JP7065517B2 - Method for producing PTF1A-positive cells - Google Patents

Description

The present application relates to a method for producing PTF1A positive cells.

The differentiation of pluripotent stem cells into pancreatic cells is induced in the order of pluripotent stem cells → endometrial cells → progenitor cells → pancreatic progenitor cells → pancreatic cells. It is known that PDX1 expression is observed in pancreatic progenitor cells in humans and mice. On the other hand, PDX1 is found not only in pancreatic progenitor cells but also in cells that differentiate into other organs.

In mouse embryology, it has been confirmed that almost all cells expressing Ptf1a after Pdx1 is expressed in the pancreatic primordium eventually differentiate into pancreatic cells, and pancreatic hypoplasia is observed in Ptf1a knockout mice. (Non-Patent Document 1). Mice with low Ptf1a expression have hypoplasia of the pancreas and impaired glucose tolerance (Non-Patent Document 2). In humans, pancreatic hypoplasia due to mutation of PTF1A gene (Non-Patent Document 3) or mutation of its enhancer (Non-Patent Document 4) has been reported.

From these findings, it is speculated that the PTF1A gene is a fate-determining gene for pancreatic development not only in mice but also in humans, and that the expression of the PTF1A gene is important for the differentiation into pancreatic cells.

Examples of the method for inducing pluripotent stem cells from pluripotent stem cells include a method for inducing differentiation of pluripotent stem cells using activin or retinoic acid (RA) (Patent Document 1, Non-Patent Document 5). From 9). In addition to this, a method of introducing PDX1 into pluripotent stem cells and culturing them (Patent Documents 2 and 3), and a method of appropriately combining low molecular weight compounds and allowing them to act on pluripotent stem cells to produce insulin-producing cells (Patent). Document 4, non-patent document 10) is known. In the method of inducing pancreatic cells from these pluripotent stem cells, PDX1 expression in pancreatic progenitor cells has been emphasized, but few have been evaluated focusing on the expression of PTF1A, and its expression control is unclear. ..

Japanese Unexamined Patent Publication No. 2009-22661 US Pat. No. 7,534,608 Japanese Unexamined Patent Publication No. 2006-075022 WO2011 / 081222 WO2015 / 020113 Gazette

Kawaguchi Y et al., Nat Genet 32: 128-134, 2002 Fukuda A et al., Diabetes 57: 2421-2431, 2008 Gabrielle SS et al., Nat Genet 36: 1301-1305, 2004 Michael NW et al., Nat Genet 46: 61-66, 2014 E.Kroon et al., Nature Biotechnology (2008) Vol.26, No.4: 443-452 K.A.D'Amour et al., Nature Biotechnology (2006) Vol.24, No.11: 1392-1401 W.Jiang, Cell Research (2007) 17: 333-344 J.H.Shim et al., Diabetologia (2007) 50: 1228-1238 R. Maehra et al., PNAS (2009), vol.106, No.37: 15768-15773 Kunisada Y et al., Stem Cell Res. (2012) vol.8, No.2: 274-284.

All of the above prior art documents form part of the present application by citation.

This application aims to provide a method for producing PTF1A positive cells in vitro. In particular, we provide a method for inducing PTF1A-positive cells from pluripotent stem cells, for example, iPS cells via PDX1-positive cells.

This application is
The process of providing PDX1-positive cells, and
PDX1-positive cells are selected from the group consisting of (a) adenylate cyclase activator, cAMP phosphodiesterase inhibitor, and at least one selected from the group consisting of cAMP relatives, (b) steroids and (c) nicotine amide. Provided is a method for producing PTF1A positive cells, which comprises a step of culturing in a medium containing one or more substances.

In a preferred embodiment, it is selected from the group consisting of (a) an adenylate cyclase activator, a cAMP phosphodiesterase inhibitor, and at least one selected from the group consisting of cAMP relatives, (b) a steroid and (c) a nicotine amide. Provided is a method comprising culturing PDX1-positive cells in a medium containing a histone deacetylase inhibitor prior to culturing in a medium containing one or more substances to be grown.

In the present application, PDX1-positive cells may be cells derived from pluripotent stem cells. Various methods for obtaining PDX1-positive cells from pluripotent stem cells have been reported, and any known method may be used.

The method of the present invention has made it possible to efficiently induce the expression of PTF1A in PDX1-positive cells using a small molecule substance. According to the method of the present invention, PTF1A-expressing cells can be efficiently produced from pluripotent stem cells.

It is a schematic diagram of the Example of this application. The time course of the expression level of PTF1A in the cells induced by the method of the present application is shown. The expression of the insulin gene and the amylase gene in the cells induced by the method of the present application is shown.

The present invention comprises at least one selected from the group consisting of (a) adenylate cyclase activator, cAMP phosphodiesterase inhibitor, and cAMP analog, (b) steroid and (c) nicotine amide, for PDX1-positive cells. Provided is a method for producing PTF1A positive cells, which comprises a step of culturing in a medium containing one or more substances selected from the above.

The medium used in the present invention is prepared by appropriately adding a substance to the basal medium. The basal medium includes, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Improved MEM (Invitrogen), Ham's F12 medium, RPMI 1640 medium, Fischer's medium. , Neurobasal Medium (Life Technologies), StemPro34 (Invitrogen) and their mixed media, such as DMEM / F12 medium (1: 1 mixed medium of DMEM and Ham's F12) and the like. The basal medium may be a serum-containing medium or a serum-free medium. If desired, the basal medium may be, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum substitutes such as collagen precursors, trace elements, 2-mercaptoethanol, 1-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.

As the PDX1-positive cells used in the present application, cell cultures derived from pluripotent stem cells are preferably used. As the culture of PDX1 gene-positive cells, it is preferable to use those cultured in a cell mass state having a three-dimensional structure.

In one aspect of the method of the present application, the step of culturing PDX1-positive cells in a medium containing one or more selected from the group consisting of (a), (b), and (c) is preferably to preliminarily obtain PDX1-positive cells. This is performed as the third step after culturing in the first step and the second step described below.

[First step of inducing PTF1A-positive cells from PDX1-positive cells]
In the first step of inducing PTF1A-positive cells from PDX1-positive cells, PDX1-positive cells are subjected to a medium containing a growth factor, a retinoic acid receptor (RAR) agonist, a hedgehog pathway inhibitor, a protein kinase C activator, and an ALK5 receptor inhibitor. Cultivate in. The concentration of each component in the medium may be appropriately determined.

In the first step of inducing PTF1A cells from PDX1-positive cells, a histone deacetylase (HDAC) inhibitor is preferably added to the medium.
Examples of histone deacetylase (HDAC) inhibitors include small molecule inhibitors such as valproic acid (VPA), tricostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg HDAC1 siRNA Smartpool®). ) (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene)) and other nucleic acid expression inhibitors, MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase-3 inhibitors (eg, PD184352, PD98059, U0126, SL327 and PD0325901). Bio and CHIR99021), DNA methyltransferase inhibitors (eg, 5-azacytidine), histone methyltransferase inhibitors (eg, small molecule inhibitors such as BIX-01294), nucleic acids such as siRNA and shRNA against Suv39hl, Suv39h2, SetDBl and G9a. Examples include sex expression inhibitors), L-channel nucleic acid agonists (eg Bayk 8644).

Sodium butyrate is preferably used as the HDAC inhibitor. When sodium butyrate is used as the HDAC inhibitor, the concentration in the medium is usually 50 μM to 1000 μM, preferably 200 μM to 800 μM, for example about 500 μM. In the first step, the cells are cultured for 1 to 3 days, preferably 1 day.

[Second step of inducing PTF1A-positive cells from PDX1-positive cells]
In the second and subsequent steps, the culture is a suspension culture, preferably using a low-adhesion plate. In the second step, the medium preferably contains a growth factor, a retinoic acid receptor (RAR) agonist, a hedgehog pathway inhibitor, a ROCK inhibitor, a protein kinase C activator, and an ALK5 receptor inhibitor in the basal medium. Used for. The concentration of each component in the medium may be appropriately determined. In the second step, the cells obtained in the first step are transferred to a medium for the second step filled with a new plate and then cultured for 1 to 3 days, preferably 1 day.

[Third step of inducing PTF1A-positive cells from PDX1-positive cells]
PDX1-positive cells are selected from the group consisting of (a) adenylate cyclase activator, cAMP phosphodiesterase inhibitor, and at least one selected from the group consisting of cAMP analogs, (b) steroids and (c) nicotine amide. Incubate in medium containing one or more substances.

The medium is a group consisting of (a) an adenylate cyclase activator, a cAMP phosphodiesterase inhibitor, and at least one selected from the group consisting of cAMP analogs, (b) steroids and (c) nicotine amide to the above-mentioned basal medium. It is made by adding one or more substances selected from. A preferred basal medium is DMEM / F12 supplemented with B-27 supplement.

Examples of substances selected from the group consisting of adenylate cyclase activator, cAMP phosphodiesterase inhibitor, and cAMP analogs include compounds having adenylate cyclase activity, compounds having cAMP phosphodiesterase inhibitory activity, and adenylate cyclase activity. And a compound having both cAMP phosphodiesterase inhibitory activity and the like. More specifically, forskolin, dibutyl cAMP, PACAP27 (pituitary adenylate cyclase activating polypeptide 27), IBMX (3-isobutyl-1-methylxanthine) and the like can be mentioned. Forskolin is preferred. The concentration of forskolin in the medium is usually 0.1 to 50 μM, preferably 2 to 50 μM, for example about 10 μM.

Examples of steroids include dexamethasone, hydrocortisone, betamethasone, beclomethasone and the like. Of these, dexamethasone is preferably used. When dexamethasone is used as a steroid, the concentration in the medium is usually 0.1 to 50 μM, preferably 2 to 50 μM, for example about 10 μM.

The concentration of nicotinamide in the medium is 0.01 to 20 mM, preferably 0.1 to 5 mM, for example about 1 mM.

In the method of the present application, the components (a), (b), and (c) may be added alone or in combination of any two types, or the components (a), (b), and (c) may be added. All may be added. Preferred are media containing any one, two or all three of forskolin, dexamethasone and nicotinamide.

In the method of the present application, the medium may further contain a growth factor, a retinoic acid receptor (RAR) agonist, a hedgehog pathway inhibitor, a protein kinase C inhibitor and an ALK5 receptor inhibitor. The concentration of each component in the medium may be appropriately determined.

As the growth factor, KGF is preferably used. KGF is a protein called Keratinocyte Growth Factor and is sometimes called FGF-7. EGF is a protein called Epidermal Growth Factor or Epidermal Growth Factor.

Retinoic acid receptor (RAR) agonists are naturally occurring retinoids, chemically synthesized retinoids, retinoic acid receptor agonist compounds that do not have a retinoid skeleton, and natural products that have retinoic acid receptor agonist activity. There may be. Examples of natural retinoids with activity as RAR agonists are retinoic acid (the stereoisomers of total trans-retinoic acid (total trans RA) and 9-cis-retinoic acid (9-cis RA) are known). Can be mentioned. Chemically synthesized retinoids are known in the art (US Pat. No. 5,234,926, US Pat. No. 4,326,055, etc.). Examples of retinoic acid receptor agonist compounds having no retinoid skeleton include Am80, AM580, TTNPB, AC55649. Examples of natural products having retinoic acid receptor agonist activity include honokiol and magnolol (Bulletin of the Institute for Biological Function Development 9: 55-61, 2009). RAR agonists are preferably retinoic acid, AM580 (4-[[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl 2-naphthalenyl] carboxyamide] benzoic acid), TTNPB (4- [[E] -2- [5,6,7,8-Tetrahydro-5,5,8,8-Tetramethyl 2-naphthalenyl] -1-propenyl] Benzoic acid), AC55649 (4'-octyl- [1 , 1'-biphenyl] -4-carboxylic acid), more preferably retinoic acid.

Hedgehog pathway inhibitor means a compound that inhibits the activity of a signal such as Sonic hedgehog, Indian hedgehog, or Desert hedgehog that binds to the membrane receptor Patched, eg, Smoothened. However, as long as the hedgehog inhibits the signal generated by binding to the receptor, it is not particularly limited, and for example, cyclopamine, jervine, 3-Keto-N- (aminoethyl-aminocaproyl-dihydro-cinnamoyl) (KAAD) -cyclopamine, Examples include CUR-61414, SANT-1, SANT-2, SANT-3, SANT-4, IPI-926, IPI-269609, GDC-0449 and NVP-LDE-225. Cyclopamine is preferred.

Examples of the protein kinase C activator include Alpha APP Modulator, for example (2S, 5S)-(E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2,4-pentadienoylamino. ) Benlactam is exemplified.

Examples of the ALK5 receptor inhibitor include ALK5 inhibitor II (2- [3- [6-methylpyridine-2-yl] -1H-pyrazol-4-yl] -1,5-naphthylidine).
In the third step, the culture is suspension culture, and it is preferable to use a low-adhesion plate. The culture period is not particularly limited, and 4 to 14 days, for example, 8 days or more, preferably about 8 days or more, after transferring the cells obtained in the previous step to the above-mentioned medium filled with a new plate at the start of the third step. Incubate for 12 days.

The culture conditions are not particularly limited, but in one embodiment, the culture may be performed under the conditions of 37 ° C., 5% CO2, and 5% O2.

The method of the present application increases the expression of PTF1A in cells. In addition, cells cultured by the method of the present application express both insulin and amylase.

In one aspect of the method of the present application, PDX1-positive cells are derived from mammalian pluripotent stem cells. Pluripotent stem cells are stem cells having pluripotency capable of differentiating into all cells existing in a living body and also having proliferative ability. For example, embryonic stem (ES) cells (J.A. Thomson et al. (1998), Science 282: 1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; J.A. Thomson et al. (1996), Biol. Reprod., 55: 254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165), obtained by nuclear transplantation Embryonic stem (ntES) cells derived from cloned embryos (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936 J. Byrne et al. (2007), Nature, 450: 497-502), sperm stem cells (“GS cells”) (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69: 612-616 K. Shinohara et al. (2004), Cell, 119: 1001-1012), Embryonic Stem Cell (“EG Cell”) (Y. Matsui et al. (1992), Cell, 70: 841-847; J.L. Resnick et al. (1992), Nature, 359: 550-551), Artificial pluripotent stem (iPS) cells (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); WO2007 / 069666), pluripotent cells (Muse cells) derived from cultured fibroblasts and bone marrow stem cells (WO2011 / 007900), etc. are included. More preferably, the pluripotent stem cells are human pluripotent stem cells, such as human ES cells and human iPS cells. More preferably, it is a human iPS cell.

Pluripotent stem cells can be commercially available pluripotent stem cells or pluripotent stem cells stored with information on the individual from which they are derived for research or transplantation medicine, even if they are produced using known methods. You may use it. A project to construct a highly versatile iPS cell bank by using a person homozygous for the HLA haplotype, which is frequently used, is currently underway in Japan (CYRANOSKI, Nature vol. 488, 139 (2012)). For example, pluripotent stem cells obtained from such an iPS cell bank may be used.

The pluripotent stem cells used in the method of the present application can be cultured as a single cell state by substantially separating (or dissociating) by an arbitrary method, or a state of a cell aggregate in which cells adhere to each other. It may be cultured in. Separation methods for separating and culturing into a single cell state include, for example, mechanical separation and separation solutions having protease activity and collagenase activity (for example, solutions containing trypsin and collagenase Accutase (TM) and Accumax. (TM) (Innovative Cell Technologies, Inc)) or separation using a separation solution having only collagenase activity. Pluripotent stem cells can be adherently cultured using a coated culture dish.

To induce PDX1-positive cells from pluripotent stem cells, first induce endoderm cells from pluripotent stem cells and then induce archenteron cells from endoderm cells. Further induce PDX1-positive cells from archenteronal cells.

In order to obtain archenteron cells from pluripotent stem cells via endoderm cells, a conventionally known method may be used. In one embodiment, pluripotent stem cells are cultured in medium containing activin receptor-like kinase-4,7 activator, GSK3 inhibitor, ROCK inhibitor and P13 kinase for 1-4 days, preferably 2 days. Then, incubate in a medium containing only the activin receptor-like kinase-4,7 activator for 1 to 4 days, preferably 2 days, and further in a medium containing a GSK3 inhibitor and a growth factor for 3 to 3 to. Incubate for 8 days, preferably 4 days. Archenteronal cells can be obtained by such an operation.

The activin receptor-like kinase-4,7 activator is a substance having an activating effect on ALK-4 and / or ALK-7, and examples thereof include activin, Nodal, and Myostatin. Preferred is activin. As activin, activin A, B, C, D and AB are known, but any activin A, B, C, D or AB can be used. As activin, activin A is particularly preferably used. Further, as activin, activin derived from any mammal such as human or mouse can be used. As the activin used in step 1, it is preferable to use activin derived from the same animal species as the pluripotent stem cells used for differentiation. For example, when human-derived pluripotent stem cells are used as a starting material, human-derived activin is used. It is preferable to use it. These activins are commercially available.

A GSK3 inhibitor is defined as a substance that inhibits the kinase activity of the GSK-3β protein (for example, the ability to phosphorylate β-catenin), and many of them are already known. Also known as GSK-3β inhibitor IX; 6-bromoindylbin 3'-oxym, SB216763 (3- (2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl), a maleimide derivative. ) -1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione) , GSK-3β inhibitor VII (4-dibromoacetophenone), a phenylαbromomethylketone compound, L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-), a cell membrane permeabilizing kinase. NH2) and CHIR99021 with high selectivity (6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] Ethyl] amino] nicotinonitrile). These compounds are commercially available from, for example, Calbiochem, Biomol, etc., and can be easily used. It may be obtained from other sources or may be made by yourself. The GSK-3β inhibitor may preferably be CHIR99021.

Then, PDX1-positive cells are induced from archenteron cells.
To induce PDX1-positive cells from proto-enterocytes, first place the proto-enterocytes in a medium containing growth factors such as KGF and EGF, retinoic acid receptor (RAR) agonists, hedgehog pathway inhibitors, and ROCK inhibitors. And cultivate. As the medium, a known basal medium may be appropriately selected and used. For example, a medium in which B27 supplement is mixed with DMEM / F12 medium is preferably used. The content of each additive in the medium may be appropriately determined.

The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK), for example, Y-27632 (eg, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000)). Narumiya et al., Methods Enzymol. 325,273-284 (2000)), Fasudil / HA1077 (eg Uenata et al., Nature 389: 990-994 (1997)), H-1152 (eg Sasaki et al). ., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (eg, Nakajima et al., Cancer Chemother Pharmacol. 52 (4): 319-324 (2003)) and their derivatives, Also included are antisense nucleic acids for ROCK, RNA interference-inducible nucleic acids (eg, siRNA), dominant negative variants, and expression vectors thereof. In addition, other known low molecular weight compounds can also be used as the ROCK inhibitor (for example, US Patent Application Publication No. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508). , 2004/0002507, 2003/0125344, 2003/0087919, and International Publication 2003/062227, 2003/059913, 2003/062225, 2002/076976 No., 2004/039796). In the present invention, one or more ROCK inhibitors can be used. A preferred ROCK inhibitor used in this step is Y-27632.

In order to induce PDX1-positive cells from archenteron cells to obtain PDX1-positive cells as a cell mass having a three-dimensional structure, the cells may be cultured in the presence of a substance that serves as a three-dimensional support, for example, Matrigel.

The step of inducing PDX1-positive cells from archenteron cells is carried out for 4 to 14 days, for example, about 8 days, while changing the medium to a new one as appropriate. This step induces PDX1-positive cells. In one aspect of the present application, the cells obtained in this step are used as PDX1-positive cells.

The invention of the present application will be described in more detail with reference to Examples. The outline of the example is shown in FIG.
Human iPS cell 201B7 strain (Cell 131: 861-872, 2007) was used.
To maintain the undifferentiated state, a mouse fibroblast line SNL treated with mitomycin as a feeder cell under 5% CO ₂ was used as a medium, and a medium for primate ES cells (reprocell) was used as a medium. 4 ng / mL human recombinant bFGF (WAKO) and 0.5% Penicillin-Streptomycin (GIBCO) were added to the medium. Medium exchange was performed daily from the day after the passage, and the passage was performed every 6 to 7 days.

First, undifferentiated iPS cells were differentiated into endoderm cells on a 24-well plate.
Feeder cells were removed from the dish of undifferentiated iPS cells and dissociated by Accutase (Innovative Cell Technologies) until they became single cells. The 24-well plate used was Matrigel-coated at room temperature from the previous day. IPS cells dispersed in RPMI medium (GIBCO) containing various differentiation-inducing factors were seeded on a 24-well plate at a density of 2 × 10 ⁴ cells per hole, and cultured at 37 ° C. and 5% CO ₂ for 2 days. Activin A (100 ng / mL) and GSK3 inhibitors CHIR99021 (3 μM), Y-27632 (10 μM), Waltmanin (100 nM), and 2% B27 supplement (GIBCO) were used as differentiation-inducing factors. After culturing for 2 days, the medium was replaced with RPMI medium containing activin A (100 ng / mL) and 2% B27 supplement, and the cells were cultured for 1 day. Further, it was replaced with the same medium and cultured for 1 day.

Next, differentiation into archenteron cells was induced. RPMI medium containing KGF (50 ng / mL) and CHIR99021 (2 μM) and 2% B27 supplement as differentiation-inducing factors was exchanged daily and cultured for a total of 4 days.

Archenteron cells were induced into PDX1-positive cells.
The cells induced so far are dissociated into single cells by Accutase, and DMEM / F12 medium containing various differentiation-inducing factors and 2.5% Matrigel and 2% B27 supplement in a 6-well plate coated with Matrigel from the previous day ( It was suspended in GIBCO) and sown.

As differentiation-inducing factors, KGF (50 ng / mL), EGF (100 ng / mL), tretinoin acid (2 μM), cyclopamine (250 nM), and Y-27632 (10 μM) were added. The cells were cultured at 37 ° C, 5% O ₂ , and 5% CO ₂ for 8 days. Subsequent cultures were all performed with the same gas and temperature settings. The same medium was exchanged once on the 4th day. Samples that have been cultured for 8 days are referred to as "D0". When the obtained cell mass was confirmed by immunostaining and quantitative RT-PCR, it was confirmed that PDX1 was expressed, but PTF1A expression was not confirmed.

<First step of inducing PTF1A expression from PDX1-positive cells>
Subsequently, in order to induce PTF1A expression, the cells were cultured for 1 day in DMEM / F12 medium containing various differentiation-inducing factors without Matrigel. As differentiation-inducing factors, sodium butyrate (500uM), KGF (50ng / mL), retinoic acid (100nM), cyclopamine (250nM), PKC activator Alpha-APP Modulator (250nM), ALK5 receptor inhibitor (1μM), 2 % B27 supplement was used. Sodium butyrate was not added in the negative control group.

<Second step of inducing PTF1A expression from PDX1-positive cells>
The cells were stripped of adherent cells with a cell scraper and floated on a low adhesion 6-hole plate. The medium was KGF (50 ng / mL), retinoic acid (100 nM), cyclopamine (250 nM), Alpha-APP Modulator (250 nM), ALK5 receptor inhibitor (1 μM), 2% B27 supplement, and Y-27632 (10 μM). ) Was added to DMEM / F12 medium, and the cells were cultured under these conditions for 1 day to form cell clumps. (Second step)

<Third step of inducing PTF1A expression from PDX1-positive cells>
Next, the obtained cell mass was newly added to KGF (50 ng / mL), retinoic acid (100 nM), cyclopamine (250 nM), Alpha-APP Modulator (250 nM), ALK5 receptor inhibitor (1 μM), and 2% B27 supplement. The medium was replaced with a medium containing horscholine (10 μM), dexamesazone (10 μM), and nicotinamide (1 mM) as differentiation-inducing factors (addition group). The culture was continued for 12 days thereafter, and the medium was replaced with the same medium every 4 days. Sampling was performed on the 6th, 10th, and 14th days. Notated as "D6", "D10", and "D14", respectively. Forskolin, dexamethasone, and nicotinamide were not added to the negative control group to which sodium butyrate was not added before the suspension culture, and this was used as the final negative control (non-addition group).
The expression levels of PTF1A in the added group and the non-added group were evaluated by quantitative RT-PCR using the B2M gene as an internal control and a human pancreatic tissue sample as a positive control. The results are shown in FIG.

In addition, the expression of insulin and amylase was confirmed by quantitative RT-PCR on the cells on the 14th day. The results are shown in FIG.

Claims (7)

A step of providing PDX1-positive cells derived from human pluripotent stem cells,
The first step of culturing the obtained PDX1-positive cells in a medium containing sodium butyrate, KGF, a retinoic acid receptor agonist, cyclopamine, a protein kinase activator and an ALK5 receptor inhibitor for 1 to 3 days.
The cells obtained in step 1 are cultured in a medium containing KGF, a retinoic acid receptor agonist, cyclopamine, a protein kinase activator and an ALK5 receptor inhibitor for 1 to 3 days, and the cells obtained in step 2 are obtained. PTF1A comprising a third step of culturing the cells in a medium containing holscholine, dexamethasone, nicotine amide, KGF, retinoic acid receptor agonist, cyclopamine, protein kinase activator and ALK5 receptor inhibitor for 4-14 days. Method for producing positive cells. The method according to claim 1, wherein the culture in the second step and the third step is a suspension culture using a low-adhesion plate. The method according to claim 1 or 2, wherein the culture is carried out for 8 days or more in the third step. The method according to any one of claims 1 to 3, wherein the culture is carried out for one day in the first step. The method according to any one of claims 1 to 4, wherein PTF1A-positive cells express insulin and amylase. The method according to any one of claims 1 to 5, wherein the pluripotent stem cell is a human ES cell or a human iPS cell. A method for producing an insulin-expressing cell and an amylase-expressing cell, which comprises the method according to any one of claims 1 to 6.

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