo.

Methods of Encapsulating Pancreatic Progenitors In some embodiments, the pluripotent, multipotent and differentiated cell compositions described herein can be encapsulated in biological and/or non-biological mechanical devices, where the encapsulated device separates and/or segregates the cell composition from the host.

The method of encapsulation is described in U.S. Patent Application No. 61/114,857, filed November 14, 2008.
No., Title: ENCAPSULATION OF PANCREATIC PROGENIT
ORS DELIVERED FROM HES CELLS, and December 12, 2008
No. 61/121,086, filed on the same day, entitled ENCAPSULATION
OF PANCREATIC ENDODERM CELLS, which are incorporated herein by reference in their entirety, and semipermeable membranes, such as Theracyte™
Alternatively, encapsulation of pancreatic endoderm cells using a Gore device is described.

In one embodiment, the hES derived cells are cultured in a biocompatible polyethylene glycol (PEG
PEG-based encapsulation is described in U.S. Pat. No. 7,427,415.
No., Title: IMPLANTATION OF ENCAPSULATED BIOLOGY
CAL MATERIALS FOR TREATING DISEASES; U.S. Patent No. 6,911,227, entitled GELS FOR ENCAPSULATION OF
BIOLOGICAL MATERIALS; and U.S. Pat. No. 6,911,227,
Nos. 5,529,914, 5,801,033, and 6,258,870, entitled GE
LS FOR ENCAPSULATION OF BIOLOGICAL MATER
Further details are given in IALS, which are incorporated herein by reference in their entirety, describing conformal coating of cell aggregates using polyethylene glycol.

In one embodiment, the encapsulation device contains pluripotent derived cells, e.g., PDX-1 positive foregut endoderm cells, PDX-1 positive pancreatic endoderm or progenitor cells, endocrine precursors such as NGN3 positive endocrine precursors, or functional differentiated hormone secreting cells such as insulin-secreting cells, glucagon-secreting cells, somatostatin-secreting cells, ghrelin-secreting cells, or pancreatic polypeptide-secreting cells, within a semi-permeable membrane that prevents the transplanted cell population from passing through and retains them within the device, while allowing the passage of certain secreted polypeptides, e.g., insulin, glucagon, somatostatin, ghrelin, pancreatic polypeptide, etc. Alternatively, the device has multiple membranes, such as a vascularized membrane.

Use of agents to enhance and promote the growth, survival, proliferation and cell-cell adhesion of human pluripotent stem cells, e.g., hES cells and iPS cells.
It can modulate biochemical pathways within cells. Protein phosphorylation represents a process by which intracellular signals are propagated from molecule to molecule, ultimately resulting in a cellular response. These signal transduction cascades are highly regulated and often overlap, as evidenced by the existence of many protein kinases and phosphatases. In humans, protein tyrosine kinases have been reported to have a key role in the progression of many pathologies, including diabetes, cancer, and are also associated with a wide variety of congenital syndromes. Serine-threonine kinases, such as Rho kinase, are a class of enzymes that, when inhibited, may be associated with the treatment of human diseases, including diabetes, cancer, and various inflammatory cardiovascular disorders and AIDS. The majority of inhibitors identified to date act at the ATP binding site. Such ATP-competitive inhibitors have demonstrated selectivity due to their ability to target less conserved regions of the ATP binding site.

The Rho kinase family of small GTP-binding proteins includes RhoA-E and Rho
RhoA kinase inhibitors include at least 10 members, including hoG, Rac1 and Rac2, Cdc42, and TC10. Inhibitors are often referred to as ROK inhibitors or ROCK inhibitors or Rho kinase inhibitors, which are used interchangeably herein.
The effector domains of Rho, RhoB, and RhoC have the same amino acid sequence and appear to have similar intracellular targets. Rho kinase operates as the first downstream mediator of Rho and exists as two isoforms: α (ROCK2) and β (ROCK1). Rho kinase family proteins have a catalytic (kinase) domain in their N-terminal domain, a coiled-coil domain in their central part, and a putative pleckstrin homology (PH) domain in their C-terminal region. The Rho-binding domain of ROCK is located in the C-terminal part of the coiled-coil domain and binds Rho in the GTP-bound form.
Binding to ho results in enhanced kinase activity. Rho/Rho kinase-mediated pathways play an important role in signal transduction initiated by many agonists, such as angiotensin II, serotonin, thrombin, endothelin-1, norepinephrine, platelet-derived growth factor, ATP/ADP and extracellular nucleotides, and urotensin II. Through modulation of its target effector/substrates, Rho kinase plays an important role in various cellular functions, such as smooth muscle contraction, actin cytoskeleton organization, cell adhesion and motility, and gene expression. Due to the role played by Rho kinase proteins in mediating several cellular functions recognized to be associated with the pathogenesis of arteriosclerosis, inhibitors of these kinases may also be useful for treating or preventing various arteriosclerotic cardiovascular diseases, and are involved in endothelial contraction.

In some embodiments, including but not limited to, the Rho kinase inhibitor Y-2763
2. Agents that promote and/or support cell growth, survival, proliferation and cell-cell adhesion are added to various cell culture medium conditions, such as fasudil (also called HA1077), H-1152P and ITS (insulin/transferrin/selenium; Gibco), Wf-536, Y-30141 (described in U.S. Pat. No. 5,478,838, which is fully incorporated herein by reference) and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), competitive peptides, antagonist peptides, inhibitory antibodies, antibody-scFV fragments, dominant negative variants and expression vectors thereof. In addition, other small molecule compounds are known as ROCK inhibitors, and such compounds or derivatives thereof can also be used in the present invention (e.g., U.S. Pat. App. No. 2005020, which is fully incorporated herein by reference).
No. 9261, No. 20050192304, No. 20040014755, No. 20
Nos. 20040002508, 20040002507, 20030125344 and 20030087919, and International Patent Publication Nos. 2003/062227, 2003/059913, 2003/062225, 2002/07
6976 and 2004/039796).

Combinations of one or two or more ROCK inhibitors can also be used in the present invention. These agents function, in part, by promoting the reassociation of dissociated hES cells, iPS or differentiated cell cultures, such as definitive endoderm, foregut endoderm, pancreatic endoderm, pancreatic epithelium, pancreatic progenitor populations, endocrine progenitors and populations. Similarly,
The agents may also function when cell dissociation is not performed. Increased growth, survival, proliferation and cell-cell adhesion of human pluripotent stem cells has been demonstrated whether the cells are generated from cell aggregates in suspension or in adherent plate cultures (with or without components of the extracellular matrix,
This was achieved independently of whether they were generated from cultured cells (with or without serum, with or without fibroblast feeders, with or without FGF, with or without activin). The increased viability of these cell populations facilitates and improves purification systems using cell sorters, thus allowing improved recovery of cells.
The use of Rho kinase inhibitors such as Y27632 may also allow the amplification of differentiated cell types by promoting their survival during stepwise passaging of dissociated single cells or from cryopreservation. Rho kinase inhibitors such as Y27632 have been shown to inhibit the proliferation of human pluripotent stem cells (e.g., hES cells)
Although Rho kinase inhibitors have been tested on pluripotent stem cells (e.g., pluripotent stem cells and iPS cells) and the cells into which they have been differentiated, Rho kinase inhibitors can be generally applied to other cell types, for example, epithelial cells, including but not limited to, intestinal, lung, thymus, kidney, and nervous system cell types as well as retinal pigment epithelium.

The concentration of the ROCK inhibitor in the cell culture medium is such that the desired effect is achieved.
For example, as long as the enhancement, increase, and/or promotion of cell growth, survival, proliferation, and cell-cell adhesion is realized, the present invention is not limited to those described in the following examples.
It will be appreciated by those skilled in the art that inhibitors may need to be optimized under different conditions. For example, when using Y-27632, the preferred concentration is about 0.01 to about 1000.
μM, more preferably from about 0.1 to about 100 μM, and even more preferably from about 1.0 to about 50 μM.
M, most preferably in the range of about 5-20 μM. When fasudil/HA1077 is used, it can be used at about 2 or 3 times the Y-27632 concentration mentioned above.
When using 1152, the amount is approximately a fraction of the amount of the Y-27632 concentration mentioned above, e.g., about 1
The concentration of ROCK inhibitor used will depend, in part, on the biological activity and potency of the inhibitor and the conditions under which it is used.

The time and duration of treatment with a ROCK inhibitor may or may not be limited depending on the desired effect, such as enhancing, increasing, and/or promoting growth, survival, proliferation (cell population) and cell-cell adhesion. However, the addition of a ROCK inhibitor can also affect differentiation in a surprising manner, as better described in Example 7. The following examples include a 12-well platelet count of approximately 12% of cells.
Human pluripotent stem cell cultures and/or differentiated cell cultures that have been treated for 1 hour, 24 hours, 48 hours, or more are described.

The density of human pluripotent stem cell cultures treated with ROCK inhibitors also increased, as
There is no limit to the density as long as the desired effects, such as enhanced, increased, and/or promoted cell growth, survival, proliferation, and cell-cell adhesion, are achieved. The cell density of the seeded cells can be adjusted depending on a variety of factors, including, but not limited to, the use of adherent or suspension cultures, the particular recipe of the cell culture medium used, the growth conditions, and the intended use of the cultured cells. Examples of cell culture densities include, but are not limited to, 1000 cells/ml
0.01 x ¹⁰ cells per ml, 0.05 x ¹⁰ cells per ml, 0.1 x ¹⁰ cells per ml, 0.5 x ¹⁰ cells per ml, 1.0 x 10 cells per ml
0 ⁵ cells per ml, 1.2 x 10 ⁵ cells per ml, 1.4 x 10 ⁵ cells per ml,
1.6 x ¹⁰ cells per liter, 1.8 x ¹⁰ cells per ml, 2.0 x ¹⁰ cells per ^ml , 3.0 x ¹⁰ cells per ml, 4.0 x 10 cells per ml
cells per ml, 5.0 x ¹⁰ cells per ml, 6.0 x ¹⁰ cells per ml, 7.0 x ¹⁰ cells per ml, 8.0 x ¹⁰ cells per ml, 9.0 x 10 cells per ml.
0×10 ⁵ cells per ml, or 10.0×10 ⁵ cells per ml, or more, e.g.
Up to ^5x107 cells per mL, or more, or any value in between, were cultured with good cell growth, survival, proliferation and cell-cell adhesion.

Uses of Agents That Activate TGFβ Receptor Family Members In yet another embodiment, agents that activate TGFβ receptor family members include members of the TGFβ superfamily of growth factors, as described herein. As used herein, "TGFβ superfamily members" or equivalents include TGFβ1, TGFβ2, TF-β3, GDF-15, GDF-9, BMP-1,
5, BMP-16, BMP-3, GDF-10, BMP-9, BMP-10, GDF-6
, GDF-5, GDF-7, BMP-5, BMP-6, BMP-7, BMP-8, BMP
-2, BMP-4, GDF-3, GDF-1, GDF11, GDF8, activin βC,
βE, βA and βB, BMP-14, GDF-14, MIS, inhibin alpha, L
It refers to more than 30 structurally related proteins, including subfamilies such as efty1, Lefty2, GDNF, Neurturin, Persephin and Artemin. Chang et al. (2002) Endocrine Rev. 23(6
): 787-823.

TGFβ family members can be replaced by or used in conjunction with TGFβ signaling pathway activators, which are compounds that stimulate one or more of the polypeptides or interactions involved in transducing or otherwise causing changes in cellular properties in response to TGFβ family members. The TGFβ signaling pathway includes the TGFβ family members themselves. TGFβ superfamily members transmit signals to the nucleus by signaling through type II and type I serine-threonine kinase receptors and intracellular effectors known as Smads. These receptors fall into two subfamilies known as type I and type II receptors that act cooperatively to bind ligands and transmit signals (Attisano et al., Mol Cel
l Biol 16(3), 1066-1073 (1996)). Ligands bind to the cell surface of type I and type II receptors and promote activation of type I receptors by phosphorylation.
This activated complex in turn activates intracellular Smads, which assemble into multisubunit complexes that regulate transcription. Members of the TGF-beta superfamily are divided into two signaling subgroups: those functionally related to TGF-β/activin, and those functionally related to BMPs.
/GDF subfamily. Most TGFβ ligands are believed to first bind to type II receptors, and then this ligand/type II receptor complex recruits or phosphorylates type I receptors (Mathews, L S, Endocrinol R
ev 15:310-325 (1994); Massague, Nature Rev:
Mol Cell Biol. 1, 169-178 (2000)). Type II receptor kinases phosphorylate and activate type I receptor kinases, which then phosphorylate and activate Smad proteins. TGFβ/activin ligands bind to TGFβ and activin type II receptors, and through this activin-type pathway, Smad-2, Sma
d-3, Nodal and Lefty signaling.
F ligand binds to BMP type II receptors and mediates the expression of Smad1, Smad5, and Smad
8 can be activated. Derynck, R. et al., Cell 95, 737-740
(1998). Upon ligand stimulation, Smads translocate into the nucleus and function as components of transcription complexes.

TGFβ signaling is positively and negatively regulated through various mechanisms. In positive regulation, the signal is amplified to a sufficient level for biological activity. TGFβ superfamily ligands bind to type II receptors, which recruit and phosphorylate type I receptors. Type I receptors then mediate the expression of receptor-regulated SMADs (R-SMADs,
R-SMAD/coSMAD complexes accumulate in the nucleus where they function as transcription factors and are involved in the regulation of target gene expression. For example, growth and differentiation factors 1, 2, and 3 are involved in the regulation of target gene expression.
3, 5, 6, 7, 8, 9, 10, 11, and 15. In a preferred embodiment, GDF8 and GDF11 are TGFβ family members, as well as TG
It also binds to the extracellular ligand-binding domain of the ActRII receptor and then forms a complex with ActRI, thereby activating the Sβ signaling pathway (positive regulation).
It acts by inducing the inhibition of mad7 negative regulators and phosphorylation of the Smad2/Smad3 complex, which associates with Smad4 to regulate the expression of specific genes.

Negative regulation of TGFβ signaling occurs at the extracellular, membrane, cytoplasmic and nuclear levels.
To suppress signal transduction initiated by GFβ, signal transduction can be disrupted. This can be accomplished by any means known in the art that antagonizes or prevents the interaction between TGFβ receptor(s) and SMAD proteins, such as proteins that block or compete with TGFβ receptor and SMAD protein interactions. Alternatively, the transcription or translation of TGFβ receptor or SMAD proteins can be altered by any means known in the art to block signal transduction along the signal transduction pathway. The positive and negative regulation of various TGFβ member proteins is further illustrated by a more detailed description of some of the factors below.

As with the use of any agent, the concentration of any TGFβ superfamily member in the cell culture medium is not limited to those described in the Examples below, so long as the concentration is capable of achieving the desired effect, such as activating a TGFβ receptor family member. For example, when using activins, such as activin A and/or activin B, or GDF8 and GDF-11, preferred concentrations are about 1:1 or about 1:1.
0 to about 300 nM, more preferably about 50 to about 200 nM, even more preferably about 75 to about
The concentration may be in the range of about 150 nM, and most preferably about 100-125 nM. One of skill in the art can readily test any concentration and determine the effectiveness of such concentrations using standard techniques in the art, for example, by assaying for genetic markers (genomic DNA) for any cell type.
Differentiation can be assessed by determining expression and non-expression of the ne maker panel.

These and other growth factors used to differentiate human pluripotent stem cells have been
No. 12/132,437, filed Jun. 3, 2008, entitled GROWTH FA
CTORS FOR PRODUCTION OF DEFINITIVE ENDOD
ERM, which is incorporated herein by reference in its entirety.

Having generally described the invention, a further understanding can be obtained by reference to certain specific examples provided herein, which are intended to be illustrative and not limiting.

It should also be understood that the above relates to preferred embodiments of the present invention, and that many modifications can be made thereto without departing from the scope of the present invention. The present invention is further illustrated in the following examples, which should not be construed as limiting its scope in any way. On the contrary, it should be clearly understood that various other embodiments, modifications and equivalents thereof can be used, which may occur to those skilled in the art after reading the description herein, without departing from the spirit of the present invention and/or the scope of the appended claims.

Example 1
Differentiation of human iPS cells into pancreatic precursors and endocrine cells via definitive endoderm and endoderm intermediates Human induced pluripotent stem (iPS) cells were differentiated in suspension aggregates using a four (4) stage procedure over approximately two weeks (or 14 days) to generate populations of pancreatic cell types, such as pancreatic precursors, endocrine precursors and hormone-expressing endocrine cells. The human iPS cell line used herein was obtained from S. Yamanaka, Kyoto University, Japan and Cellular Dynamism Center, Tokyo, Japan.
The data was provided by CDI.

The iPS cells described herein were provided initially by Shinya Yamanaka and subsequently by CDI. Undifferentiated iPS cells were grown on mitotically inactivated mouse embryonic fibroblasts or preferably feeder-free (no fibroblast feeder cell layer) in DMEM/F12 containing 20% knockout serum replacement. Differentiation was initiated by dissociating undifferentiated iPS cells into single cells using Accutase and cell samples were taken for RNA isolation and analysis. Cells were incubated with 1:5000 dilution of insulin-transferrin-selenium (ITS), activin A (100 ng/mL), wnt
The cells were resuspended at 1-2 million cells per milliliter in RPMI + 0.2% v/v FBS containing 3a (50 ng/mL) and rho kinase or ROCK inhibitor, Y-27632 at 10 μM, placed in ultra-low attachment 6-well plates placed on a rotating platform and spun at approximately 100 rpm. Cultures were spun at 100 rpm with daily medium changes for the remainder of the differentiation process. Growth, passaging and expansion of iPSCs were performed substantially as described in U.S. Pat. Nos. 7,961,402 and 8,211,699.
No. 6,313,633, the disclosure of which is incorporated herein by reference in its entirety.

The methods described herein for producing aggregated suspension cultures of pluripotent cells, e.g., hES or iPS cells, and cells derived from pluripotent cells, are substantially similar to those described in US Pat.
The title of the application filed on the 3rd is Compositions and Methods for
Culturing Differential Cells PCT/US200
No. 7/062755, filed on October 20, 2008, entitled Methods
and compositions for feeder-free pluripo
tent stem cell media containing human se
rum, PCT/US2008/080516, which is incorporated herein by reference in its entirety.

The methods described herein can be facilitated by first coating the culture vessel with an extracellular matrix, for example, as described in U.S. Patent No. 6,800,480 to Bodnar et al., assigned to Geron Corporation. As with other methods for culturing other pluripotent stem cells, e.g., hES and iPS cells, this method is substantially similar to that described in U.S. Patent No. 6,800,480 to Bodnar et al., filed on Oct. 20, 2008, entitled "Methods and Methods for Producing Cells and Cell Cultures," which is incorporated herein by reference in its entirety.
positions for feeder-free pluripotent st
em cell media containing human serum,
No. 60/339,933, filed on Oct. 13, 2008. No. 60/339,935, filed on Oct. 13, 2008.
16, the cells can be cultured using soluble human serum.

The methods described herein are based on the Wisconsin Alumni Research
This can be facilitated by exogenously added fibroblast growth factors (FGFs) provided from sources other than solely a fibroblast feeder layer, as described in U.S. Patent No. 7,005,252 to Thomson, J., assigned to the World Aids Research Foundation (WARF), which is incorporated herein by reference in its entirety.

During the first approximately 24 hours of rotation, the single cells that were attached to each other formed cell aggregates, and the RNA
Sufficient cell samples were obtained for isolation and analysis of iPS cells. The cell aggregates were approximately 60 to 120 microns in diameter. The iPS cell samples were placed on a rotating platform for approximately 1 day (
or 24 hours later, the cultures were treated with 1:5000 dilution of ITS, activin A (100-2
RPMI+ containing 0.000 ng/mL), and Wnt3a (50-100 ng/mL)
0.2% v/v FBS for about 1 day (day 0 to day 1) and for an additional day;
or received the same medium (days 1 to 2) but without Wnt3a. Daily cell samples were taken for RNA isolation and analysis. After 2 days of differentiation, cultures were diluted with 1:10
00 dilution of ITS, KGF (or FGF7, 25 ng/mL) and TGFβ inhibitor
RPMI + 0.2% v/v FBS containing o.4 (2.5 μM) for 1 day (or 24 h, days 2 to 3). For the next 2 days (days 3 to 5), TGFβ
The iPS cell aggregate suspension was fed with the same growth factor cocktail medium except that the inhibitor was omitted from the medium. Again, at the end of this stage (stage 2 or day 5), cell samples were taken for RNA isolation. For stage 3 (days 5 to 8), TTNPB[4-[
E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid] (3 nM), KAAD-cyclopamine (0.25
The cell culture medium was replaced with DMEM + 1% v/v B27 supplement containing Noggin (50 ng/mL), KGF (50 ng/mL) and Noggin (50 ng/mL). Again, at the end of this stage (stage 3 or day 8), cell samples were taken for RNA isolation and analysis. For stage 4 (days 8 to 14), Noggin (50 ng/mL), KGF (50 ng/mL) and EGF (
The medium was replaced with DMEM + 1% v/v B27 supplement containing 50 ng/mL of ribosomal RNA (50 ng/mL). Again, at the end of stage 4 (or day 14), cell samples were taken for RNA isolation and analysis.

Real-time PCR was performed to measure the gene expression of various marker genes during differentiation. Gene expression of specific markers or genes was first normalized to the average expression levels of housekeeping genes, cyclophilin G and TATA binding protein (TBP) expression. The normalized relative gene expression was then displayed in a bar graph against the expression levels in undifferentiated iPS cells, thus representing the fold upregulation of various differentiation markers. OCT
For 4, gene expression was normalized to set the lowest sample in the data set (day 14) and therefore represents fold downregulation during differentiation.

2A-L show the expression levels of identified genes (e.g., Oct4, Brachyury, Cer1, GSC, FOXA2, FO) compared to the expression levels of the same genes in undifferentiated iPS cells.
1 is a bar graph showing the relative gene expression of OC4+, OC5+, OC6+, OC7+, OC8+, OC9+, OC10+, OC11+, OC12+, OC13+, OC14+, OC15+, OC16+, OC17+, OC18+, OC19+, OC20+, OC21+, OC22+, OC23+, OC24+, OC25+, OC30+, OC31+, OC32+, OC33+, OC45+, OC55+, OC6+, OC6+, OC7+, OC8+, OC9+, OC11+, OC12+, OC15+, OC16+, OC25+, OC26+, OC31+, OC4+, OC5+, OC27+, OC28+, OC31+, OC32+, OC4+, OC5+, OC27+, OC28 ...29+, OC31+, OC4+, OC5+, OC5+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+, OC2+,
For T4 (FIG. 2A), gene expression was normalized and the sample with the lowest level of expression was set to 1 (day 14). Thus, as shown by FIG. 2A, the relative expression levels of OCT4 represent the fold downregulation (y-axis) during differentiation (x-axis, stages 0 to 4, or days 0 to 14).

As shown in Figure 2A, OCT4 (POU5F1) is expressed at high levels in undifferentiated iPS cells and then downregulated during differentiation (days 0 to 14). By day 14, OCT4 expression levels had decreased more than 3000-fold from those observed in undifferentiated cells. In contrast, the brachyury gene (
There was a transient upregulation of BRACHYURY (FIG. 2B) expression. The transient upregulation of brachyury was the result of directed differentiation of pluripotent/iPS cells to mesoendoderm by application of activin A and wnt3a.
Endodermal mesoderm was further differentiated into definitive endoderm during days 2 and 3 by continuous exposure to activin A, as indicated by upregulation of ER1, GSC and FOXA2 (Fig. 2C-E). During stage 2, definitive endoderm was further induced to differentiate into gut endoderm, as indicated by upregulation of FOXA1, maintenance of FOXA2 expression and downregulation of CER1 and GSC by day 6 of differentiation (Fig. 2C-F). At stage 3, definitive endoderm was further induced to differentiate into gut endoderm, as indicated by upregulation of HNF6 and PDX1 by day 8 (Fig. 2G-H).
During the first few days, exposure to the retinoids, cyclopamine and noggin, resulted in the intestinal endoderm forming the posterior foregut/
They were further induced to differentiate into PDX1-expressing endoderm.
As indicated by the upregulation of KX6-1, NGN3 and INS ( Fig. 2I-L ).
During stage 4, exposure to KGF and EGF resulted in the posterior foregut/PDX1-expressing endoderm being further induced to differentiate into pancreatic precursors, endocrine precursors and hormone-expressing endocrine cells.

Example 2
Rho Kinase Inhibitors Promote iPS Cell Growth, Survival, Proliferation and Cell-Cell Adhesion Methods for differentiating various hES and iPS cell lines are substantially as described herein and in Example 1. In addition to the culture conditions described for stages 1, 2, 3, 4 and 5, apoptosis inhibitors and/or Rho kinase or ROCK inhibitors were added to the medium to enhance and promote growth, survival, proliferation and cell-cell adhesion during differentiation. Typically, about 10 μM of a Rho kinase inhibitor, such as Y-27632, was added to the cell culture at each stage. Alternatively, at least stages 1 and 2, and stage 4 were added to the medium.
and 5, or any combination thereof, a Rho kinase inhibitor was added. The morphology and gene marker expression profile of differentiated iPS suspension (aggregate) cell cultures are substantially similar to that of suspension cell cultures derived from hES cells.

3 and 4 show immunocytochemistry (IC) of iPS cell cultures from stages 4 and 5, respectively.
FIG. 3 shows cell aggregates from stage 4 expressing typical gene markers characteristic of PDX1 positive pancreatic endoderm (also called pancreatic epithelium or pancreatic precursors), such as PDX1/NKX6.1 co-positive cells. Although not shown in FIG. 3, stage 4 cells do not express hormone secretory proteins or gene markers more characteristic of stage 5 cells, such as insulin (INS), glucagon (GCG), somatostatin (SST) and pancreatic polypeptide (PP). FIG. 4 shows cell aggregates of hormone expressing cells from stage 5. These ICC results were further confirmed using QPCR. However, QPCR was not performed.
Because PCR is a population study of the total levels of RNA expressed in a sample or cell culture, it cannot definitively show that any one cell expresses multiple markers.

Example 3
Encapsulation of iPS-derived pancreatic progenitors To date, methods for generating iPS cells and sources for generating iPS cells have been reported. However, there is no sufficient description of the differentiation of any iPS cells into any functional differentiated cells for potential use in cell therapy to treat certain diseases, such as diabetes.

To determine whether stage 4 PDX1 positive pancreatic endoderm or pancreatic progenitor cell cultures derived from human iPS cells are fully capable of developing and maturing in vivo into glucose sensitive insulin secreting cells, pancreatic progenitor populations substantially as described in Examples 1 and 2 were cultured in U.S. Patent Application Serial No. 12/618,659, filed November 13, 2009, entitled ENCAPSULATION OF HIGH-GROWTH PANCREATIC ENDODERM OR PANCREATIC PROGENITOR CELLS, which is incorporated by reference in its entirety.
PANCREATIC LINEAGE CELLS DERIVED FROM H
UMAN PLURIPOTENT STEM CELLS; and U.S. Pat.
Nos. 34,608 and 7,695,965, entitled METHODS OF PRODUCTION
The ^cells were then added to a macroencapsulation device substantially similar to that described in CING PANCREATIC HORMONES. Briefly, 5-10-
20 μL of gravity settled cell suspension aggregates were added to each device.

The encapsulated cells in the device are then prepared for implantation into a mammal, e.g., an immunodeficient SCID/Bg mouse, but can be implanted into larger animals, such as rats, pigs, monkeys, or human patients. The method and device for implanting encapsulated cells is essentially the same as that of GELFOA.
Pancreatic progenitor cells implanted in an M matrix and transplanted under the epididymal fat pad (EFP), as described in US patent application Ser. No. 12/618,659, US Pat. Nos. 7,534,608 and 7,695,965.

Although immunosuppression was not required in these studies, it may be required for certain mammals for an initial intermediate period until the precursors in the device are fully mature and glucose responsive. In some mammals, the immunosuppression regimen lasts for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 79, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 123, 134, 146, 150, 165, 170, 186, 197, 198, 199
It may be six weeks or more, depending on the mammal.

The transplanted cells were allowed to differentiate and further mature in vivo. To determine whether the transplanted cells have normal physiological function, e.g., as naturally occurring beta cells, the levels of human insulin are determined by examining the levels of human C-peptide.
The peptide is cleaved or processed from human proinsulin, and thus specific detection of human C-peptide, but not endogenous mouse C-peptide, indicates that insulin secretion is derived from the transplanted (exogenous) cells. The graft-bearing animals are administered human C-peptide every 2, 3, or 4 weeks by bolus injection of arginine or glucose, preferably glucose, into them.
The levels of the peptide are examined. The mature beta cells (derived from differentiated pluripotent iPS cells) should then be physiologically functional and glucose responsive, similar to naturally occurring or endogenous beta cells. Generally, an amount of human C-peptide above 50 pM or the average basal (threshold) level is an indication of the function of the now beta cells from the transplanted precursors.

Encapsulated pancreatic progenitors derived from hIPS cells are expected to mature into functional islet clusters with endocrine, acinar and ductal cells similar to those of naturally occurring islets, as described in Kroon et al. (2008), supra, U.S. Patent Application 12/618,659, U.S. Patent Nos. 7,534,608; 7,695,965 and 7,993,920, all of which are fully incorporated by reference herein. Purified or enriched pancreatic progenitors derived from hIPS cells prior to transplantation are also expected to mature and develop into functional islets, in
It is anticipated that the cells will produce insulin in vivo. Specific embodiments for purifying and enriching various differentiated cell populations are described in U.S. patent application Ser. No. 12/134, filed Apr. 8, 2008.
07,020, Title: METHODS FOR PURIFYING ENDODERM
AND PANCREATIC ENDODERM CELLS DERIVED FR
OM HES CELLS, now U.S. Patent No. 8,338,170, the disclosure of which is incorporated herein by reference in its entirety. It is further expected that pancreatic progenitors derived from cryopreserved hIPS cells can be thawed and adapted to culture prior to transplantation, such that they can mature in vivo to produce insulin. In diabetic-induced animals with transplanted pancreatic progenitors derived from hIPS cells,
It can improve hypoglycemia.

In summary, fully encapsulated pancreatic progenitor cells derived from hIPS cells in macroencapsulation devices are expected to mature into physiologically functional pancreatic islets and produce insulin in response to glucose in vivo.

Example 4
Pancreatic Progenitor and Hormone-Secreting Cell Composition Using flow cytometry, differentiated hIPS cell populations were classified into PDX1-positive pancreatic endoderm or pancreatic progenitor cells (stage 4), as shown in Tables 5a, 5b, and 6, respectively.
and endocrine or endocrine precursor cell (stage 5) content. Table 5b is the same data set as in Table 5a, but presented similarly to that in Table 9 for comparison.
The total PDX1+ and NKX6.1+ counts overlap with that of the NKX6.1+/PDX1+/CHGA- cell population (column 5 of Table 5a), so the Table 5a populations overlap with each other, e.g., total cell counts are greater than 100%. Table 5b includes PDX1+ only and triple negative (residual) data, which are not shown in Table 5a. To determine in vivo function, these i
Certain of the PEC grafts, as well as others using substantially similar formulations, were transplanted into animals, but human serum C-peptide levels were not stable enough for any potential therapeutic purposes (data not shown). Values shown are the percentage of total cells belonging to a given population. The number of pancreatic precursors (NKX6.1(+)/PDX1(+)/chromogranin A(-)) in the suspension cell aggregates and the very small population of NKX6.1+/PDX1-/CHGA- were consistent with that observed in pancreatic progenitor suspension aggregates derived from hES cells, with a 200% increase in the number of pancreatic precursors.
U.S. patent application Ser. No. 12/264,760, filed Nov. 4, 2008, entitled STEM CEL
L AGGREGATE SUSPENTION COMPOSITIONS AND
METHODS OF DIFFERENTIATION THEREOF,
The levels of endocrine and/or endocrine precursor cells were also measured using the ESC stage, as described herein in detail in the present specification, which is incorporated by reference in its entirety.
U.S. Patent Application 12/107,020, filed April 8, 2008, entitled METHODS
FOR PURIFYING ENDODERM AND PANCREATIC EN
hES in DODERM CELLS DERIVED FROM HES CELLS
Similar to the suspension aggregates of hES-derived cells, varying the concentration of different growth factors in the medium at specific stages of differentiation (e.g., stage 4) should increase and/or decrease specific populations of pancreatic endoderm, endocrine, PDX1-positive endoderm or non-pancreatic cell types.

Example 5
PEC receptor tyrosine kinase The above methods are substantially the same as those described below in Table 7, which is adapted from Schulz et al. (2012) supra. These and other methods described herein may be used in conjunction with U.S. Patent Nos. 7,964,402; 8,233,663; and 8,366,711, the disclosures of which are incorporated herein by reference in their entireties.
Nos. 211,699; 8,334,138; 8,008,07; and 8,15
These and other related patents and non-patent publications can be found in Applicant's many patents and non-patent publications, such as Applicant's US Pat. No. 3,429.

When applying the above methods to iPS cells and pancreatic progenitors implanted in animals, Applicants have not consistently obtained the same robust in vivo function as compared to applying the same methods to hESCs and hES-derived pancreatic progenitors.
The morphology and gene expression pattern of SCs were observed in vitro and in vivo.
This was surprising given that human pluripotent stem cells are capable of forming teratomas in vivo, and have been shown to be capable of generating cells from all three germ layers (see, at least, e.g., Yu et al. (2007), supra; U.S. Patent Application Publication No. 2006/0233612, supra).
09/0047263, International Patent Application Publication No. WO 2005/80598; U.S. Patent Application Publication No. 2008/0233610; and International Patent Application Publication No. WO 2008/11882,
See, These references state that iPS cells meet the defining criteria of ESCs.
It is therefore anticipated that iPS cells can substitute for ESCs in in vitro differentiation protocols that provide hES-derived pancreatic progenitor cells that further mature and develop in vivo into fully functional glucose-responsive cells. However, in light of the inconsistent in vivo functional data using the above methods, Applicants sought to explore differentiation medium formulations specific for pancreatic precursor and/or pancreatic endoderm cells (PECs), i.e., stage 4-derived cells from hiPSCs (or "iPECs") that could provide substantially similar and stable levels of in vivo function consistently observed in PECs derived from hESCs.

Applicants have previously reported that the endocrine (CHGA+ cells) cells present in PECs are multihormonal endocrine cells and are not a subpopulation of cells in PECs that generate islets with glucose-responsive insulin-secreting cells in vivo. See Kelly et al. (2011), supra. Rather, it is a non-endocrine cell population (CHGA- cells), specifically, the islets that are multihormonal endocrine cells that are multihormonal endocrine cells and are not a subpopulation of cells in PECs that generate islets with glucose-responsive insulin-secreting cells in vivo.
It is those that co-express NKX6.1 and PDX-1 that are believed to be the PECs that actually generate functional islets in vivo. Applicants therefore believe that modulating, altering or shifting the relative ratios of endocrine and non-endocrine subpopulations may be a potential mechanism for subsequent in vivo cell death.
We investigated whether it would affect function.

Previous efforts to decipher receptor-ligand signaling in hESCs have been successful in identifying growth factors that promote self-renewal, allowing the development of defined media culture conditions.
(2007). Wang et al. identified heregulin-1β as a ligand that binds to ERBB3 and induces dimerization with ERBB2, thereby affecting hESC self-renewal. ERBB is a receptor tyrosine kinase (RTK); RTKs
RTKs are widely expressed transmembrane proteins that act as receptors for growth factors and other extracellular signaling molecules. Upon ligand binding, they undergo tyrosine phosphorylation at specific residues in their cytoplasmic tails, initiating a signaling cascade for the binding of other protein substrates involved in RTK-mediated signaling. RTK functions in several developmental processes, including regulation of cell survival, proliferation and motility, and their role in cancer formation are well documented. ERBB tyrosine kinase receptors were also known to be expressed throughout the developing fetal human pancreas, but the specific roles of particular ERBB receptors and their ligands are unknown. Mari-Anne Huotari et al. (2002) ERBB
Signaling Regulations Lineage Determinati
on of Developing Pancreatic Islet Cells
in Embryonic Organ Culture, Endocrinology
143(11):4437-4446.

The self-renewal of pluripotent stem cells in the fetal human pancreas and their expression as demonstrated by Wang et al. (2007) above, and the expression of ERB in ERBB RTK in the human fetal pancreas.
Because of the role of BRTK signaling, Applicants next hypothesize that BRTK signaling may be involved in PEC specification during differentiation, or proliferation by promoting self-renewal, or in vivo induction of islet hormone-secreting cells that function physiologically.
In an effort to identify receptors and ligands that may enhance some other unknown mechanism that promotes in vivo maturation, we set out to investigate the potential activation of RTKs in in vitro pancreatic endoderm cells (PECs) derived from hESCs. PECs were generated in suspension of differentiating aggregates substantially as described in Table 8, with the following modifications.

Four PEC samples were generated for RTK blot analysis: db-N50 K50
"Steady-state" samples of PEC aggregates at E50 were collected at the end of stage 4 (or d13). "Fasted" samples were cultured in db (DMEM high glucose or 0.5xB-27
d12 PEC aggregates were fed with DMEM high glucose supplemented with supplements (Life Technologies) medium alone (no growth factors) and harvested on d13.
The two "pulse" samples were cultured in db medium on d12, then on d13,
They were fed either db-K50 E50 medium or db medium containing 2% FBS for 15 minutes before harvesting. Such conditions were intended to detect RTKs that were active in stage 4 conditions, and what responses could be elicited with a pulse of KGF, EGF and insulin (present in B27 supplement) or serum. The serum pulse was used to detect RTKs that were present in PEC and could be activated but not stimulated in this stage 4 condition.
We aimed to use broad-spectrum growth factor stimulation to potentially identify TK.

RTK analysis was performed essentially as previously described in Wang et al. (2007), supra. Briefly, Proteome Profiler™ human phospho-RTK antibody arrays (R&D Systems) were used as per the manufacturer's instructions. 1% NP-40, 2
0 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2
Protein lysates were prepared with 0 mM EDTA, 1.0 mM sodium orthovanadate, 10 μg/mL aprotinin, and 10 μg/mL leupeptin.
Fresh protein lysates of 1000 ng/ml were incubated overnight with nitrocellulose membranes dotted with duplicate spots for 42 anti-RTK antibodies and 5 negative control antibodies, as well as 8 anti-phosphotyrosine positive control spots (Figure 5A). The arrayed antibodies capture the extracellular domains of phosphorylated and non-phosphorylated RTKs, and bound phospho-RTKs are detected with a pan-anti-phosphotyrosine antibody conjugated to horseradish peroxidase (HRP) using chemiluminescence. See Figure 5 for the RTK array layout and Table 8 below for a list of RTKs in the array.

Analysis of RTK blots (FIG. 6A) showed that the insulin receptor and IGF1 receptor (IR and IGF1R, respectively) were phosphorylated and activated in all conditions, similar to what was previously observed in hESCs. See Wang et al. (2007) supra.
The EGF receptor (EGFR, also known as ERBB1) was phosphorylated under steady-state conditions, which was expected given the presence of EGF in stage 4 medium. Indeed, low levels of phosphorylation of ERBB2 were detected under both steady-state and fasting conditions.
Phosphorylation of both GFR and ERBB2 was elevated in each pulsing condition, confirming the ability of the assay to detect activation in response to pulsing of ligand.
R3 was also detected in all conditions and was elevated in pulsed samples, suggesting that PECs produce endogenous VEGFR3 ligands, with likely candidates being VEGF-C and D. Serum pulsing appeared to activate additional receptors, including low levels of ERBB3 phosphorylation. Detection of phosphorylated ERBB2/3 suggests that heregulin-like EGF family members can activate signaling in PECs. TI
E-2 is one of two angiopoietin receptors and appeared to be phosphorylated at low levels in response to serum. Angiopoietin 1 and angiopoietin 4 are known to be activating ligands of Tie-2, whereas angiopoietin 2 and angiopoietin 3 function as scene-dependent competitive antagonists. HGF receptor (HGFR) was also phosphorylated in response to serum pulsing, suggesting that hepatocyte growth factor can also elicit signaling in PEC. Finally, low levels of phosphorylation of ephrinB2 RTK (EPHB2) were detected, but ephrin/Eph signaling is a membrane-bound cell-cell signaling system that does not appear to be readily exploited in PEC differentiation. Interestingly, ERBB4
was not phosphorylated. Thus, RTK analysis highlighted several receptors that are phosphorylated in PECs or can be phosphorylated in response to different conditions, e.g., serum. These results suggest that several soluble ligands can elicit RTK signaling in PECs, potentially affecting cell proliferation, differentiation and/or specification and thus subsequent in vivo maturation into functional islets.

Example 6
Heregulin and FGF2 growth factors affect PECs derived from hESC compositions. As described in Example 5 above, certain RTKs are activated (or phosphorylated) under certain conditions.
In light of the RTK analysis demonstrating that ERBB2 and ERBB
Because heregulin 3 appeared to be activated in PEC (after 13 days of differentiation from stages 1-4), applicants sought to determine the effect of heregulin when applied to stage 3 and 4 cells.

Preliminary studies were performed using heregulin and FGF. Certain of these studies included the Rho kinase inhibitor, Y-27632. These preliminary studies were performed at 10 ng/mL.
Treatment of pluripotent stem cells at stage 1 for 1 day with mL heregulin-1β (same concentration and heregulin isomer as disclosed in Wang et al. (2007)) reduced heregulin-1β (
The results showed that Hrg1β increased the cell aggregate size of hES-derived cell aggregates in suspension culture compared to the aggregate size of hES-derived cell aggregates in suspension culture without Hrg1β. Increasing cell aggregate size is advantageous in providing a larger cell mass for subsequent transplantation and functional testing in animals. Furthermore, aggregate disk size increased when Hrg1β was increased from 10 ng/mL to 50 ng/mL at stage 3. This result was also observed when 50 ng/mL of another growth factor, FGF2, was used at stage 3 compared to culture in the absence of FGF2. The increase in cell aggregate size was observed when stage 3 cultures were exposed to FGF2 for additional days, e.g., 2 days.
This was also observed when exposed to 50 ng/mL FGF2 for 3 days compared with 1 day.

Table 9 provides a summary of flow cytometry analysis of PEC cells treated with Hrg1β and FGF2 at stage 3 and/or stage 4. Endocrine cells were CHGA positive (
Endocrine (CHGA+) and non-endocrine (CHGA-) cells are designated as triple negative or residual cells (CHGA+, CHGA-) cells. Endocrine (CHGA+) and non-endocrine (CHGA-) cells can stain positive for other markers, for example staining positive for PDX1 and/or NKX6.1. Cells that do not stain for any of the markers tested are designated as triple negative or residual cells (
This is expressed as CHGA-/NKX6.1-/PDX1).

To determine whether the increase in cell aggregate size affects the PEC subpopulation,
The composition of the C population was analyzed by flow cytometry.
Compared to control cultures in which 1β and FGF2 were not used, the PEC non-endocrine subpopulation (C
HGA-) increased from 54.01% to 61.2% with the addition of 10 ng/mL Hrg1β at stage 3, and increased from 54.01% to 61.2% with the addition of 50 ng/mL Hrg1β at stage 3.
The endocrine subpopulation (CHGA+) had an HrgO of 10 ng/mL or higher.
Treatment with 1β had little effect, but more so at 50 ng/mL.
The relative levels of residual cells were reduced, more so at 50 ng/mL Hrg1β, and thus the increase in cell aggregate size with Hrg1β treatment was largely attributable to an increase in non-endocrine subpopulations compared with endocrine and residual subpopulations.

The effect of FGF2 in stage 3 cultures was similar, but more pronounced than that of Hrg1β. For example, the PEC non-endocrine subpopulation (CHGA-) was increased, as was Hrg1β. The major effect of FGF2 in these cultures was a substantial decrease in the endocrine subpopulation; in some cases, these cells were nearly undetectable (from 32.9% to 0.33%) after 3 days of treatment. Thus, the increase in cell aggregate size in cultures treated with FGF2 was mostly attributable to an increase in the non-endocrine, and in some cases residual, cell subpopulation (from 13.1% to 22.7% after 3 days at stage 3).

Thus, heregulin and/or FGF2 appear to play a role in cell specification in the PEC population, which is surprising given that Wang et al. (2007), supra, reported that heregulin alone plays a role in cell regeneration when used in the context of pluripotent stem cells.

(Example 7)
Methods for improving in vivo graft function of PECs by treatment of iPS-derived cell cultures with Heregulin Because the methods according to Table 7 when applied to iPSCs to generate iPECs did not provide stable in vivo function in animals, Applicants explored other methods for iPEC generation. Modifications of the standard method shown in Table 7 include, but are not limited to, the following:
Optimization of any iPSC passage number; modulating the level of BMP signaling; modulating iPSC suspension aggregation parameters during expansion and differentiation (e.g., shear force, rotation speed, etc.); optimization of the concentration, timing and duration of use of growth factors such as Wnt, activin and rho kinase inhibitors; and treatment with other growth factors (e.g., ERBB ligands) as candidates to improve cell quantity, proliferation, differentiation, survival, etc. at various stages 1-4 of the differentiation protocol. A number of these iterative experiments were examined alone or in combination to determine how the differentiation method of iPSCs during stages 1-4 could be optimized. Such optimized differentiation methods would generate iPEC populations that, when transplanted, yielded stable glucose-responsive insulin-secreting cells in vivo similar to those observed and reported for hESCs. Table 10 below lists the baseline conditions with and without heregulin that were demonstrated to differentiate iPSCs into iPECs that subsequently matured into glucose-responsive islet cells in vivo. Baseline conditions were similar to those described in Examples 1, 2, and 5 and Table 7 herein, except that heregulin was added at stages 3 and 4. 30 ng/mL of Heregulin
1β was used, but concentrations of 10 ng/mL to 50 ng/mL or even greater than 50 ng/mL are suitable. Additionally, as described in Example 2, the Rho kinase inhibitor, Y
Addition of -27632 was maintained in differentiation cultures.

To determine the effect of the addition of heregulin or heregulin and a rho kinase inhibitor on stage 3 and 4 cell subpopulations, iPEC populations were analyzed by flow cytometry. Table 11 shows the results of the combinations shown in Table 10 and the addition of activin at a concentration of 200 ng/ml.
Table 11 provides a summary of flow cytometry analysis of various iPEC populations using formulations modified by increasing L. Additionally, Table 11 shows the general conditions used in each set of experiments (baseline with and without Heregulin) and the relative percentages of cell types (endocrine, non-endocrine, PDX1 only and triple negative or residual cell subpopulations) in the iPEC populations. Table 11 also discloses data regarding the in vivo functionality of cells generated in each experiment.

Under certain conditions, PECs (hESCs, E2354) and iPECs (E2314,
E2344, E2347) populations were altered. For example, sometimes the percentage of endocrine (CHGA+) cells decreased and the percentage of non-endocrine cells (CHGA-/NKX6.1+/PDX1+) increased compared to baseline (no heregulin) conditions. Heregulin appeared to be responsible for the change in the proportion of endocrine cells compared to non-endocrine cells in these PEC and iPEC populations, however, in experiment #2380 (E2380), the addition of heregulin increased, rather than decreased, the level of endocrine (CHGA+) cells.

To determine whether changes in the composition of the PEC and iPEC populations affected in vivo function, PEC and iPEC grafts from most of the experiments described in Table 11 were implanted into mice substantially as previously described herein and in Schulz et al. (2012) and Kroon et al. (2008) supra, and in Applicant's other patent and non-patent publications, including U.S. Pat. Nos. 7,534,608; 7,695,965; 7,993,920 and 8,278,106 supra. Briefly, the PEC and iPEC populations were all encapsulated in biodegradable, semi-permeable cell encapsulation devices, some of which contained microperforations. The devices were manufactured by Applicant and described in U.S. Pat. No. 8,278,106, filed Nov. 13, 2009, entitled ENCAPSULATION OF PANCREATIC CARCINOMA AND ITS USE IN HUMAN ...
CELLS FROM HUMAN PLURIPOTENT STEM CELLS
The disclosure of which is fully incorporated herein by reference. Glucose stimulated insulin secretion (GSIS) assays were performed starting about 56 days after transplantation. Blood was collected at combinations before glucose challenge (fasting) and 30 and/or 60 minutes after glucose challenge. Graft function was assessed by measuring serum human C-peptide concentrations in response to glucose challenge.

The amount of human C-peptide released into the serum is an indicator of the amount of insulin released.
The peptide is a short 31 amino acid peptide that connects or links the A and B chains of proinsulin and preproinsulin, and is secreted by functional beta or insulin secreting cells. As previously described by Kroon et al. (2008) and others, supra, measurement of human C-peptide is suitable for assessing the release of newly produced insulin by transplanted cells. Thus, the levels of human C-peptide in the serum of these animals are consistent with the expression of mature PECs and iPECs.
It is a measure of in vivo function of the graft. Human C-peptide was detected in serum by at least 8 weeks after transplantation. Additional weeks of transplantation and fasting increased glucose-stimulated C-peptide levels, shifting the peak C-peptide level from 60 minutes to 30 minutes after glucose challenge, indicative of a faster response to glucose challenge following insulin cell maturation. There were a few mice that failed to demonstrate function or were sacrificed due to poor health. However, these mice were in a cohort of animals that otherwise demonstrated high function, thus suggesting graft failure rather than an inability of the transplanted cells to differentiate and function.

Figures 7A-C show human C-peptide levels in serum following glucose administration for all of the experiments shown in Table 11 except E2344. Figures 7A-C show that grafts resulting from heregulin treatment generally had higher levels of human C-peptide in serum compared to baseline controls. For example, in Figure 7A, in experiment 2380, grafts resulting from heregulin treatment had approximately 5% higher levels of human C-peptide in serum compared to those prepared without heregulin (baseline).
There is a fold increase (933 pM: 200 pM 60 minutes after glucose administration).
Heregulin inhibits P peptide generated from hESCs, as (FIG. 7C) does not show higher levels of serum C-peptide in grafts resulting from heregulin treatment compared to baseline controls.
Furthermore, hESC-derived PECs (C
When comparing iPEC derived from iPSCs (iT203) with iPECs, iPEC grafts showed
C grafts have the same functions in vivo (see, for example, Figs. 7A and 7B (iPS
Compare Fig. 7C (C grafts) with Fig. 7C (CyT203 hESCs). Thus, iPEC grafts are as robust as PEC grafts. Furthermore, iPECs from E2380, which did not have the same shift in endocrine and non-endocrine subpopulations, also showed superior function, suggesting that iPECs are more robust than PEC grafts.
The relative ratio of endocrine to non-endocrine cells, which appeared to affect some of the C populations (eg, E2314, E2347 and E2354), did not appear to affect in vivo function (see Figures 7A-C).

In addition to testing glucose-stimulated insulin secretion, mature iPEC grafts were tested to determine whether they alone could maintain euglycemia similar to that maintained by hESC-derived PECs when the host animal's beta cells were destroyed. This required destroying the beta cells of the transplanted mice using streptozotocin (STZ), a beta cell toxin that exhibits greater cytotoxicity to mouse beta cells compared to human beta cells. Random non-fasting blood glucose was measured for each mouse before and after STZ treatment. Explantation of the iPEC grafts 13 days after STZ treatment resulted in the resumption of hyperglycemia (noted by a spike in blood glucose), demonstrating control of glycemia by the iPEC grafts and not the endogenous mouse pancreas (see Figures 8A and 8B).

Furthermore, there appeared to be a synergistic effect when heregulin and rho kinase inhibitor were given during stages 1-4 of differentiation (see Table 10). For example, iPSCs treated with heregulin at stages 3 and 4 without the rho kinase inhibitor resulted in visibly inferior cell mass, rendering transplantation impossible. Further supporting evidence of a synergistic effect of heregulin and rho kinase inhibitor was evident in some of the experiments, e.g., E2356, E2380, where baseline conditions with rho kinase inhibitor alone did not function as stably as grafts with rho kinase inhibitor and heregulin (see Figures 7A and B). Treatment with heregulin and rho kinase inhibitor did not appear to be additive, as the addition of heregulin alone provided insufficient cell mass for transplantation and the addition of rho kinase inhibitor alone (baseline conditions) had poor in vivo function. Thus, providing only heregulin or only rho kinase inhibitor is not substantially equivalent to the total effect of the two combined. That is, while neither alone produced a stable glucose responsiveness in vivo, in combination they produced glucose responsiveness comparable to that of hES-derived cells. Thus, provision of both heregulin and a rho-kinase inhibitor appeared to be synergistic as their combined effect was greater than the sum of each effect separately. That is, iPECs treated with a rho-kinase inhibitor and heregulin matured in vivo, exhibited glucose-stimulated insulin secretion, and were able to maintain normoglycemia in a diabetic mouse model (see Figures 7A-B and 8A-B).

ERBB functionality requires ligand binding, receptor dimerization, and receptor trafficking. Variations in each process can result in differential regulation of the receptors and the downstream signals they control. For example, different ERBB ligands bind to ERBB receptors with different affinities,
Thereby altering the pattern and dynamics of ERBB dimer formation.
Many different possible combinations of ligand and receptor binding complexes are shown. A review of the complexity of this system is given in Oda et al. (2005) A comprehensive pathway
ay map of epidermal growth factor receive
or signaling, Mol. Syst. Biol., 1 (2005) and La
Zzzara et al. (2009) Quantitative modeling perspe
tives on the ERBB system of cell regulation
Tory processes, Experimental Cell Research
h 315(4):717-725, the disclosure of which is incorporated herein by reference in its entirety.

Huotari et al. have shown that neuregulin-4 can modulate the relative levels of endocrine cell subpopulations by increasing the number of somatostatin (delta) cells at the expense of glucagon (alpha) cells, and that neuregulin-4 mediates the expression of endocrine (e.g., β-insulin, α-glucagon, δ-somatostatin, PP-pancreatic polypeptide) cells.
These studies, however, were performed by incubating neuregulin-4 on whole mount organotypic tissue cultures obtained from mice at day E12.5. These mouse explant cell populations were more differentiated than the stage 3 (e.g., PDX1-negative foregut endoderm) and/or stage 4 (PDX1-positive foregut endoderm) cell populations described herein. Since neuregulin-4 binds only to the ERBB4 RTK, only the endocrine subpopulations of whole mount mouse cultures can be modulated by neuregulin-4 in this context. Thus, Hrg1 binds ERBB3 to upregulate ERBB2/3
Since it has already been shown that ERBB induces dimerization of Hrg1, it would not be expected that treatment of stage 3 (PDX1-negative foregut endoderm) and/or stage 4 (PDX1-positive foregut endoderm) cells with different ERBB ligands, such as Hrg1, as described herein, would modulate the relative endocrine subpopulations as in Huotari. However, due to the low levels of expression of ERBB2 and 3 in PEC as shown in Figure 6, it was unclear whether stage 3 and 4 cells express low or high levels of ERBB2 and 3 and bind Hrg1.

Furthermore, on a different occasion, Applicants have described that Hrg1 binds to ERRB2/3 and promotes self-renewal of pluripotent stem cells (see Wang et al. (2007)).
While it is possible that heregulin could act in the same capacity at the stage 3 and 4 setting, applicants have previously noted that the majority of cell expansion for the generation of PECs occurs at the pluripotent stem cell stage (stage 0). During stage 0, hESCs are grown, passaged and expanded for approximately two (2) weeks. Thus, most of the cell proliferation or self-renewal that results in cell growth does not occur during stages 1-4. See Schulz et al. (2012), supra. Furthermore, given that ERRB2/3 is present during stages 3 and 4, one would expect heregulin to have a similar effect on pluripotent stem cells (self-renewal) as opposed to influencing induced differentiation. The difference in function then appears to depend on the setting, i.e., pluripotent stem cells versus cell types of endoderm or pancreatic lineage.

In summary, in vitro provision of heregulin or heregulin and a rho-kinase inhibitor to foregut endoderm (stage 3) and PDX1-expressing pancreatic endoderm cells (end of stage 3 and stage 4) generated PEC and iPEC populations that matured in vivo when transplanted and developed into glucose-responsive insulin-secreting cells (see Figures 7 and 8). Such use of heregulin or heregulin and a rho-kinase inhibitor is reported here for the first time. Such uses and effects are not discernible from those previously described in patent or non-patent literature.

It will be appreciated that the Q-PCR results described herein can be further confirmed by immunocytochemistry (ICC), which can be readily performed by one of skill in the art.

The methods, compositions and devices described herein are representative of currently preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention. Those skilled in the art will envision modifications thereto and other uses that are encompassed by the spirit of the present invention and are defined by the scope of the disclosure. Thus, it will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the present invention.

In the claims below and throughout this disclosure, the phrase "consisting essentially of" is meant to include any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements. Additionally, in embodiments in which numerical values, such as amounts, concentrations, percentages, proportions, or ranges are recited, it is understood that the value recited may be "at least about" that numerical value, "about" that numerical value, or "at least" that numerical value.

EMBODIMENTS Embodiment 1. In vitro human pancreatic endoderm cell cultures.
Embodiment 2. The cell culture of embodiment 1, wherein the pancreatic endoderm cells are derived from pluripotent cells.
Embodiment 3. The cell culture of embodiment 1 or 2, wherein the cell culture is contacted with an ERBB tyrosine kinase receptor activating agent.
Embodiment 4. The ERBB tyrosine kinase receptor activating agent is selected from the group consisting of EGF (epidermal growth factor), AREG (amphiregulin), TGF-alpha (transforming growth factor-alpha), Btc (betacellulin), HBEGF (heparin-binding EGF), Er
The cell culture of embodiment 3, wherein the antigen-binding domain is eg (epiregulin), neuregulin or heregulin.
Embodiment 5. The pancreatic endoderm cells include pancreatic progenitor cells and multihormonal endocrine cells.
5. The cell culture of any one of embodiments 1 to 4.
Embodiment 6. The cell culture of embodiment 5, wherein the pancreatic progenitor cells express NKX6.1 but do not express CHGA.
Embodiment 7. The cell culture of embodiment 5, wherein the multihormonal endocrine cells express CHGA.
Embodiment 8. The cell culture of any one of embodiments 1-4, wherein the pancreatic endoderm cells comprise CHGA positive and CHGA negative cells.
Embodiment 9. The cell culture of any one of embodiments 1-8, wherein at least 30% of the pancreatic endoderm cells are CHGA-negative cells.
Embodiment 10. The cell culture of any one of embodiments 1-9, wherein at least 50% of the pancreatic endoderm cells are PDX1 positive cells.
Embodiment 11 The cell culture of embodiment 3, wherein the ERBB tyrosine kinase receptor activating agent is heregulin-4.
Embodiment 12. The cell culture of any one of embodiments 1 to 11, wherein the cell culture is in contact with a fibroblast growth factor (FGF).
Embodiment 13. The cell culture of embodiment 12, wherein the FGF is FGF-7.
Embodiment 14. The cell culture of any one of embodiments 1-13, wherein the cell culture is contacted with an ERBB tyrosine kinase receptor activating agent and an FGF.
Embodiment 15. The cell culture of any one of embodiments 1-15, wherein the cell culture is contacted with an ERBB tyrosine kinase receptor activating agent and a rho kinase inhibitor.
Embodiment 16. ERBB tyrosine kinase receptor activating agent, FGF and rho
17. The cell culture of any one of embodiments 1-16, wherein the cell culture is in contact with a kinase inhibitor.
Embodiment 17. An in vitro human pancreatic endoderm population.
Embodiment 18. The cell population of embodiment 17, wherein the pancreatic endoderm cells are derived from pluripotent cells.
Embodiment 19. The cell population of embodiment 18, wherein the pluripotent cells are human embryonic stem cells or dedifferentiated genetically reprogrammed cells.
Embodiment 20. The cell population of any one of embodiments 17-19, wherein the cell culture is contacted with an ERBB tyrosine kinase receptor activating agent.
Embodiment 21. The ERBB tyrosine kinase receptor activating agent is selected from the group consisting of EGF (epidermal growth factor), AREG (amphiregulin), TGF-alpha (transforming growth factor-alpha), Btc (betacellulin), HBEGF (heparin-binding EGF), E
reg (epiregulin), neuregulin or heregulin.
Embodiment 22. The cell population of any one of embodiments 17-21, wherein the pancreatic endoderm cells comprise pancreatic progenitor cells and multihormonal endocrine cells.
Embodiment 23. The cell population of embodiment 22, wherein the pancreatic progenitor cells express NKX6.1 but do not express CHGA.
Embodiment 24. The cell population of embodiment 22, wherein the multihormonal endocrine cells express CHGA.
Embodiment 25. The cell population of any one of embodiments 17-24, wherein the pancreatic endoderm cells comprise CHGA-positive and CHGA-negative cells.
Embodiment 26 The cell population of any one of embodiments 17-25, wherein at least 30% of the pancreatic endoderm cells are CHGA-negative cells.
Embodiment 27. At least 50% of pancreatic endoderm cells are PDX1 positive.
27. The cell population according to any one of claims 7 to 26.
Embodiment 28 The cell population of embodiment 20, wherein the ERBB tyrosine kinase receptor activating agent is heregulin-4.
Embodiment 29. The cell population of any one of embodiments 17-28, wherein the cell culture is contacted with a fibroblast growth factor (FGF).
Embodiment 30. The cell population of embodiment 29, wherein the FGF is FGF-7.
Embodiment 31. The cell culture is administered an ERBB tyrosine kinase receptor activating agent and F
31. The cell population of any one of embodiments 17 to 30, in contact with a GF.
Embodiment 32. The cell culture is administered an ERBB tyrosine kinase receptor activating agent and
31. The cell population of any one of embodiments 17-30, which is in contact with a ho kinase inhibitor.
Embodiment 33. The cell culture is administered an ERBB tyrosine kinase receptor activating agent, FGF
and a rho kinase inhibitor.
Embodiment 34. An in vitro cell population comprising human pancreatic endoderm cells in contact with an ERBB tyrosine kinase receptor activating agent.
Embodiment 35. The cell population of embodiment 34, wherein the pancreatic endoderm cells are derived from pluripotent cells.
Embodiment 36. The cell population of embodiment 35, wherein the pluripotent cells are human embryonic stem cells or dedifferentiated genetically reprogrammed cells.
Embodiment 37. The ERBB tyrosine kinase receptor activating agent is selected from the group consisting of EGF (epidermal growth factor), AREG (amphiregulin), TGF-alpha (transforming growth factor-alpha), Btc (betacellulin), HBEGF (heparin-binding EGF), E
reg (epiregulin), neuregulin or heregulin, embodiments 34-36.
13. The cell population according to any one of claims 1 to 12.
Embodiment 38. The cell population of any one of embodiments 34-37, wherein the pancreatic endoderm cells comprise pancreatic progenitor cells and multihormonal endocrine cells.
Embodiment 39. The cell population of embodiment 38, wherein the pancreatic progenitor cells express NKX6.1 but do not express CHGA.
Embodiment 40. The cell population of embodiment 38, wherein the multihormonal endocrine cells express CHGA.
Embodiment 41. The cell population of any one of embodiments 34-40, wherein the pancreatic endoderm cells comprise CHGA-positive and CHGA-negative cells.
Embodiment 42 The cell population of any one of embodiments 34-41, wherein at least 30% of the pancreatic endoderm cells are CHGA-negative cells.
Embodiment 43. At least 50% of the pancreatic endoderm cells are PDX1 positive.
43. The cell population according to any one of 4 to 42.
Embodiment 44 The cell population of any one of embodiments 34-36, wherein the ERBB tyrosine kinase receptor activating agent is heregulin-4.
Embodiment 45. The human pancreatic endoderm cells are contacted with fibroblast growth factor (FGF).
45. The cell population of any one of embodiments 34 to 44.
Embodiment 46 The cell population of embodiment 45, wherein the FGF is FGF-7.
Embodiment 47. The cell population of any one of embodiments 34-46, wherein the human pancreatic endoderm cells are contacted with an ERBB tyrosine kinase receptor activating agent and an FGF.
Embodiment 48. The cell population of any one of embodiments 34-47, wherein the human pancreatic endoderm cells are contacted with an ERBB tyrosine kinase receptor activating agent and a rho kinase inhibitor.
Embodiment 49. Any one of embodiments 34 to 48, wherein the human pancreatic endoderm cells are contacted with an ERBB tyrosine kinase receptor activating agent, an FGF, and a rho kinase inhibitor.
1. The cell population according to claim 1.
Embodiment 50. A method for producing insulin, comprising:
a. contacting foregut endoderm cells with an ERBB tyrosine kinase receptor activating agent, thereby generating a cell population comprising pancreatic endoderm;
b. transplanting and maturing the pancreatic endoderm of step (a) in vivo, thereby obtaining insulin-secreting cells, wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.
Embodiment 51. A method for producing insulin, comprising:
a. contacting foregut endoderm cells derived from dedifferentiated genetically reprogrammed cells with an ERBB tyrosine kinase receptor activating agent in vitro, thereby generating a cell population comprising endocrine and non-endocrine subpopulations;
b. transplanting and maturing the subpopulation of step (a) in vivo, thereby obtaining insulin-secreting cells, which secrete insulin in response to glucose stimulation.
Embodiment 52. A method for producing insulin, comprising:
A method comprising the steps of: transplanting and maturing pancreatic endoderm cells in vivo, thereby obtaining insulin-secreting cells, which secrete insulin in response to glucose stimulation.
Embodiment 53 The method of embodiment 52, wherein the pancreatic endoderm cells are produced by contacting foregut endoderm cells with an ERBB tyrosine kinase receptor activating agent.
Embodiment 54. The ERBB tyrosine kinase receptor activating agent is selected from the group consisting of EGF (epidermal growth factor), AREG (amphiregulin), TGF-alpha (transforming growth factor-alpha), Btc (betacellulin), HBEGF (heparin-binding EGF), E
reg (epiregulin), neuregulin or heregulin, embodiments 50-52.
53. The method according to any one of claims 1 to 53.
Embodiment 55. The method of any one of embodiments 50-52 and 53-54, wherein the foregut endoderm cells are further contacted with fibroblast growth factor (FGF).
Embodiment 56 The method of embodiment 55, wherein the FGF is FGF-7.
Embodiment 57 The method of any one of embodiments 50-52 and 53-56, wherein the foregut endoderm cells are further contacted with a rho kinase inhibitor.
Embodiment 58 The method of any one of embodiments 50-52 and 53-57, wherein the foregut endoderm cells are further contacted with a rho kinase inhibitor and an FGF.
Embodiment 59. The rho kinase inhibitor is Y-27632, fasudil, H-1152
P, Wf-536, Y-30141, ROCK antisense nucleic acid, RNA interference-inducing nucleic acid, competitive peptide, antagonist peptide, inhibitory antibody, antibody-ScFV fragment, dominant negative variant thereof, derivative and expression vector;
59. The method according to any one of embodiments 57 to 58.
Embodiment 60. The rho kinase inhibitor is Y-27632, fasudil, H-1152
60. The method of any one of embodiments 57-59, wherein the IL-11 is selected from the group consisting of P, Wf-536 and Y-30141 and derivatives thereof.
Embodiment 61. The Rho kinase inhibitor is selected from the group consisting of Y-27632, fasudil and H-11
52P, and derivatives thereof.
Embodiment 62 The method of embodiment 50, wherein the pancreatic endoderm comprises endocrine and non-endocrine cell subpopulations.
Embodiment 63 The method of embodiment 62, wherein the endocrine cell subpopulation is CHGA positive (CHGA+) cells.
Embodiment 64. The method of embodiment 6, wherein the non-endocrine cell subpopulation is CHGA negative (CHGA-).
2. The method according to claim 2.
Embodiment 65 The method of embodiment 62, wherein the non-endocrine cell subpopulation expresses NKX6.1.
Embodiment 66 The method of embodiment 50, wherein at least 30% of the pancreatic endoderm is CHGA negative.
Embodiment 67 The method of embodiment 50, wherein at least 50% of the pancreatic endoderm is PDX1 positive.
Embodiment 68. A method for producing insulin, comprising:
a. contacting dedifferentiated genetically reprogrammed cells in vitro with a first medium comprising an agent that activates a TGFβ receptor family member;
b. culturing in vitro the cells of step (a) in a second culture medium lacking an agent that activates a TGFβ receptor family member, thereby generating foregut endoderm cells;
c. contacting the foregut endoderm cells of (b) with an ERBB tyrosine kinase receptor activating agent, thereby generating a cell population comprising endocrine and non-endocrine cell subpopulations;
d. transplanting and maturing the cell population of (c) in vivo, thereby obtaining insulin-secreting cells, wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.
Embodiment 69. The method of embodiment 68, wherein the non-endocrine cells are CHGA-negative (CHGA-) cells.
The method according to
Embodiment 70. The method according to embodiment 68, wherein the endocrine cells are CHGA positive (CHGA+) cells.
69. The method according to any one of claims 69 to 70.
Embodiment 71 The method of embodiment 69, wherein CHGA-negative (CHGA-) cells express NKX6.1.
Embodiment 72 The method of embodiment 69, wherein the foregut endoderm cells of (b) are contacted with a rho kinase inhibitor.
Embodiment 73 The method of embodiment 69, wherein the foregut endoderm cells of (b) are contacted with FGF.
Embodiment 74 The method of embodiment 69, wherein the foregut endoderm cells of (b) are contacted with a rho kinase inhibitor and FGF.
Embodiment 75. The rho kinase inhibitor is Y-27632, fasudil, H-1152
P, Wf-536, Y-30141, ROCK antisense nucleic acid, RNA interference-inducing nucleic acid, competitive peptide, antagonist peptide, inhibitory antibody, antibody-ScFV fragment, dominant negative variant thereof, derivative and expression vector;
75. The method according to any one of embodiments 72 to 74.
Embodiment 76. A method for increasing the proportion of non-endocrine cells in a pancreatic endoderm cell population relative to endocrine cells, comprising:
2. A method comprising the step of contacting foregut endoderm cells with an ERBB tyrosine kinase receptor activating agent, thereby increasing the proportion of non-endocrine cells in a pancreatic endoderm cell population.
Embodiment 77 The method of embodiment 76, wherein the foregut endoderm cells are contacted with FGF.
Embodiment 78 The method of embodiment 76, wherein the foregut endoderm cells are contacted with a rho kinase inhibitor.
Embodiment 79 The method of embodiment 76, wherein the foregut endoderm cells are further contacted with an FGF and a rho-kinase inhibitor.
Embodiment 80. A method for improving glucose responsiveness of transplanted pancreatic endoderm, comprising:
contacting foregut endoderm cells with an ERBB tyrosine kinase receptor activating agent, thereby generating a cell population comprising pancreatic endoderm;
and transplanting and maturing pancreatic endoderm in vivo, thereby obtaining insulin-secreting cells, the insulin-secreting cells secreting insulin better than insulin-secreting cells derived from pancreatic endoderm produced without contacting foregut endoderm cells with an ERBB tyrosine kinase receptor activating agent.
Embodiment 81 The method of embodiment 80, wherein the pancreatic endoderm cells are derived from dedifferentiated genetically reprogrammed cells.
Embodiment 82. A method for producing insulin, comprising the steps of: (a) contacting living cells with an ERBB tyrosine kinase receptor activating agent; and (b) transplanting and maturing the cells at the end of step a, thereby obtaining insulin-secreting cells, which secrete insulin in response to glucose stimulation.
Embodiment 83. The method of embodiment 8, wherein the living cells are foregut endoderm or PDX1-negative foregut endoderm.
2. The method according to claim 2.
Embodiment 84. The method of any one of embodiments 82-83, wherein the living cells are derived from dedifferentiated genetically reprogrammed cells.
Embodiment 85. A cell population comprising endocrine and non-endocrine cell subpopulations.
Embodiment 86. The cell population of embodiment 85, wherein the endocrine and non-endocrine cell subpopulations are derived from pluripotent cells.
Embodiment 87. The cell population of embodiment 86, wherein the pluripotent cells are human embryonic stem cells or dedifferentiated genetically reprogrammed cells.
Embodiment 88. The ERBB tyrosine kinase receptor activating agent is selected from the group consisting of EGF (epidermal growth factor), AREG (amphiregulin), TGF-alpha (transforming growth factor-alpha), Btc (betacellulin), HBEGF (heparin-binding EGF), E
reg (epiregulin), neuregulin or heregulin, embodiments 85-87.
13. The cell population according to any one of claims 1 to 12.
Embodiment 89. The cell population of any one of embodiments 85-88, wherein the non-endocrine cells express NKX6.1 but do not express CHGA.
Embodiment 90. The cell population of any one of embodiments 85-89, wherein the endocrine cells express CHGA.
Embodiment 91. At least 30% of the cells are CHGA-negative cells, according to embodiments 85-9.
10. The cell population according to any one of claims 1 to 9.
Embodiment 92. The cell population of any one of embodiments 85-91, wherein at least 50% of the cells are PDX1 positive.
Embodiment 93 The cell population of any one of embodiments 85-92, wherein the ERBB tyrosine kinase receptor activating agent is heregulin-4.
Embodiment 94. The cell population of any one of embodiments 85-93, wherein the cell culture is contacted with a fibroblast growth factor (FGF).
Embodiment 95. The cell population of embodiment 94, wherein the FGF is FGF-7.
Embodiment 96. The cell culture is administered an ERBB tyrosine kinase receptor activating agent and F
96. The cell population of any one of embodiments 85-95, in contact with a GF.
Embodiment 97. The cell culture is administered an ERBB tyrosine kinase receptor activating agent and
97. The cell population of any one of embodiments 85-96, which is contacted with a ho kinase inhibitor.
Embodiment 98. The cell culture is administered an ERBB tyrosine kinase receptor activating agent, FGF
and a rho kinase inhibitor.
Embodiment 99. A method of producing insulin, comprising the steps of: (a) contacting in vitro dedifferentiated genetically reprogrammed cells with a first culture medium comprising an agent that activates a TGFβ receptor family member; and (b) in vitro contacting the cells of step (a) with a second culture medium lacking the agent that activates a TGFβ receptor family member.
(c) contacting the cells of (b) with an ERBB tyrosine kinase receptor activating agent, thereby generating a cell population comprising endocrine and non-endocrine cell subpopulations; and (d) transplanting and maturing the cell population of (c) in vivo, thereby obtaining insulin-secreting cells, wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.
Embodiment 100. A method of producing insulin, comprising: (a) transfecting PDX1 positive cells with the ER
(b) contacting the cell population of (a) with a BB tyrosine kinase receptor activating agent; and (b) transplanting and maturing the cell population of (a) in vivo, thereby obtaining insulin-secreting cells, wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.
Embodiment 101 The method of embodiment 68 or 99, wherein a rho kinase inhibitor is added in steps a, b, or c.
Embodiment 102 The method of embodiment 68 or 99, wherein a rho kinase inhibitor is added in steps a, b, and c.
Embodiment 103. The method of any one of embodiments 68 or 99-100, wherein a rho kinase inhibitor is added in steps a and b.
Embodiment 104 The method of embodiment 68 or 99, wherein an ERBB tyrosine kinase receptor activating agent is added in step a or c.
Embodiment 105 The method of embodiment 68 or 99, wherein an ERBB tyrosine kinase receptor activating agent is added in steps a and c.
Embodiment 106. A method for generating a cell population capable of maturing in vivo into glucose-responsive insulin-secreting cells, comprising:
A population of PDX1-negative foregut endoderm, or at least a population of PDX1-positive pancreatic endoderm cells, is isolated from the ER.
with a BB receptor tyrosine kinase activating agent, thereby generating a cell population capable of maturing in vivo into glucose-responsive insulin-secreting cells.
Embodiment 107. A method for generating pancreatic endoderm, comprising:
a. contacting foregut endoderm cells with an ERBB tyrosine kinase receptor activating agent, thereby producing a cell population comprising pancreatic endoderm.
Embodiment 108 The method of embodiment 107, further comprising contacting the foregut endoderm cells with fibroblast growth factor (FGF).
Embodiment 109. The method of embodiment 108, wherein the FGF is FGF-7.
Embodiment 110. The method of any one of embodiments 107-109, further comprising contacting the foregut endoderm cells with a rho kinase inhibitor.
Embodiment 111 The method of any one of embodiments 107-109, further comprising contacting the foregut endoderm cells with a rho kinase inhibitor and FGF.
Embodiment 112. At least 30% of the pancreatic endoderm is CHGA negative.
7. The method according to any one of claims 7 to 111.
Embodiment 113. At least 50% of the pancreatic endoderm is PDX1 positive.
7 to 112. The method according to any one of claims 7 to 112.
Embodiment 114. The foregut endoderm cells are derived from pluripotent cells, according to embodiments 50, 76,
114. The method according to any one of claims 107 to 113.
Embodiment 115. The method of any one of embodiments 50, 76, 107-114, wherein the pluripotent cells are human embryonic stem cells or dedifferentiated genetically reprogrammed cells.
Embodiment 116 The cell culture of embodiment 1, wherein the pluripotent cells are human embryonic stem cells or dedifferentiated genetically reprogrammed cells.
Embodiment 117. An in vitro method for the treatment of a genetically reprogrammed cell comprising: a differentiated cell derived from a dedifferentiated genetically reprogrammed cell; and an ERBB receptor tyrosine kinase activating agent.
o A human pancreatic endoderm cell population.
Embodiment 118 The pancreatic endoderm cell population of embodiment 117, wherein the ERRB receptor tyrosine kinase activating agent comprises an EGF growth factor or ligand.
Embodiment 119. The EGF growth factor or ligand is heregulin-1, heregulin-2
and heregulin-3 and heregulin-4.
Embodiment 120. The pancreatic endoderm cell population of embodiment 119, wherein the heregulin isoforms comprise heregulin-1 and heregulin-4.
Embodiment 121 The pancreatic endoderm cell population of embodiment 119, wherein the ligand heregulin isoform comprises heregulin-4.
Embodiment 122. A method for producing insulin, comprising:
a. contacting a foregut endoderm cell culture derived from dedifferentiated genetically reprogrammed cells with an ERBB receptor tyrosine kinase activating agent in vitro;
thereby generating a cell population comprising endocrine cells and non-endocrine cell subpopulations;
b. maturing the subpopulation of step (a) in vivo, thereby obtaining insulin-secreting cells, wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.
Embodiment 123 The method of embodiment 122, further comprising contacting the foregut endoderm cell culture with a rho kinase inhibitor.
Embodiment 124. The rho kinase inhibitor is Y-27632, fasudil, H-115
2P, Wf-536, Y-30141, ROCK antisense nucleic acid, RNA interference-inducing nucleic acid, competitive peptide, antagonist peptide, inhibitory antibody, antibody-ScFV fragment, dominant negative variant, derivatives thereof, and expression vectors thereof.
Embodiment 125. The rho kinase inhibitor is Y-27632, fasudil, H-115
2P, Wf-536, Y-30141 and derivatives thereof;
124. The method of embodiment 123.
Embodiment 126. The Rho kinase inhibitor is Y-27632, fasudil, H-115
126. The method of embodiment 125, wherein the aryl group is selected from the group consisting of 2P and derivatives thereof.
Embodiment 127. A method for producing insulin, comprising:
a. contacting dedifferentiated genetically reprogrammed cells in vitro with a first medium comprising an agent that activates a TGFβ receptor family member;
b. culturing in vitro the cells of step (a) in a second culture medium lacking an agent that activates a TGFβ receptor family member, thereby generating foregut endoderm cells;
c. contacting the foregut endoderm cells of step (b) with an ERBB receptor tyrosine kinase activating agent, thereby generating a cell population comprising endocrine cells and a non-endocrine cell subpopulation;
and d. maturing the cell subpopulation of step (c) in vivo, thereby obtaining insulin-secreting cells, wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.
Embodiment 128. The method of embodiment 1, wherein the non-endocrine cells are CHGA-negative (CHGA-) cells.
27. The method according to claim 27.
Embodiment 129. The method of embodiment 12, wherein the endocrine cells are CHGA positive (CHGA+) cells.
7. The method according to claim 7.
Embodiment 130. CHGA-negative (CHGA-) cells further express NKX6.1.
128. The method of embodiment 127.
Embodiment 131 The method of embodiment 127, further comprising contacting the foregut endoderm cells with a rho kinase inhibitor.
Embodiment 132. The rho kinase inhibitor is Y-27632, fasudil, H-115
132. The method of embodiment 131, wherein the antisense nucleic acid of ROCK, RNA interference-inducing nucleic acid, competitive peptide, antagonist peptide, inhibitory antibody, antibody-ScFV fragment, dominant negative variant, derivatives thereof, and expression vectors thereof.

Claims (14)

A composition comprising an in vitro cell population comprising human pancreatic endoderm cells and heregulin in an aqueous medium , said heregulin comprising heregulin-1β, heregulin-1, heregulin-2, heregulin-3, and/or heregulin-4 . The composition of claim 1 , wherein the heregulin comprises heregulin-1 and heregulin-4. The composition of claim 1 , wherein the heregulin comprises heregulin-4. The composition of any one of claims 1 to 3 , further comprising a rho kinase inhibitor. The composition according to any one of claims 1 to 4, further comprising at least one additional ERBB receptor tyrosine kinase activating agent. 1. An in vitro method for generating human pancreatic endoderm, comprising culturing foregut endoderm cells in a medium comprising heregulin, thereby generating a cell population comprising human pancreatic endoderm , said heregulin comprising heregulin-1β, heregulin-1, heregulin-2, heregulin-3, and/or heregulin-4. The in vitro method of claim 6 , wherein the medium further comprises a rho kinase inhibitor. The in vitro method of claim 7 , wherein the rho kinase inhibitor is selected from the group consisting of Y-27632, fasudil, H-1152P, Wf-536, Y-30141, and an antisense nucleic acid of ROCK. The foregut endoderm cells are
The in vitro method of any one of claims 6 to 8, wherein the pluripotent cells are generated by: a) contacting in vitro the pluripotent cells with a first medium comprising an agent that activates a TGFβ receptor family member, and b ) culturing in vitro the cells of step (a) in a second medium lacking said agent that activates a TGFβ receptor family member. The in vitro method according to any one of claims 6 to 9 , wherein the pancreatic endoderm comprises an endocrine cell subpopulation and a non-endocrine cell subpopulation. The in vitro method of claim 10 , wherein the non-endocrine cells in the non-endocrine cell subpopulation are CHGA-negative (CHGA-) cells. 12. The in vitro method of claim 10 or 11 , wherein the endocrine cells in the endocrine cell subpopulation are CHGA positive (CHGA+) cells. The in vitro method of claim 11 , wherein the CHGA-negative (CHGA-) cells further express NKX6.1. 1. A method of producing a composition comprising a cell population comprising human pancreatic endoderm for use in a method of treating diabetes and/or controlling blood glucose levels in a human in vivo , said method of treatment comprising transplanting and maturing said pancreatic endoderm in vivo, thereby obtaining insulin-secreting cells, said insulin-secreting cells secreting insulin in response to glucose stimulation ;
The method of producing comprises carrying out an in vitro method according to any one of claims 6 to 13 to generate a cell population comprising human pancreatic endoderm .