Method for producing pancreatic endoderm cells

Abstract

The present invention provides: a method for producing pancreatic endoderm cells, the method comprising a step for culturing pancreatic endoderm cells in a culture medium containing a ROCK inhibitor and KGF and / or EGF; and pancreatic endoderm cells produced by said method.

Description

Method for producing pancreatic endoderm cells

[0001] The present invention relates to a method for producing pancreatic endoderm cells. More specifically, the present invention relates to a method for producing pancreatic endoderm cells, which comprises a step of culturing pancreatic endoderm cells in the presence of a ROCK inhibitor, and pancreatic endoderm cells produced by the method.

[0002] The pancreas functions as an exocrine gland, secreting digestive enzymes such as pancreatic lipase, trypsin, elastase, and pancreatic amylase, and as an endocrine gland, secreting pancreatic hormones such as glucagon, insulin, somatostatin, and pancreatic polypeptide (PP). Recently, it has been reported that the gastric hormone ghrelin is also secreted by pancreatic endocrine cells. These pancreatic hormones are produced by cell clusters called pancreatic islets, which consist primarily of four types of cells: alpha cells, beta cells, delta cells, and PP cells. Diabetes mellitus (DDM) is a disease caused by insulin deficiency or loss of insulin function, and is difficult to cure once it develops. Diabetes can be broadly classified into two types: type 1 diabetes (insulin-dependent diabetes) and type 2 diabetes (non-insulin-dependent diabetes).

[0003] Type 2 diabetes is a chronic disease that develops due to a decline in insulin secretion and the acquisition of insulin resistance. It is believed that lifestyle factors, such as obesity caused by overeating and lack of exercise, and stress, are involved in its onset. Type 1 diabetes, on the other hand, occurs when beta cells (insulin-producing cells) are destroyed by autoimmune disease or viral infection, resulting in a lack of insulin secretion. Symptomatic treatment involves administering insulin. Another potential treatment for type 1 diabetes is to induce insulin-producing cells ex vivo from patient-derived cells and then transplant the induced insulin-producing cells into the patient's body. Insulin-producing cells can be obtained, for example, by extracting and differentiating cells derived from the patient's pancreatic duct epithelium ex vivo.

[0004] However, extracting cells from patients is invasive, and the number of cells obtained is insufficient. Therefore, efforts are being made to develop methods for inducing differentiation of pluripotent stem cells, such as induced pluripotent stem cells (iPS cells), into β-like cells, which are necessary for cell therapy for diabetes. Methods for inducing differentiation of β-like cells have been reported (e.g., Patent Literature 1 and Non-Patent Literature 1). However, these methods require six to seven steps (approximately four to five weeks) to generate β-like cells from pluripotent stem cells, as shown in Figure 1 (stepwise differentiation induction method).

[0005] Furthermore, methods for expanding and culturing posterior foregut cells derived from human pluripotent stem cells have also been developed (e.g., Non-Patent Documents 1 and 2). Non-Patent Documents 1 and 2 describe the expansion of PDX1, which corresponds to the posterior foregut cells in Figure 1. + / SOX9 + / NKX6.1 - Methods for expanding pancreatic progenitor cells (PDX1) have been reported. However, these methods do not allow for the differentiation of PDX1 cells. + / SOX9 + / NKX6.1 - The proliferation efficiency of pancreatic progenitor cells cannot be said to be high, and it is known that the induction efficiency decreases when inducing differentiation of pancreatic progenitor cells into NKX6.1-positive pancreatic endoderm cells.

[0006] International Publication No. 2020 / 059892

[0007] Kimura et al., Cell Chemical Biology 2020 Dec 17;27(12):1561-1572.e7Konagaya et al., Scientific Reports. 2019 Jan 24;9(1):640

[0008] Therefore, the objective of the present invention is to develop a method for proliferating (i.e., expanding) pancreatic endoderm cells, which are cells at a stage of differentiation more advanced than posterior foregut cells, while maintaining their differentiation potential.

[0009] To solve the above problems, the present inventors focused on senescence-related reagents and screened them for reagents that could potentially expand pancreatic endoderm cells. They found that the ROCK inhibitor Y-27632 significantly increased the number of pancreatic endoderm cells, and that other ROCK inhibitors besides Y-27632 also exerted similar effects on pancreatic endoderm cell proliferation. Based on these findings, further testing demonstrated that pancreatic endoderm cells could be expanded by adding Y-27632 to the culture medium at a concentration of 10 μM or 50 μM.

[0010] In cell culture, a low concentration (e.g., 10 μM) of Y-27632 is sometimes used exclusively on the first day of reseeding to suppress apoptosis. However, it is known that long-term use of ROCK inhibitors such as Y-27632 can alter the differentiation state of cells (Maldonado M. et al., Stem Cell Res 17; 222-227; 2016), and therefore the use of ROCK inhibitors for more than one day has been avoided. Therefore, even when Y-27632 is used at a high concentration of 10 μM or more, and even when Y-27632 is used for a long period during culture, pancreatic endoderm cells can be efficiently expanded over a long period while maintaining their differentiation potential (in one embodiment, 1 × 10 cells / ml can be expanded over 60 days or more of culture). 5 It was quite surprising that the cells could multiply by more than twofold.

[0011] The present inventors further investigated the mechanism behind the proliferation effect of ROCK inhibitors on pancreatic endoderm cells. It was suggested that the primary mechanism of this effect is not due to anti-apoptosis, but rather to the suppression of cell senescence and the associated suppression of fibrosis or epithelial-mesenchymal transition. Based on these findings, further research led to the completion of the present invention.

[0012] That is, the present invention provides the following: [1-1] A method for producing pancreatic endoderm cells, comprising the step of culturing pancreatic endoderm cells in a medium containing a ROCK inhibitor and KGF and / or EGF. [1-2] The method described in [1-1], wherein the medium contains both KGF and EGF. [1-3] The method described in [1-1] or [1-2], wherein the medium contains nicotinamide. [2] The method described in any one of [1-1] to [1-3], wherein the culture is carried out for two days or more. [3] The method described in any one of [1-1] to [2], wherein the ROCK inhibitor is selected from the group consisting of Y-27632, GSK269962, GSK429286A, fasudil hydrochloride, H1152, and thiazovivin. [4] The method described in any one of [1-1] to [2], wherein the ROCK inhibitor is Y-27632. [5] The method according to any one of [1-1] to [4], wherein the culture medium contains a TGFβ inhibitor and / or a retinoic acid receptor agonist. [6] The method according to [5], wherein the TGFβ inhibitor is 2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine. [7] The method according to [5] or [6], wherein the retinoic acid receptor agonist is retinoic acid. [8] The method according to any one of [1-1] to [7], wherein the pancreatic endoderm cells are derived from pluripotent stem cells. [9] The method according to any one of [1-1] to [8], wherein the pancreatic endoderm cells are cells derived from a patient with a genetic pancreatic disease. [10-1] The method according to any one of [1-1] to [9], wherein the culture is under feeder-free conditions. [10-2] The method according to any one of [1-1] to [10-1], wherein the culture is under xeno-free conditions.

[11] Pancreatic endoderm cells produced by the method according to any one of [1-1] to [10-2]. [12-1] A kit for expanding pancreatic endoderm cells, comprising a ROCK inhibitor and KGF and / or EGF. [12-2] The kit according to [12-1], comprising both KGF and EGF. [12-3] The kit according to [12-1] or [12-2], comprising nicotinamide.[13-1] The kit according to any one of [12-1] to [12-3], which contains a TGFβ inhibitor and / or a retinoic acid receptor agonist. [13-2] The kit according to [13-1], which contains both a TGFβ inhibitor and a retinoic acid receptor agonist. [13-3] The kit according to [13-1] or [13-2], wherein the TGFβ inhibitor is 2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine. [13-4] The kit according to any one of [13-1] to [13-3], wherein the retinoic acid receptor agonist is retinoic acid. [14-1] A method for producing β-like cells or their precursor cells, which comprises a step of inducing differentiation of the pancreatic endoderm cells according to

[11] into β-like cells or their precursor cells. [14-2] Cells produced by the method according to [14-1].

[15] A cell transplantation therapy agent comprising the cells according to

[11] or [14-2].

[16] The agent according to

[15] for treating diabetes. [17-1] A method for treating a pancreatic disease in a mammal, comprising administering to the mammal an effective amount of the cells according to

[11] or [14-2] or the agent according to

[15] or

[16] . [17-2] The method according to [17-1], wherein the pancreatic disease is diabetes. [18-1] The cells according to

[11] or [14-2] or the agent according to

[15] or

[16] , for use in treating a pancreatic disease. [18-2] The cells or agent according to [18-1], wherein the pancreatic disease is diabetes. [19-1] Use of the cells according to

[11] or [14-2] or the agent according to

[15] or

[16] for the manufacture of a therapeutic agent for a pancreatic disease. [19-2] The cells or agent according to [19-1], wherein the pancreatic disease is diabetes.

[0013] The present invention enables the efficient proliferation of pancreatic endoderm cells. The cells thus proliferated, as well as pancreatic endocrine and exocrine cells, including pancreatic beta cells, obtained by inducing differentiation from these cells, are also important for clinical applications. Furthermore, these cells are useful in regenerative medicine and drug discovery screening for pancreatic diseases, replacing human pancreatic islets and exocrine cells and tissues, which are difficult to obtain as research samples.

[0014] Overview of the stepwise differentiation induction method for β-like cells. Schematic diagram of senescence-associated reagent screening. NKX6.1-positive pancreatic endoderm cells were seeded in 24-well plates and screened using conventional stage 4 medium plus various senescence-associated reagents. One week later, the number of cells was counted using a fluorescence microscope. Number of NKX6.1-positive cells after culture with various senescence-associated reagents. Number of replicates = 3, cell count = mean of triplicate results, Dunnett's test. Error bars indicate standard error. Percentage of NKX6.1-positive cells after culture with various senescence-associated reagents. Number of replicates = 3, fraction = mean of triplicate results, Dunnett's test. Error bars indicate standard error. Percentage of β-gal-positive cells after culture with various senescence-associated reagents. Number of replicates = 3, fraction = mean of triplicate results, Dunnett's test. Error bars indicate standard error. Schematic diagram of an experiment investigating other ROCK inhibitors. NKX6.1-positive pancreatic endoderm cells were seeded in 24-well plates and cultured in conventional stage 4 medium supplemented with various ROCK inhibitors. One week later, the number of cells was counted under a fluorescent microscope. Total cell counts when cultured with each ROCK inhibitor. Experiments repeated 3 times, one-way analysis of variance was used. Error bars indicate standard error. NKX6.1-positive cell counts and percentages when cultured with each ROCK inhibitor. Experiments repeated 3 times, one-way analysis of variance was used. Error bars indicate standard error. Schematic diagram of expansion of pancreatic endoderm cells using Y-27632 (50 μM). NKX6.1-positive pancreatic endoderm cells were seeded in plates and cultured in medium containing Y-27632 (50 μM) for one week. After one week, the cells were replated and passaged and expanded repeatedly. Changes in total cell number during expansion with Y-27632 (50 μM). n=3. Changes in the number of pancreatic endoderm cells due to expansion culture using Y-27632 (50 μM). n=3. Immunostained image of pancreatic endoderm cells after expansion culture using Y-27632 (50 μM). Flow cytometry scatter plot of NKX6.1 expression and Ki67 expression in cells after expansion culture using Y-27632 (50 μM). Changes in the Ki67-positive rate due to expansion culture using Y-27632 (50 μM).Number of replicates = 3, error bars indicate standard deviation. Immunostained image of pancreatic islet-like cell clusters induced to differentiate from pancreatic endoderm cells after two rounds of expansion culture. Transition of cell number during expansion culture of pancreatic endoderm cells derived from ES cells (KhES-3 line). PDX1 cells after expansion culture using Y-27632 (0, 10, or 50 μM). + / NKX6.1 + Changes in pancreatic endoderm cell and total cell numbers. Immunostained images of pancreatic endoderm cells derived from the KhES-3 cell line after five passages (five weeks) of culture using Y-27632 (10 or 50 μM). Quantitative evaluation of nuclear morphology in all cells using NKX6.1 immunostaining in Figure 5B (n=616 cells (10 μM Y-27632); n=519 cells (50 μM Y-27632); biological replicate=1). Eccentricity (circularity) was compared between the two groups using the Mann-Whitney U test. Immunostained images of α-SMA in pancreatic endoderm cells at the third passage, with and without Y-27632 (50 μM) administration. Percentage of NKX6.1-positive cells when cultured with each screening reagent. Accumulated NKX6.1 cells were obtained by expansion culture using DMSO group (Y-27632 + DMSO) or ALK5 inhibitor (ALK5i) + retinoic acid (RA) group (Y-27632 + ALK5i + RA). + Changes in the number of pancreatic endoderm cells. Number of replicates = 4. Percentage of β-like cells induced from pancreatic endoderm cells after five rounds of expansion culture in the DMSO group (Y-27632 + DMSO) or ALK5i + RA group (Y-27632 + ALK5i + RA). Compared to the control group, in which β-like cells were induced from unpassaged (P0) pancreatic endoderm cells. Number of replicates = 3. Error bars indicate standard deviation.

[0015] 1. Method for Producing Pancreatic Endodermal Cells The present invention provides a method for producing pancreatic endoderm cells using a medium containing a ROCK inhibitor. Specifically, the present invention provides a method for producing pancreatic endoderm cells (hereinafter sometimes referred to as the "production method of the present invention"), which comprises the step of culturing pancreatic endoderm cells in a medium containing a ROCK inhibitor and KGF and / or EGF. The production method of the present invention may also include the step of isolating the produced pancreatic endoderm cells.

[0016] The method of the present invention allows pancreatic endoderm cells to self-proliferate, producing homogeneous cells. That is, the method of the present invention can also be interpreted as a method for proliferation or expansion of pancreatic endoderm cells, which comprises the step of culturing pancreatic endoderm cells in a medium containing a ROCK inhibitor and KGF and / or EGF.

[0017] As used herein, "pancreatic endoderm cells" refers to cells that have the potential to differentiate into at least β-like cells and express at least PDX1 and NKX6.1. Pancreatic endoderm cells may also express one or more gene markers, such as SOX9 and GATA4. Furthermore, as used herein, "β-like cells" refers to cells that are induced to differentiate in vitro from endocrine precursor cells or immature β cells, have properties identical to or similar to those of in vivo pancreatic β cells, and express and / or secrete insulin.

[0018] In this specification, unless otherwise specified, the term "cell" includes a "cell population." A cell population may be composed of one type of cell, or may be composed of two or more types of cells.

[0019] As used herein, "expansion culture" refers to culturing a desired cell population for the purpose of expanding the cell population and increasing the cell number. The increase in cell number may be achieved by the increase in cell number due to cell proliferation exceeding the decrease in cell number due to cell death, and does not require proliferation of all cells in the cell population. The increase in cell number may be 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, 3000-fold, 5000-fold, 10000-fold, 100000-fold, 1000000-fold, or 1000000-fold or more compared to before the start of expansion culture.

[0020] The basal medium used in the production method of the present invention is not particularly limited, but includes StemFit® AK02 medium (Ajinomoto Co., Inc.), StemFit® AK03 medium (Ajinomoto Co., Inc.), StemFit® Basic03 medium, CTS® KnockOut SR XenoFree Medium (Gibco), mTeSR1 medium, TeSR1 medium (Stem Cell Technologies), Iscove's modified Dulbecco's medium (GE Healthcare), and Improved MEM (Thermo Fisher Scientific). Among these, Improved MEM medium is preferred. These media can also be used for culture under feeder-free and xeno-free conditions. Other basal media include RPMI-1640 medium, EagleMEM (EMEM), Dulbecco's modified MEM, Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, DMEM, Hybridoma Serum-free medium, and KnockOut. TMDMEM, Advanced TM medium (e.g. Advanced MEM, Advanced RPMI, Advanced DMEM / F-12), Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, DMEM / F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's Medium (e.g. Waymouth's MB752 / 1), CMRL medium (e.g. CMRL-1066), Williams' medium E, Brinster's BMOC-3 Medium, E8 Medium, StemPro 34, MesenPRO RS (Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (ReproCELL Co., Ltd.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (Lonza), EX-CELL302 medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMed TM Examples of suitable medium include, but are not limited to, iPSC Medium (ReproCell Corporation) and mixtures thereof.

[0021] As shown in the Examples below, various types of ROCK inhibitors have been shown to have a proliferative effect on pancreatic endoderm cells. That is, inhibition of Rho-kinase (ROCK) function is important for the proliferation of pancreatic endoderm cells, and any ROCK inhibitor can be used in the production method of the present invention as long as it can inhibit the function. Examples of ROCK inhibitors include Y-27632 (see, e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil / HA1077 (see, e.g., Uenata et al., Nature 389: 990-994 (1997)), SR3677 (see, e.g., Feng Y et al., J Med Chem. 51: 6642-6645 (2008)), GSK269962 (see, e.g., Stavenger RA et al., J Med Chem. 50: 2-5 (2007) or WO2005 / 037197), GSK429286A, H1152 (see, e.g., Sasaki et al., J Med Chem. 50: 2-5 (2007) or WO2005 / 037197), and the like. al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (see, e.g., Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)), thiazovivin, and salts or derivatives thereof. Other ROCK inhibitors include antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof. Other known low molecular weight compounds and their salts or derivatives can also be used as ROCK inhibitors (see, for example, U.S. Patent Application Publication Nos. 2005 / 0209261, 2005 / 0192304, 2004 / 0014755, 2004 / 0002508, 2004 / 0002507, 2003 / 0125344, 2003 / 0087919, and International Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796).Among these, Y-27632, GSK269962, GSK429286A, fasudil hydrochloride, H1152 and thiazovivin are preferred, and Y-27632 is particularly preferred. In the production method of the present invention, only one ROCK inhibitor may be used, or two or more ROCK inhibitors may be used.

[0022] Examples of the salts of the compounds include inorganic base salts such as alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, etc.), aluminum salts, and ammonium salts; base addition salts such as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine; and acid addition salts such as inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate; and organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate.

[0023] When Y-27632 or a salt thereof (e.g., Y-27632 dihydrochloride, etc.) is used as a ROCK inhibitor, the concentration in the medium is usually 1 μM or higher (e.g., 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, or higher) and 500 μM or lower (400 μM, 300 μM, 200 μM, 150 μM, 100 μM, or lower). Furthermore, when Y-27632 is used, the concentration in the medium may be 0.1 μM to 1000 μM, 1 μM to 300 μM, or 10 μM to 100 μM. In one embodiment, it is 50 μM.

[0024] When GSK269962 or a salt thereof (e.g., GSK269962 hydrochloride, etc.) is used as a ROCK inhibitor, the concentration in the medium is generally 0.02 μM to 100 μM, preferably 0.2 μM to 50 μM, more preferably 2 μM to 10 μM. In one embodiment, it is 2 μM.

[0025] When GSK429286A or a salt thereof is used as a ROCK inhibitor, the concentration in the medium is generally 0.02 μM to 100 μM, preferably 0.2 μM to 50 μM, more preferably 2 μM to 10 μM. In one embodiment, it is 2 μM.

[0026] When fasudil or a salt thereof (e.g., fasudil hydrochloride, etc.) is used as the ROCK inhibitor, the concentration in the medium is generally 0.1 μM to 500 μM, preferably 1 μM to 200 μM, more preferably 10 μM to 50 μM, and in one embodiment, 50 μM.

[0027] When H1152 or a salt thereof (e.g., H1152 dihydrochloride, etc.) is used as a ROCK inhibitor, the concentration in the medium is generally 0.02 μM to 100 μM, preferably 0.2 μM to 50 μM, more preferably 2 μM to 10 μM. In one embodiment, it is 2 μM.

[0028] When thiazovivin or a salt thereof is used as a ROCK inhibitor, the concentration in the medium is generally 0.02 μM to 100 μM, preferably 0.2 μM to 50 μM, more preferably 2 μM to 10 μM, and in one embodiment, 2 μM or 10 μM.

[0029] KGF is a protein known as keratinocyte growth factor, and is also known as FGF-7. Commercially available KGF, for example, from R&D Systems, Inc., can be used. The concentration of KGF in the medium is usually 1 ng / ml to 1 μg / ml, preferably 5 ng / ml to 500 ng / ml, and more preferably 10 ng / ml to 200 ng / ml (e.g., 100 ng / ml).

[0030] EGF is a protein known as epidermal growth factor. Commercially available EGF, for example, from R&D Systems, Inc., can be used. The concentration of EGF in the medium is usually 1 ng / ml to 1 μg / ml, preferably 5 ng / ml to 500 ng / ml, and more preferably 10 ng / ml to 100 ng / ml (e.g., 50 ng / ml).

[0031] The medium may contain nicotinamide. The concentration of nicotinamide in the medium is usually 0.1 mM to 200 mM, preferably 1 mM to 100 mM, and more preferably 5 mM to 50 mM (e.g., 10 mM).

[0032] If necessary, the medium may contain medium additives other than those described above, for example, one or more serum substitutes such as fetal bovine serum (FBS), horse serum, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol, and may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone, and putrescine.

[0033] The culture in the production method of the present invention may be performed under feeder-free conditions and / or xeno-free conditions for all or part of the period. From the perspective of clinical use, the production method of the present invention is preferably performed under feeder-free and xeno-free conditions for the entire period. As used herein, "feeder-free" refers to a medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play a supporting role and are used to establish the culture conditions for the cells to be cultured. Furthermore, "xeno-free" refers to a medium or culture conditions that do not contain components derived from organisms other than the biological species of the cells to be cultured.

[0034] The culture in the production method of the present invention may be either suspension culture or adherent culture as long as the desired cells can grow, but adherent culture is preferred. As used herein, "suspension culture" refers to culture carried out under conditions that maintain cells or cell aggregates suspended in the culture medium, i.e., culture under conditions that do not allow the formation of strong cell-substratum junctions between the cells or cell aggregates and the culture vessel. Furthermore, as used herein, "adhesion culture" refers to culture under conditions that allow the formation of strong cell-substratum junctions between the cells or cell aggregates and the cultureware, etc.

[0035] Culture vessels used in adhesion culture include those whose surfaces have been artificially treated to improve cell adhesion (e.g., coating with basement membrane preparations, extracellular matrices such as fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, synthemax, vitronectin, etc., or polymers such as polylysine or polyornithine, or surface treatments such as positive charge treatment). Of these, culture vessels coated with laminin or fragments thereof are preferred.

[0036] Examples of laminin or fragments thereof used in the present invention include laminin-111 and fragments thereof containing its E8 region, laminin-211 and fragments thereof containing its E8 region (e.g., iMatrix-211), laminin-121 or fragments thereof containing its E8 region, laminin-221 or fragments thereof containing its E8 region, laminin-332 or fragments thereof containing its E8 region, laminin-3A11 or fragments thereof containing its E8 region, laminin-411 or fragments thereof containing its E8 region (e.g., iMatrix-411), laminin-421 or fragments thereof containing its E8 region, and laminin-511 or fragments thereof containing its E8 region (e.g., iMatrix-511, iMatrix-511). silk), laminin-521 or a fragment thereof containing its E8 region, laminin-213 or a fragment thereof containing its E8 region, laminin-423 or a fragment thereof containing its E8 region, laminin-523 or a fragment thereof containing its E8 region, laminin-212 / 222 or a fragment thereof containing its E8 region, and laminin-522 or a fragment thereof containing its E8 region.

[0037] The culture vessel used for suspension culture is not particularly limited as long as it is capable of "suspension culture," and can be appropriately determined by one skilled in the art. Examples of such culture vessels include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. Another example of a vessel for suspension culture is a bioreactor. These culture vessels are preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive culture vessels that can be used include those whose surfaces have not been artificially treated (e.g., coated with an extracellular matrix) to improve cell adhesion.

[0038] The culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0039] Since the method of the present invention allows for the production of target cells over a long period of time, the culture period is not particularly limited, but is typically 2 days or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 20 days or more) and 100 days or less (e.g., 90, 80, 70, 60 days or less). In one embodiment, since the method of the present invention allows for the proliferation of pancreatic endoderm cells for more than 100 days, the culture period may be longer than 100 days (e.g., 110, 120 days or more).

[0040] The cell culture density is not particularly limited as long as the cells can grow. 2 ~1.0×10 7 cells / cm 2 , preferably 1.0 x 10 3 ~1.0×10 6 cells / cm 2 , more preferably 1.0 × 10 4 ~1.0×10 5 cells / cm 2 is.

[0041] The pancreatic endoderm cells used in the production method of the present invention may be isolated from a living body or may be commercially available cell lines, but are preferably cells derived from pluripotent stem cells. In one embodiment, the pancreatic endoderm cells used as starting cells in the production method of the present invention are derived from a patient with a genetic pancreatic disease. Examples of pancreatic diseases include acute pancreatitis, chronic pancreatitis, type 1 diabetes, type 2 diabetes, pancreatic tumors, and tumors of the islets of Langerhans. Among pancreatic diseases, genetic pancreatic diseases are those caused by genetic abnormalities. Specific examples of genetic pancreatic diseases include, but are not limited to, hereditary pancreatitis, familial pancreatic tumors, and cystic fibrosis. Pancreatic endoderm cells derived from a patient with a genetic pancreatic disease may be isolated from the patient, but are preferably prepared by reprogramming patient-derived somatic cells to establish iPS cells and inducing differentiation from the iPS cells using a method known per se. Pancreatic endoderm cells derived from patients with hereditary pancreatic diseases and cells such as β-like cells induced to differentiate from such cells can be used as pancreatic disease models that reflect the pathology of the disease, and are therefore suitable for, for example, screening of therapeutic or preventive drugs for pancreatic diseases.

[0042] "Pluripotent stem cells" refer to stem cells that can differentiate into tissues and cells with various different morphologies and functions in the body and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells). Preferably, iPS cells (more preferably, human iPS cells) are used. When the pluripotent stem cells are ES cells or any cells derived from human embryos, they may be produced by or without the destruction of the embryo, but preferably, they are produced without the destruction of the embryo.

[0043] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by isolating the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from a developmentally arrested embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).

[0044] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). Specifically, nt ES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To generate nt ES cells, nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (see above) (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg is then cultured for several hours to reprogram the embryo.

[0045] Examples of ES cell lines that can be used in the present invention include mouse ES cell lines established by, for example, inGenious targeting laboratory, Inc., RIKEN (Riken), etc., and human ES cell lines established by, for example, the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, and Cellartis, Inc. Specific examples of human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, H1 and H9 strains distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 strains distributed by RIKEN.

[0046] iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of iPS cells, including iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPSCs established by selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPSCs created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.), etc. can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells created by Sakurada et al. (JP Patent Publication No. 2008-307007) can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7, 795-797), or patent publications (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008-2336610 A, US 2009-047263 A, WO 2007-069666 A, WO 2008-118220 A, WO 2008-124133 A, WO 2008-151058 A, WO 2009-006930 A, WO 2009-006997 A, WO 2009-007852 A) and known in the art can be used.

[0047] As induced pluripotent stem cell lines, various iPSC lines established by NIH, RIKEN, Kyoto University, etc. can be used. For example, human iPSC lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 lines, and Kyoto University's 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 201B7, 409B2, 454E2, 585A1, 606A1, 610B1, 648A1, 1231A3, and FfI-01s04 lines, with 585A1 being preferred.

[0048] mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lineages, e.g., when transplanted into mouse blastocysts, chimeric mice can be generated (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004) Cell, 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, germline stem cells can be obtained by repeated passage under culture conditions similar to those for ES cells (Takebayashi M. et al. (2008) Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0049] EG cells are derived from embryonic primordial germ cells (PGCs) and have pluripotency similar to that of ES cells. They can be established by culturing PGCs in the presence of LIF, bFGF, stem cell factor, and other substances (Matsui Y. et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0050] The species from which the pluripotent stem cells are derived is not particularly limited, and may be cells from, for example, rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The preferred species is human.

[0051] Pluripotent stem cells can be induced to differentiate into pancreatic endoderm cells by known methods such as those described in Toyoda T, et al. Stem Cell Reports 2017, Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, and International Publication No. 2017 / 047797. Specifically, differentiation can be induced by a method including, for example, A) a step of inducing differentiation from pluripotent stem cells into definitive endoderm cells, B) a step of inducing differentiation from definitive endoderm cells into primitive gut cells, C) a step of inducing differentiation from primitive gut cells into posterior foregut cells, or D) a step of inducing differentiation from posterior foregut cells into pancreatic endoderm cells.

[0052] The basal medium and medium additives used in steps A) to D) can be the same as those used in the production method of the present invention. The culture temperature in steps A) to D) is typically about 30 to 40°C, preferably about 37°C, and the culture is performed in a CO2-containing air atmosphere, with a CO2 concentration of preferably about 2 to 5%. Steps A) to D) are preferably performed by adherent culture. The definition and method of adherent culture are as described above.

[0053] The differentiation into definitive endoderm cells in step A) can be carried out, for example, by culturing pluripotent stem cells in a medium containing a low dose of activin A. The medium may further contain a ROCK inhibitor or a GSK3β inhibitor. The culture period is usually 2 to 8 days.

[0054] The concentration of activin A in the medium used in step A) is, for example, 5 to 1000 ng / mL, preferably 20 to 500 ng / mL, more preferably 50 to 150 ng / mL.

[0055] Examples of GSK3β inhibitors used in step A) include CHIR98014, CHIR99021, TDZD-8, SB216763, TWS-119, kenpaullone, 1-azakempaullone, SB216763, SB415286, AR-AO144-18, CT99021, and CT20026. Among these, CHIR99021 is preferred. When using CHIR99021, the concentration in the medium is usually 0.5 to 5 μM, preferably 1 to 4 μM.

[0056] The ROCK inhibitor used in step A) may be the same as that used in the production method of the present invention. When Y-27632 is used as the ROCK inhibitor, the concentration in the medium is usually 1 to 20 μM, preferably 5 to 15 μM.

[0057] The medium can further contain insulin. The concentration of insulin in the medium is usually 0.01 to 20 μM, preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM. The concentration of insulin in the medium may be, but is not limited to, the concentration of insulin contained in the added B-27 supplement.

[0058] The differentiation into primitive intestinal cells in step B) can be carried out, for example, by culturing the definitive endoderm cells obtained in step A) in a medium containing growth factors. The culture period is usually 2 to 8 days.

[0059] The growth factors used in step B) are preferably EGF, KGF, or FGF10, more preferably EGF and / or KGF, and even more preferably KGF. The concentration of the growth factor in the medium is appropriately determined depending on the type of growth factor used, but is generally about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / ml (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / ml (i.e., about 0.8 to 160 nM), and more preferably about 10 to 1000 ng / ml (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / ml (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM), and more preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM). For example, when KGF is used as a growth factor, the concentration is usually 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL.

[0060] The differentiation into posterior foregut cells in step C) can be carried out, for example, by culturing the primitive intestinal cells obtained in step B) in a medium containing a growth factor, a retinoic acid receptor agonist such as a retinoic acid derivative, a Hedgehog signal inhibitor, and a BMP inhibitor. The culture period is usually 1 to 5 days.

[0061] The types and concentrations of growth factors used in step C) are the same as those described in step B).

[0062] Examples of the retinoic acid receptor agonist used in step C) include retinoic acid (all-trans-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoic acid (Cas No.: 302-79-4)), retinoic acid salts, retinoic acid precursors, and retinoic acid derivatives. Examples of retinoic acid salts include sodium retinoate, potassium retinoate, and calcium retinoate. Examples of retinoic acid precursors include β-carotene, retinol esters, retinol, and retinal. The retinoic acid derivative means an artificially modified retinoic acid that retains the function of natural retinoic acid, and examples thereof include 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-benzoic acid (AM580) (Tamura K, et al., Cell Differ. Dev. 32: 17-26 (1990)), 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-benzoic acid (TTNPB) (Strickland S, et al., Cancer Res. 43: 5268-5272). (1983)), retinol palmitate, retinol, retinal, 3-dehydroretinoic acid, 3-dehydroretinol, 3-dehydroretinal, etc. Among these, TTNPB is preferred. When TTNPB is used, the concentration in the medium is usually 1 to 50 nM, preferably 5 to 15 nM.

[0063] Examples of hedgehog pathway inhibitors used in step C include cyclopamine, jervine, 3-Keto-N-(aminoethyl-aminocaproyl-dihydro-cinnamoyl) (KAAD)-cyclopamine, CUR-61414, SANT-1, SANT-2, SANT-3, SANT-4, IPI-926, IPI-269609, GDC-0449, and NVP-LDE-225. Among these, SANT-1 is preferred. When SANT-1 is used, its concentration in the medium is usually 100 to 500 nM, preferably 50 to 150 nM.

[0064] Examples of BMP inhibitors used in step C) include proteinaceous inhibitors such as chordin, noggin, and follistatin, dorsomorphin (i.e., 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), its derivatives (PB Yu et al. (2007), Circulation, 116:II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904), and LDN-193189. Among these, LDN-193189 is preferred. When using LDN-193189, its concentration in the medium is usually 10 to 1000 nM, preferably 100 to 300 nM.

[0065] The differentiation into pancreatic endoderm cells in step D) can be carried out, for example, by culturing the posterior foregut cells obtained in step C) in a medium containing a growth factor and a BMP inhibitor. The medium may also contain a non-muscle myosin II inhibitor, a TGFβ inhibitor, nicotinamide, etc. The culture period is usually 2 to 10 days.

[0066] When performing step D), the posterior foregut cells obtained in step C) may be treated with 0.25% trypsin-EDTA, dispersed by pipetting, and then suspended by centrifuging the 0.25% trypsin-EDTA, according to a previous report (Toyoda et al., Stem Cell Research (2015) 14, 185-197).

[0067] The types and concentrations of the growth factors and BMP inhibitors used in step D) are the same as those described in steps B) and C).

[0068] Examples of non-muscle myosin II inhibitors used in step D) include blebbistatin A3, calphostin C, Goe6976, Goe7874, Fasudil / HA1077, hypericin, K-252a, KT5823, ML-7, ML-9, piceatannol, staurosporine, W-5, W-7, W-12, W-13, and wortmannin. Among these, blebbistatin is preferred. When blebbistatin is used as the non-muscle myosin II inhibitor, its concentration in the medium is 1 μM to 200 μM, preferably 10 μM to 100 μM.

[0069] The TGFβ inhibitor used in step D) is not particularly limited as long as it is a substance that inhibits signal transduction from the binding of TGFβ to the receptor to SMAD, and inhibits the binding to the ALK family receptor, or inhibits the phosphorylation of SMAD by the ALK family. Examples of the TGFβ inhibitor include Lefty-1, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276, A-83-01 (WO 2009146408), ALK5 inhibitor II (ALK5 inhibitor II), and the like. Examples of such inhibitors include TGFβRI kinase inhibitor II (2-[3-[6-methylpyridine-2-yl]-1H-pyrazole-4-yl]-1,5-naphthyridine; CAS: 446859-33-2), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline), DMH1, and derivatives thereof. ALK5 inhibitor II is preferred. When ALK5 inhibitor II is used as the TGFβ inhibitor, the concentration in the medium is usually 0.1 μM to 50 μM, more preferably 1 μM to 20 μM.

[0070] The concentration of nicotinamide in the medium used in step D) is usually 1 mM to 100 mM, preferably 5 mM to 50 mM.

[0071] In step D), the culture may be performed in the presence of a ROCK inhibitor for the first day, and thereafter in a medium containing no ROCK inhibitor. The ROCK inhibitor may be the same as that used in the production method of the present invention. When Y-27632 is used as the ROCK inhibitor, the concentration in the medium is usually 1 to 20 μM, preferably 5 to 15 μM.

[0072] As shown in the Examples below, culturing pancreatic endoderm cells in the presence of a specific low-molecular-weight compound or protein (hereinafter, sometimes referred to as "expansion-promoting factor") has been shown to improve the proliferation efficiency of the cells. Therefore, the method of the present invention may include a step of culturing pancreatic endoderm cells in a medium containing an expansion-promoting factor. Examples of such expansion-promoting factors include TGFβ inhibitors, retinoic acid receptor agonists, growth factors (e.g., NGF, βcellurin, TGFα, PDGFAA, LIF, IGF-1, FGF9, FGF10, etc.), and bone morphogenetic proteins (BMPs) (e.g., BMP4, BMP7, etc.), with TGFβ inhibitors and retinoic acid receptor agonists being preferred. More specific examples of expansion-promoting factors include those shown in Figure 7. One or more types of expansion-promoting factors may be used. In one embodiment of the present invention, the medium used in the production method of the present invention contains a TGFβ inhibitor and / or a retinoic acid receptor agonist, and preferably contains both a TGFβ inhibitor and a retinoic acid receptor agonist.

[0073] The TGFβ inhibitor may be the same as the TGFβ inhibitor used in step D). Among the TGFβ inhibitors, ALK5 inhibitor II, SB431542, A-83-01, LY2109761, and DMH1 are preferred, with ALK5 inhibitor II being more preferred. When ALK5 inhibitor II is used as the TGFβ inhibitor, its concentration in the medium is typically 0.1 μM to 50 μM, more preferably 1 μM to 20 μM. In one embodiment, it is 10 μM.

[0074] The retinoic acid receptor agonist can be the same as the retinoic acid receptor agonist used in step C). Among them, retinoic acid and TTNPB are preferred, with retinoic acid being more preferred. When retinoic acid or a salt thereof is used as the retinoic acid receptor agonist, the concentration in the medium is usually 100 nM to 10 μM, more preferably 500 nM to 5 μM. In one embodiment, it is 1 μM.

[0075] In another aspect of the present invention, pancreatic endoderm cells obtained by the method of the present invention (hereinafter also referred to as "pancreatic endoderm cells of the present invention") are also provided. Such pancreatic endoderm cells have the ability to differentiate into at least β-like cells and express at least PDX1 and NKX6.1. The pancreatic endoderm cells may further express one or more gene markers, such as SOX9 and GATA4.

[0076] As used herein, "expressing" various gene markers such as PDX1 or "positive" for the marker means at least "production of mRNA encoded by the gene," unless otherwise specified, but preferably also means "production of protein encoded by the mRNA." Therefore, when the production of mRNA encoded by the gene is detected at least by quantitative RT-PCR, it can be said that the gene is expressed. On the other hand, when the production of mRNA encoded by the gene is not detected by quantitative RT-PCR (i.e., below the detection limit) or is at background levels, it can be said that the gene is not expressed or is negative.

[0077] 2. Pancreatic Endoderm Cell Expansion Kit The present invention further provides a pancreatic endoderm cell expansion kit (hereinafter sometimes referred to as the "expansion kit of the present invention") comprising a ROCK inhibitor and KGF and / or EGF. The "pancreatic endoderm cell expansion kit" can also be interpreted as a "pancreatic endoderm cell proliferation kit" or a "pancreatic endoderm cell production kit." The expansion kit of the present invention preferably contains nicotinamide. The expansion kit of the present invention also preferably contains a TGFβ inhibitor and / or a retinoic acid receptor agonist.

[0078] Furthermore, the expansion culture kit of the present invention may contain at least one of a basal medium, a medium additive, a culture vessel, and pancreatic endoderm cells and their precursor cells (e.g., pluripotent stem cells, definitive endoderm cells, primitive gut cells, posterior foregut cells, etc.). The definitions and specific examples of each of the constituent substances contained in the expansion culture kit of the present invention, such as a ROCK inhibitor, a TGFβ inhibitor, and a retinoic acid receptor agonist, are all incorporated by reference in the description above under "1. Method for producing pancreatic endoderm cells."

[0079] 3. Method for Producing β-Like Cells As described above, the pancreatic endoderm cells of the present invention have at least the ability to differentiate into β-like cells. Therefore, in another aspect, a method for producing β-like cells or their precursor cells, which includes inducing the differentiation of pancreatic endoderm cells of the present invention into β-like cells or their precursor cells (hereinafter, sometimes referred to as the "method for producing β-like cells of the present invention"), and β-like cells or their precursor cells obtained by this method (hereinafter, also referred to as the "β-like cells of the present invention") are provided. Examples of precursor cells for β-like cells include endocrine cells that express NGN3 and immature β cells that express insulin and NKX6.1. From the perspective of clinical use, it is preferable that all steps of the method for producing β-like cells of the present invention be performed under feeder-free and xeno-free conditions.

[0080] Induction of differentiation of pancreatic endoderm cells into β-like cells or their precursor cells can be carried out by known methods such as those described in Patent Document 1 and Non-Patent Documents 1 and 2. Specifically, differentiation can be induced by a method including, for example, Step E) a step of inducing differentiation from the pancreatic endoderm cells of the present invention into endocrine precursor cells, and Step F) a step of inducing differentiation from the endocrine precursor cells into β-like cells.

[0081] The basal medium and medium additives used in steps E) and F) can be the same as those used in the production method of the present invention. The culture temperature in steps E) and F) is typically about 30-40°C, preferably about 37°C, and the culture is performed in a CO2-containing air atmosphere, with a CO2 concentration of preferably about 2-5%. Steps E) and F) are preferably performed by suspension culture. The definition and method of suspension culture are described in "1. Method for producing pancreatic endoderm cells" above.

[0082] The differentiation into endocrine precursor cells in step E) can be carried out, for example, by culturing the pancreatic endoderm cells of the present invention in a medium containing a γ-secretase inhibitor and a TGFβ inhibitor. The medium may also contain thyroid hormone, growth factors, hedgehog pathway inhibitors, retinoic acid derivatives, BMP inhibitors, etc. The culture period is usually 1 to 5 days.

[0083] The types and concentrations of the TGFβ inhibitor, growth factor, hedgehog pathway inhibitor, retinoic acid derivative, and BMP inhibitor used in step E) are the same as those described in steps C) and D).

[0084] Examples of γ-secretase inhibitors used in step E) include RO4929097, DAPT (GSI-IX), semagacestat (LY450139), and dibenzazepine (YO-01027). Among these, RO4929097 is preferred. When RO4929097 is used, its concentration in the medium is generally 0.1 to 10 μM, preferably 0.5 to 5 μM.

[0085] Examples of thyroid hormones used in step E) include triiodothyronine (T3) and thyroxine (T4). Among these, triiodothyronine is preferred. When triiodothyronine is used, its concentration in the medium is usually 0.1 to 10 μM, preferably 0.5 to 5 μM.

[0086] The differentiation into β-like cells in step F) can be carried out, for example, by culturing the endocrine precursor cells obtained in step E) in a medium used in step E) from which a γ-secretase inhibitor has been removed. The culture period is usually 4 to 10 days.

[0087] Other culture conditions, culture methods, additives to the medium, specific examples of culture vessels, surface treatments of the culture vessels, etc. are the same as those described above in "1. Method for producing pancreatic endoderm cells."

[0088] Pancreatic exocrine cells can also be produced from the pancreatic endoderm cells of the present invention by methods known per se. Examples of such methods include those described in International Publication No. 2014 / 104403. Specifically, pancreatic exocrine cells can be produced by culturing the pancreatic endoderm cells of the present invention in a culture medium supplemented with a histone deacetylase inhibitor and / or a Notch signaling ligand protein, and a protein kinase C activator. The contents of International Publication No. 2014 / 104403 are incorporated by reference in their entirety for the specific types and production methods of histone deacetylase inhibitors, Notch signaling ligand proteins, and protein kinase C activators.

[0089] 4. Cell Transplantation Therapy The pancreatic endoderm cells of the present invention and the β-like cells of the present invention (collectively referred to as "cells of the present invention") can be induced to differentiate into pancreatic islet-like cells by transplantation into a mammalian organism. Therefore, the cells of the present invention can be suitably used in cell transplantation therapy. Therefore, in another aspect of the present invention, a cell transplantation therapy agent (hereinafter, sometimes referred to as "cell transplantation therapy agent of the present invention") containing the cells of the present invention is provided. The present invention also encompasses a method for treating pancreatic diseases, in which an effective amount of the cells of the present invention is administered or transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) to be treated. Examples of pancreatic diseases that can be treated include acute pancreatitis, chronic pancreatitis, type 1 diabetes, type 2 diabetes, pancreatic tumors, and islet tumors of Langerhans.

[0090] The cells or cell transplantation therapeutic agent of the present invention can be used by transplanting them into the living body of a patient in need thereof. Transplantation is preferably performed in an area of ​​the body where the cells can be fixed at a fixed position, such as subcutaneously, intraperitoneally, into the peritoneal epithelium, omentum, adipose tissue, muscle tissue, or under the capsule of various organs such as the pancreas and kidney. Subcutaneous transplantation, which is less invasive, is preferred. The cells to be transplanted should be administered in a therapeutically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the recipient, and are not particularly limited, but may be, for example, 10 x 10 4 Cell ~10×10 11 It can be as small as a cell.

[0091] When the cells of the present invention are used in cell transplantation therapy, it is desirable to use cells derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to avoid rejection. Here, "substantially the same" means that the HLA genotype is identical to that of the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants, e.g., somatic cells with an HLA type that matches the three HLA loci (HLA-A, HLA-B, and HLA-DR) or the four HLA loci (HLA-C). If sufficient cells cannot be obtained due to age, constitution, or other reasons, they can be transplanted in a state that avoids rejection by embedding them in capsules or porous containers made of polyethylene glycol or silicone.

[0092] The cells of the present invention are prepared as parenteral formulations such as injections, suspensions, and infusions by mixing with a pharmaceutically acceptable carrier according to conventional methods. Thus, in one embodiment, there is also provided a method for producing a cell transplantation therapy agent, which includes a step of formulating the cells of the present invention. Such a production method may include a step of preparing the cells of the present invention. Furthermore, it may also include a step of preserving the cells of the present invention.

[0093] Pharmaceutically acceptable carriers that can be contained in such parenteral formulations include aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), etc. The cell transplantation therapeutic agent of the present invention may be formulated with, for example, a buffer (e.g., phosphate buffer, sodium acetate buffer), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc.

[0094] The cell transplantation therapeutic agent of the present invention is provided in a cryopreserved state under conditions typically used for cryopreserving cells, and can be thawed immediately before use. In this case, it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be about 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0095] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0096] <Materials and Methods> Screening of aging-related reagents. After differentiation to the pancreatic endoderm cell level by a stepwise differentiation induction method, 2 × 10 cells were cultured in a 24-well plate. 5The cells were replated at 1 / well. The cultured cells were then cultured for 1 week in the following medium: Control medium was improved MEM supplemented with 0.5x B-27 Supplement, 100 U / ml Penicillin / Streptomycin, 100 ng / ml KGF / FGF7, 50 ng / ml EGF, and 10 mM Nicotinamide. Intervention groups were cultured in the same medium supplemented with Y-27632, Terreic acid, Daidzein, PD98059, Metformin, BPTES, ABT263, ARV825, or 17-DMAG. After fixation with 4% PFA, cells were stained with β-gal and immunostained with NKX6.1 antibody. Fluorescent images were captured using a BZ-800 (Keyence). The number of NKX6.1-positive cells, the NKX6.1-positive rate, and the β-gal-positive rate were calculated using the BZ-H4CM / macro cell counting function of the microscope. An outline of this procedure is shown in Figures 2-A and 3-A.

[0097] Expansion-promoting reagent screening: Cells on the 21st day of expansion culture were cultured for 6 days in a medium containing Y-27632 plus various growth factors and small molecule compounds. Other procedures were the same as in the senescence-related reagent screening.

[0098] PDX1 + / NKX6.1 + Pancreatic endoderm cell expansion culture 1. PDX1 + / NKX6.1 + Preparation of pancreatic endoderm cells: Using the method of Toyoda et al. (Toyoda T, et al. Stem Cell Reports 2017), PDX1 cells were cultured in a 6-well plate from iPS cells (585A1 line) or ES cells (KhES-3 line). + / NKX6.1 + Pancreatic endoderm cells were prepared. Briefly, 1 × 10 undifferentiated cells were cultured in a 6-well plate coated with iMatrix-511 silk. 6Cells were seeded at 1 / well and cultured for 4 days in S1 medium (RPMI supplemented with 1x B-27 supplement, 100 U / ml penicillin / streptomycin, 100 ng / ml activin A, and CHIR99021 (3 μM on day 1, 1 μM on days 2-3, and 0 μM on day 4)), 5 days in S2 medium (improved MEM supplemented with 0.5x B-27 supplement, 100 U / ml penicillin / streptomycin, and 50 ng / ml KGF / FGF7), and 2 days in S3 medium (improved MEM supplemented with 0.5x B-27 supplement, 100 U / ml penicillin / streptomycin, 50 ng / ml KGF / FGF7, 0.2 μM LDN-193189, 0.1 μM SANT-1, and 10 nM TTNPB). The cells were detached from the 6-well plate using trypsin and then plated in S3 medium onto a 6-well plate coated with iMatrix-511 silk at a density of 1.5 × 10 cells / well. 6 The next day, the medium was changed to S4 medium (improved MEM supplemented with 0.5x B-27 Supplement, 100 U / ml penicillin / streptomycin, 100 ng / ml KGF / FGF7, 50 ng / ml EGF, 10 mM nicotinamide, and 50 μM Y-27632) and cultured for 4 days.

[0099] 2. PDX1 + / NKX6.1 +Pancreatic endoderm cell expansion culture (1) After washing the cells prepared in step 1 above with PBS(-), 1 ml of 0.25% trypsin-EDTA was added and incubated for 5 minutes at 37°C and 5% CO2. (2) Adherent cells were dissociated by pipetting and added to a 50 ml centrifuge tube. (3) 4 ml of DMEM (Dulbecco's Modified Eagle Medium) / 10% FBS / PS (Penicillin-Streptomycin) was added to the centrifuge tube. (4) The tube was centrifuged at 400G for 3 minutes and the supernatant was removed. (5) The cells were suspended in Improved MEM (containing 10 μM Y-27632, 0.5x B-27 Supplement, and 100 U / ml penicillin / streptomycin) and counted. (6) When seeding onto a 6-well plate, 1 x 10 6 The required amount of suspension was collected to obtain cells / well and centrifuged at 400 G for 3 minutes (2 × 10 cells / well for seeding in a 24-well plate). 5 (7) After removing the supernatant, the cells were resuspended in improved MEM (containing 100 ng / ml KGF, 50 ng / ml EGF, 10 mM nicotinamide, 50 μM Y-27632, 0.5× B-27 Supplement, and 100 U / ml penicillin / streptomycin) to a suspension volume of 2 ml / well, and then seeded. One hour before seeding, a 6-well plate was coated with a mixture of 10 μl of iMatrix-511 silk and 1.5 ml of PBS(-) per well, and incubated at 37°C in 5% CO2 for 1 hour.

[0100] (8) The next day (Day 1), the cells were washed with improved MEM and then replaced with improved MEM containing 100 ng / ml KGF, 50 ng / ml EGF, 10 mM nicotinamide, 50 μM Y-27632, 0.5× B-27 Supplement, and 100 U / ml penicillin / streptomycin (5 ml / well). (9) Three days later (Day 4), the cells were washed with improved MEM and then replaced with the same medium as in step (7) (5 ml / well). (10) Three days later (Day 7), steps (1) to (7) were repeated for the next passage (expansion culture). From the following day, steps (8), (9), and (10) were repeated for expansion and subculture. One passage (expansion culture) corresponds to 7 days (the above procedure). An overview of this procedure is shown in Figure 4-A. When using an ALK5 inhibitor (Fujifilm Wako Chemicals; 018-23023; ALK5 inhibitor II (CAS: 446859-33-2)) (concentration in medium: 10 μM) and retinoic acid (Sigma; R2625; CAS: 302-79-4)) (concentration in medium: 1 μM), the ALK inhibitor and RA, or DMSO (concentration in medium: 0.1% (v / v)) were added to the medium in steps (7) to (9) above.

[0101] After expansion, the pancreatic endoderm cells were induced to differentiate into β-like cells using a modified version of the method of Kimura et al. (Non-Patent Document 1). Briefly, the pancreatic endoderm cells were detached from a 6-well plate using trypsin and then cultured in S4 medium at a concentration of 3 × 10 5 The cells were suspended at 3 × 10 cells / ml in a 96-well bottom plate. 4Cells were seeded at 100 μl / well. After incubation at 37°C and 5% CO2 for 1 day, the medium was changed to S5 medium (improved MEM supplemented with 0.5x B-27 supplement, 100 U / ml penicillin / streptomycin, 10 μM ALK5 inhibitor II (CAS:446859-33-2), 1 μM triiodothyronine (T3), 1 μM RO4929097, and 20 ng / ml betacellulin) and cultured for 1 week. The medium was then changed to S6 medium (improved MEM supplemented with 0.5x B-27 supplement, 100 U / ml penicillin / streptomycin, 10 μM ALK5 inhibitor II, and 1 μM T3) and cultured for another week.

[0102] Evaluation Method 1. Immunostaining Expanded cultured cells were washed twice with PBS(-) and fixed with 4% PFA for 20 minutes at 4°C. Blocking was performed for 30 minutes at room temperature using blocking solution (5% donkey serum and 0.4% Triton X-100 in PBS(-)). Primary antibody solution diluted in blocking solution was used for overnight incubation at 4°C. After washing out the primary antibody solution, fluorescent secondary antibody solution diluted in blocking solution was used for one hour of incubation at room temperature. Immunofluorescent staining images were captured using a BZ-710 or BZ-800 (Keyence).

[0103] 2. Flow cytometry analysis. Expanded cells were dissociated with 0.25% trypsin-EDTA and fixed using the Cytofix / Cytoperm Kit (BD Biosciences) according to the protocol. Blocking was performed with permeabilization solution containing 2% donkey serum. Primary antibody diluted in blocking solution was used and incubated overnight at 4°C. After washing out the primary antibody solution, fluorescent secondary antibody diluted in blocking solution was used and incubated for one hour at room temperature. Stained cells were analyzed using a FACSAria II (BD Biosciences).

[0104] 3. Quantitative evaluation of cell nuclear morphology Cells fixed in 4% PFA on plastic bottom plates were stained with Hoechst. Fluorescent stained images were acquired using a BZ-800 (Keyence) and cell morphology evaluation values, including eccentricity (circularity), were obtained using the MeasureObjectSizeShape function in CellProfiller software.

[0105] Statistical analysis: The Mann-Whitney U test was used as a nonparametric method for comparing two groups. For comparative analyses of three or more groups, one-way analysis of variance (ANOVA) was used. If the ANOVA showed a significant difference, the Dunnett's test was used for multiple comparisons between one control group and another treatment group. A P value of less than 0.05 was considered significant.

[0106] Information on the reagents used in the examples is as follows:

[0107]

[0108]

[0109] Example 1: Search for a reagent capable of proliferating pancreatic endoderm cells We searched for a reagent capable of proliferating human iPS cell-derived pancreatic endoderm cells from among senescence-related reagents. The senescence-related reagents selected were Y-27632, Terreic Acid, Daidzein, PD98059, Metformin, BPTES, BPTES, ABT263, ARV825, and 17-DMAG. We then cultured NKX6.1 cells in a medium containing each senescence-related reagent. + S4d4 cells containing pancreatic endoderm cells were cultured, and (1) the number of NKX6.1-positive cells or (2) the percentage of NKX6.1-positive cells and β-gal-positive cells were measured after culture. The results are shown in Figures 2B to 2D. Figure 2B shows that while other senescence-related reagents did not significantly increase pancreatic endoderm cells, Y-27632 significantly increased the target cells, and 50 μM was the most suitable concentration for proliferation. Figure 2C shows that, as in Figure 2B, 50 μM Y-27632 was the most effective when the percentage of pancreatic endoderm cells was used as the outcome. Furthermore, Figure 2D shows that Y-27632 also reduced the percentage of β-gal-positive cells (senescent cells). On the other hand, the percentage of β-gal-positive cells also decreased with other aging-related reagents. Considering this together with Figure 2-B, it is speculated that the decrease in the percentage of β-gal-positive cells caused by BPTES, ABT263, etc. is due to toxicity.

[0110] Example 2: Verification of the proliferation effect of ROCK inhibitors other than Y-27632 on pancreatic endoderm cells. Since Example 1 demonstrated that the ROCK inhibitor Y-27632 has a proliferation effect on pancreatic endoderm cells, it was confirmed that other ROCK inhibitors also have a similar effect on human iPS cell-derived PDX1 + / NKX6.1 + We examined whether or not a proliferation effect on pancreatic endoderm cells was observed. The experiment was performed in the same manner as in Example 1. The results are shown in Figures 3-B and 3-C. As shown in Figures 3-B and 3-C, ROCK inhibitors other than Y-27632 (GSK269962, GSK429286A, fasudil hydrochloride, H1152, and thiazovivin) also had a proliferation effect on pancreatic endoderm cells, similar to that of Y-27632.

[0111] These findings strongly suggest that ROCK inhibitors exert a broad effect on pancreatic endoderm cell proliferation, regardless of the type of cell.

[0112] Example 3: Verification of expansion culture of pancreatic endoderm cells using Y-27632 (50 μM) We verified whether or not pancreatic endoderm cells derived from human iPS cells could be expanded using Y-27632. The results are shown in Figures 4-B to 4-H. Figure 4-B shows that expansion culture using Y-27632 (50 μM) resulted in 1 x 10 5 As shown in Figure 4C, expansion culture using Y-27632 (50 μM) increased the total cell number by more than 1 × 10 5 PDX1 up to more than double + / NKX6.1 + The number of pancreatic endoderm cells was confirmed to increase. Figure 4D shows immunostaining of cells after expansion with Y-27632 (50 μM) confirming that the cells maintained expression of PDX1 and NKX6.1, as well as Ki67. Figure 4E shows that expansion with Y-27632 (50 μM) maintained the proportion of pancreatic endoderm cells and the proportion of Ki67-positive cells. Figure 4F shows that expansion with Y-27632 (50 μM) maintained the proliferation potential (= proportion of Ki67-positive cells). Figure 4G shows that pancreatic endoderm cells after two rounds of expansion maintained their ability to differentiate into β-like cells. Figure 4H shows that expansion of pancreatic endoderm cells derived from ES cells (KhES-3 line) was also possible, but expansion was difficult without the use of Y-27632.

[0113] These results demonstrate that Y-27632 (50 μM) enables efficient expansion of pancreatic endoderm cells for at least 60 days, and that the pancreatic endoderm cells maintain their ability to differentiate into β-like cells after expansion.

[0114] Example 4: Verification of expansion of pancreatic endoderm cells using Y-27632 at different concentrations Since Example 3 demonstrated that expansion of pancreatic endoderm cells was possible using 50 μM Y-27632, the effect on expansion was verified when the concentration was reduced to 10 μM. The results are shown in Figures 5-A to 5-C. Figure 5-A shows that when Y-27632 (10 μM) was used, the expansion rate of 1 × 10 cells was increased over 40 days, similar to when Y-27632 (50 μM) was used. 4 PDX1 + / NKX6.1 + It was confirmed that the number of pancreatic endoderm cells and total cell number increased. On the other hand, Figure 5-B and Figure 5-C show that when Y-27632 (10 μM) was used, the morphology of pancreatic endoderm cells derived from the KhES-3 line changed (becoming elongated and oval) after five passages (five weeks).

[0115] From the above, it was shown that expansion of Y-27632 is possible even at a concentration of at least 10 μM in the medium, but from the viewpoint of cell quality, it is preferable to use it at 50 μM.

[0116] Example 5: Verification of the mechanism by which Y-27632 enables the expansion of pancreatic endoderm cells. The mechanism by which Y-27632 enables the expansion of pancreatic endoderm cells was verified. At the third passage of expansion, the group not treated with Y-27632 showed an increase in α-SMA-positive cells compared to the group treated with Y-27632 at a concentration of 50 μM (Figure 6). In other words, administration of Y-27632 to cells suppressed fibrosis and epithelial-mesenchymal transition.

[0117] These findings suggest that the primary mechanism by which Y-27632 enables the expansion of pancreatic endoderm cells is not through anti-apoptosis, but through the inhibition of cell senescence and the associated suppression of fibrosis or epithelial-mesenchymal transition.

[0118] Example 6: Search for reagents that promote proliferation of pancreatic endoderm cells. Repeated induction in the expansion culture medium containing Y-27632 tended to result in a gradual decline in the NKX6.1-positive rate and the efficiency of induction into β-like cells. Therefore, we attempted to improve the expansion culture method by screening using proteins such as growth factors and low-molecular-weight compounds.

[0119] Pancreatic endoderm cells were passaged three times in medium supplemented with Y-27632 (50 μM), and then cultured at 2.0 × 10 cells per well on a 24-well plate coated with iMatrix. 5 Cells were seeded at 1000 cells / well. Each screening reagent was added to medium containing Y-27632 (50 μM) and cultured. After 6 days, the percentage of NKX6.1-positive cells was examined by immunohistochemistry. The results identified ALK5 inhibitors (ALK5i) and retinoic acid receptor agonists as potential factors for improving expansion (Figure 7).

[0120] Example 7: Verification of ALK5 inhibitors (ALK5i) and retinoic acid receptor agonists We attempted to determine whether long-term expansion culture was possible while maintaining the NKX6.1-positive rate by adding the two candidate factors to the expansion culture method using Y-27632 (50 μM).

[0121] Pancreatic endoderm cells were passaged weekly and counted. Immunohistochemical staining was performed at each passage to determine the NKX6.1-positive cell percentage, and the cumulative NKX6.1 increase was calculated from these values. This calculation method was performed for two conditions: the DMSO group (Y-27632 + DMSO) and the ALK5i + RA group (Y-27632 + ALK5i + RA). The results showed that the addition of ALK5i and RA improved proliferation efficiency while maintaining the NKX6.1-positive cell percentage compared to the Y-27632 alone group (Figure 8).

[0122] Example 8: Verification of β-like cell induction efficiency To confirm the function of the expanded pancreatic endoderm cells, the β-like cell induction efficiency after expansion was examined.

[0123] Pancreatic endoderm cells that had been passaged five times using an expansion culture method with either "Y-27632+DMSO" or "Y-27632+ALK5i+RA" were induced to differentiate into β-like cells. The efficiency of β-like cell induction was then compared with cell masses induced from unpassaged pancreatic endoderm cells. The improved expansion culture method ("Y-27632+ALK5i+RA") demonstrated a higher efficiency of β-like cell induction than the unimproved expansion culture method ("Y-27632+DMSO"), and the induction efficiency was comparable to that of pancreatic endoderm cells before expansion culture (Figure 9).

[0124] The methods of the present invention, and pancreatic endoderm cells produced by the present invention, or pancreatic endocrine cells and pancreatic exocrine cells, including β-like cells derived from these cells, can be applied to the development of cell therapy for pancreatic diseases (particularly type 1 and type 2 diabetes), to drug discovery screening systems for pancreatic diseases, and to the creation of pancreatic disease models using iPS cells derived from patients with hereditary pancreatic diseases.

[0125] This application is based on patent application No. 2022-153013 filed in Japan (filing date: September 26, 2022), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing pancreatic endodermal cells, comprising the step of culturing pancreatic endodermal cells in a medium containing a ROCK inhibitor and KGF and / or EGF.

2. The method according to claim 1, wherein the culture is carried out for two days or more.

3. The method according to claim 1, wherein the ROCK inhibitor is selected from the group consisting of Y-27632, GSK269962, GSK429286A, fasudil hydrochloride, H1152, and thiazovibin.

4. The method according to claim 1, wherein the ROCK inhibitor is Y-27632.

5. The method according to claim 1, wherein the culture medium comprises a TGFβ inhibitor and / or a retinoic acid receptor agonist.

6. The method according to claim 5, wherein the TGFβ inhibitor is 2-[3-[6-methylpyridine-2-yl]-1H-pyrazole-4-yl]-1,5-naphthiridine.

7. The method according to claim 5, wherein the retinoic acid receptor agonist is retinoic acid.

8. The method according to claim 1, wherein the pancreatic endoderm cells are derived from pluripotent stem cells.

9. The method according to claim 1, wherein the pancreatic endodermal cells are cells derived from a patient with a hereditary pancreatic disease.

10. The method according to claim 1, wherein the culture is performed under feeder-free conditions.

11. Pancreatic endoderm cells produced by the method described in any one of claims 1 to 10.

12. A kit for expanding pancreatic endodermal cell culture, comprising a ROCK inhibitor and KGF and / or EGF.

13. The kit according to claim 12, comprising a TGFβ inhibitor and / or a retinoic acid receptor agonist.

14. A method for producing β-like cells or their progenitor cells, comprising the step of inducing differentiation of pancreatic endodermal cells according to claim 11 into β-like cells or their progenitor cells.

15. A cell transplantation therapy agent comprising pancreatic endodermal cells as described in claim 11.

16. A cell transplantation therapy agent comprising cells produced by the method of Claim 14.

17. The agent according to claim 15 for the treatment of diabetes.

18. The agent according to claim 16 for the treatment of diabetes.