Method for producing pancreatic endoderm cells

Abstract

Provided are: a method for producing pancreatic endoderm cells, which comprises 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 the method.

Description

Technical Field

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

[0002] The pancreas plays a role as an exocrine gland that secretes digestive enzymes such as pancreatic lipase, trypsin, elastase, and pancreatic amylase, and an endocrine gland that secretes pancreatic hormones such as glucagon, insulin, somatostatin, and pancreatic polypeptide (PP). In recent years, it has been reported that ghrelin, a gastric secretory hormone, is also secreted by the endocrine cells of the pancreas. The pancreatic hormone is produced by a cell mass called the islet of Langerhans, which is mainly composed of 4 cells of α cells, β cells, δ cells, and PP cells in the pancreas. Diabetes is a disease that develops due to insufficient insulin or loss of its function, and is a disease that is difficult to cure once it develops. Diabetes can be classified into two major categories: type 1 diabetes (insulin-dependent diabetes) and type 2 diabetes (non-insulin-dependent diabetes).

[0003] Type 2 diabetes is a chronic disease caused by reduced insulin secretion ability and acquired resistance to insulin. It is a diabetes whose pathogenesis is believed to be caused by obesity or stress caused by overeating or lack of exercise. On the other hand, type 1 diabetes is a disease caused by the destruction of β cells (insulin-producing cells) due to autoimmune diseases or viral infections, etc., and the failure to secrete insulin in the body. Symptomatic treatment by administering insulin is mainly carried out. In addition, as a treatment method for type 1 diabetes, it is being studied to induce insulin-producing cells themselves from cells derived from patients in vitro, and to transplant the induced insulin-producing cells into the patient's body. Insulin-producing cells can be obtained, for example, by taking out cells derived from the pancreatic duct epithelium of the patient and differentiating them in vitro.

[0004] However, the removal of cells from patients is accompanied by invasion, and the number of cells obtained cannot be said to be sufficient. Therefore, methods for differentiating and inducing β-like cells required for cell therapy for diabetes from pluripotent stem cells such as artificial pluripotent stem cells (iPS cells) are being developed, and methods for actually differentiating and inducing β-like cells have also been reported (e.g., Patent Document 1, Non-Patent Document 1). However, as Figure 1 As shown, in these methods, 6 to 7 steps (about 4 to 5 weeks) are required to produce β-like cells from pluripotent stem cells (stepwise differentiation induction method).

[0005] In addition, methods for expanding and culturing posterior foregut cells derived from human pluripotent stem cells are also being developed (e.g., Non-Patent Documents 1 and 2). Figure 1 PDX1 in posterior foregut cells + / SOX9 + / NKX6.1 - However, in these methods, PDX1 + / SOX9 + / NKX6.1 - The proliferation efficiency of pancreatic progenitor cells cannot be said to be high, and it is known that when pancreatic progenitor cells are induced to differentiate into NKX6.1-positive pancreatic endoderm cells, the induction efficiency is reduced.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2020 / 059892

[0009] Non-patent literature

[0010] Non-patent literature 1: Kimura et al., Cell Chemical Biology 2020 Dec 17; 27(12): 1561-1572.e7

[0011] Non-patent document 2: Konagaya et al., Scientific Reports. 2019 Jan 24; 9(1): 640 Summary of the invention

[0012] Problems to be solved by the invention

[0013] Therefore, the present invention aims to develop a method for growing (ie, expanding) pancreatic endoderm cells, which are cells at a more advanced stage of differentiation than posterior foregut cells, while maintaining differentiation ability.

[0014] Means for solving problems

[0015] In order to solve the above problems, the present inventors focused on aging-related reagents and screened for reagents that have the potential to expand the culture of pancreatic endoderm cells from aging-related reagents. As a result, it was found that the ROCK inhibitor Y-27632 significantly increased the number of pancreatic endoderm cells, and further found that ROCK inhibitors other than Y-27632 also exerted the same pancreatic endoderm cell proliferation effect. Based on this finding, further verification was carried out, and it was confirmed that pancreatic endoderm cells can be expanded and cultured by adding Y-27632 to a concentration of 10μM or 50μM in the culture medium.

[0016] In cell culture, low concentrations of Y-27632 (e.g., 10 μM) are sometimes used only on the first day of reseeding from the viewpoint of inhibiting apoptosis. However, long-term use of ROCK inhibitors such as Y-27632 is known to change the differentiation state of cells, etc. (Maldonado M. et al., Stem Cell Res 17; 222-227; 2016), so the use of ROCK inhibitors for more than 1 day is avoided. Therefore, it is very surprising that even with high concentrations of Y-27632 of more than 10 μM, and further long-term use of Y-27632 in culture, pancreatic endoderm cells can be expanded and cultured efficiently for a long time while maintaining differentiation ability (in one protocol, 1×10 cells can be proliferated in culture for more than 60 days). 5 times or more).

[0017] The inventors further verified the mechanism of the proliferation effect of pancreatic endoderm cells caused by ROCK inhibitors. It was shown that the main mechanism of this effect is not through anti-apoptosis, but through the inhibition of cell aging and the fibrosis or epithelial-mesenchymal transition associated therewith. Based on these insights, further repeated studies were conducted, and the present invention was completed as a result.

[0018] That is, the present invention is as follows.

[0019] [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.

[0020] [1-2] The method of [1-1], wherein the culture medium contains both KGF and EGF.

[0021] [1-3] The method of [1-1] or [1-2], wherein the culture medium contains nicotinamide.

[0022] [2] The method according to any one of [1-1] to [1-3], wherein the culturing is performed for 2 days or more.

[0023] [3] The method according to any one of [1-1] to [2], wherein the ROCK inhibitor is selected from Y-27632, GSK269962, GSK429286A, fasudil hydrochloride, H1152 and Thiazovivin.

[0024] [4] The method according to any one of [1-1] to [2], wherein the ROCK inhibitor is Y-27632.

[0025] [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.

[0026] [6] The method of [5], wherein the TGFβ inhibitor is 2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine.

[0027] [7] The method of [5] or [6], wherein the retinoic acid receptor agonist is retinoic acid.

[0028] [8] The method according to any one of [1-1] to [7], wherein the pancreatic endoderm cells are derived from pluripotent stem cells.

[0029] [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 hereditary pancreatic disease.

[0030] [10-1] The method according to any one of [1-1] to [9], wherein the culture is culture under feeder-free conditions.

[0031] [10-2] The method according to any one of [1-1] to [10-1], wherein the culture is culture under xeno-free conditions.

[0032]

[11] Pancreatic endoderm cells produced by the method described in any one of [1-1] to [10-2].

[0033] [12-1] A kit for expanding and culturing pancreatic endoderm cells, comprising a ROCK inhibitor and KGF and / or EGF.

[0034] [12-2] The kit described in [12-1], comprising both KGF and EGF.

[0035] [12-3] The kit described in [12-1] or [12-2], comprising nicotinamide.

[0036] [13-1] The kit according to any one of [12-1] to [12-3], comprising a TGFβ inhibitor and / or a retinoic acid receptor agonist.

[0037] [13-2] The kit described in [13-1], comprising both a TGFβ inhibitor and a retinoic acid receptor agonist.

[0038] [13-3] The kit described in [13-1] or [13-2], wherein the TGFβ inhibitor is 2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine.

[0039] [13-4] The kit according to any one of [13-1] to [13-3], wherein the retinoic acid receptor agonist is retinoic acid.

[0040] [14-1] A method for producing β-like cells or their precursor cells, comprising the step of inducing the pancreatic endoderm cells described in

[11] to differentiate into β-like cells or their precursor cells.

[0041] [14-2] Cells produced by the method described in [14-1].

[0042]

[15] A cell transplantation therapy agent comprising the cells described in

[11] or [14-2].

[0043]

[16] The agent of

[15] , which is used for treating diabetes.

[0044] [17-1] A method for treating pancreatic diseases in mammals, characterized by administering an effective amount of the cells described in

[11] or [14-2] or the agent described in

[15] or

[16] to the mammal.

[0045] [17-2] The method described in [17-1], wherein the pancreatic disease is diabetes.

[0046] [18-1] The cell described in

[11] or [14-2] or the agent described in

[15] or

[16] , for use in the treatment of pancreatic diseases.

[0047] [18-2] The cell or agent described in [18-1], wherein the pancreatic disease is diabetes.

[0048] [19-1] Use of the cell described in

[11] or [14-2] or the agent described in

[15] or

[16] for the manufacture of a therapeutic drug for pancreatic diseases.

[0049] [19-2] The cell or agent described in [19-1], wherein the pancreatic disease is diabetes.

[0050] Effects of the Invention

[0051] The present invention enables efficient proliferation of pancreatic endoderm cells. Pancreatic endocrine cells or pancreatic exocrine cells, including cells proliferated in this way or pancreatic β cells obtained by differentiation induction of the cells, are also important in clinical applications. In addition, these cells are useful in regenerative medicine for pancreatic diseases and drug development screening, etc., instead of human pancreatic islets or exocrine cells and tissues that are difficult to obtain as research samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] [ Figure 1 ]Schematic diagram of the method for inducing the staged differentiation of β-like cells.

[0053] [ Figure 2-A ]Schematic diagram of aging-related reagent screening. NKX6.1-positive pancreatic endoderm cells were seeded in a 24-well plate, and various aging-related reagents were added to the conventional stage 4 culture medium for screening. After one week, the number of cells was counted using a fluorescence microscope.

[0054] [ Figure 2-B ] The number of NKX6.1-positive cells when cultured with various aging-related agents. Number of replicates = 3, number of cells = average of the results of 3 times, using Dunnett's test. Error bars show standard errors.

[0055] [ Figure 2-C ] The proportion of NKX6.1-positive cells when cultured with various aging-related agents. Number of replicates = 3, Fraction = average proportion of 3 results, using Dunnett's test. Error bars show standard errors.

[0056] [ Figure 2-D ] The proportion of βgal-positive cells caused by various aging-related agents. Number of replicates = 3, Fraction = average proportion of 3 results, using Dunnett's test. Error bars show standard error.

[0057] [ Figure 3-A ]Schematic diagram of the research experiment on other ROCK inhibitors. NKX6.1-positive pancreatic endoderm cells were seeded in 24-well plates, and various ROCK inhibitors were added to the conventional stage 4 culture medium to conduct the experiment. After 1 week, the number of cells was counted using a fluorescence microscope.

[0058] [ Figure 3-B ] Total cell number when cultured with each ROCK inhibitor. Number of replicates = 3, one-way ANOVA was used. Error bars show standard error.

[0059] [ Figure 3-C] The number of NKX6.1-positive cells and the ratio of NKX6.1-positive cells when cultured with each ROCK inhibitor. The number of replicates = 3, one-way ANOVA was used. Error bars show standard errors.

[0060] [ Figure 4-A ]Schematic diagram of the expansion culture of pancreatic endoderm cells using Y-27632 (50 μM). NKX6.1-positive pancreatic endoderm cells were seeded on a plate and cultured in a medium containing Y-27632 (50 μM) for 1 week. After 1 week, they were re-seeded and the subculture and expansion culture were repeated.

[0061] [ Figure 4-B ] Changes in the total cell number by expansion culture using Y-27632 (50 μM). n=3.

[0062] [ Figure 4-C ] Changes in the number of pancreatic endoderm cells in expanded culture using Y-27632 (50 μM). n=3.

[0063] [ Figure 4-D ] Immunostaining images of pancreatic endoderm cells after expansion culture using Y-27632 (50 μM).

[0064] [ Figure 4-E ] Flow cytometry dot plots of NKX6.1 expression and Ki67 expression in cells expanded with Y-27632 (50 μM).

[0065] [ Figure 4-F ] Changes in the Ki67-positive ratio by expansion culture using Y-27632 (50 μM). The number of replicates = 3, and the error bars indicate standard deviations.

[0066] [ Figure 4-G ] Immunostaining of islet-like cell clusters induced by differentiation of pancreatic endoderm cells after two rounds of expansion culture.

[0067] [ Figure 4-H ] Changes in the number of cells in pancreatic endoderm cells derived from ES cells (KhES-3 strain) during expansion culture.

[0068] [ Figure 5-A ] PDX1 cells were expanded by using Y-27632 (0, 10, or 50 μM) + / NKX6.1 + Transition of pancreatic endoderm cell number and total cell number.

[0069] [ Figure 5-B] Immunostaining images of pancreatic endoderm cells derived from KhES-3 strain after culturing for 5 passages (5 weeks) using Y-27632 (10 or 50 μM).

[0070] [ Figure 5-C ] Figure 5-B Quantitative evaluation of nuclear morphology of all cells in NKX6.1 immunostaining (n=616 cells (10 μM Y-27632); n=519 cells (50 μM Y-27632); biological replicate = 1). The Mann-Whitney U test was used to compare the eccentricity (circularity) between the two groups.

[0071] [ Figure 6 ] Immunostaining images of α-SMA against pancreatic endoderm cells in the third passage of pancreatic endoderm cells with and without administration of Y-27632 (50 μM).

[0072] [ Figure 7 ] The ratio of NKX6.1-positive cells when cultured with each screening agent.

[0073] [ Figure 8 ] The cumulative NKX6.1 in the expanded culture was expressed by using the DMSO group (Y-27632+DMSO) or the ALK5 inhibitor (ALK5i) + retinoic acid (RA) group (Y-27632+ALK5i+RA). + Transition of pancreatic endoderm cell number. Number of replicates = 4.

[0074] [ Fig. 9 ] The ratio of the number of β-like cells induced from pancreatic endoderm cells after 5 expansions using the DMSO group (Y-27632+DMSO) or the ALK5i+RA group (Y-27632+ALK5i+RA). Compared with the control group of β-like cells induced from pancreatic endoderm cells without passage (P0). The number of repeated experiments = 3, and the error bars show the standard deviation. DETAILED DESCRIPTION

[0075] 1. Preparation of pancreatic endoderm cells

[0076] The present invention provides a method for producing pancreatic endoderm cells using a culture 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") comprising a step of culturing pancreatic endoderm cells in a culture medium containing a ROCK inhibitor and KGF and / or EGF. The production method of the present invention may also include a step of isolating the produced pancreatic endoderm cells.

[0077] The method of the present invention allows pancreatic endoderm cells to self-proliferate and produce cells of the same species. That is, the method of the present invention may also be referred to as a method for proliferating pancreatic endoderm cells or a method for expanding pancreatic endoderm cells, which includes culturing pancreatic endoderm cells in a culture medium containing a ROCK inhibitor and KGF and / or EGF.

[0078] In this specification, "pancreatic endoderm cells" means cells that have at least the ability to differentiate into β-like cells and express at least PDX1 and NKX6.1. Furthermore, pancreatic endoderm cells can also be cells that express one or more gene markers such as SOX9 and GATA4. In addition, in this specification, "β-like cells" means cells that express and / or secrete insulin, which are cells induced by in vitro differentiation from endocrine precursor cells or immature β cells and have the same or similar properties as pancreatic β cells in vivo.

[0079] In this specification, unless otherwise specified, "cell" includes "cell population." A cell population may be composed of one type of cell or two or more types of cells.

[0080] In this specification sheets, "enlarged culture" means to cultivate for the purpose of making the desired cell mass propagation and increasing the number of cells. The increase of the number of cells is achieved by exceeding the number of reductions caused by death as long as the increase caused by cell proliferation, and all cells of the cell mass are not required to propagate. The increase of the number of cells can be 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, 300 times, 500 times, 1000 times, 3000 times, 5000 times, 10000 times, 100000 times or more than 1000000 times compared with before the enlarged culture starts.

[0081] The basal culture medium used in the preparation method of the present invention is not particularly limited, and examples thereof include StemFit (registered trademark) AK02 culture medium (Ajinomoto Co., Ltd.), StemFit (registered trademark) AK03 culture medium (Ajinomoto Co., Ltd.), StemFit (registered trademark) Basic03 culture medium, CTS (registered trademark) knockout SR exogenous-free culture medium (Gibco), mTeSR1 culture medium, TeSR1 culture medium (Stem Cell Technologies), Iscove modified Dulbecco's medium (GE HealthCare), modified MEM (Thermo Fisher Scientific Inc.), etc. Among them, modified MEM culture medium is preferred. These culture media can also be used for culture without a feeder layer and without exogenous conditions. In addition, other basal culture media include RPMI-1640 culture medium, EagleMEM (EMEM), Dulbecco's modified MEM, Glasgow's MEM (GMEM), α-MEM, 199 culture medium, IMDM, DMEM, hybridoma serum-free culture medium, KnockOut TM DMEM, Advanced TM medium (e.g., AdvancedMEM, 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 (all from Thermo Fisher Scientific Inc.), ReproFF2, primate ES cell culture medium, ReproStem (all from REPROCELL Inc.), ProculAD (ROHTO Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (all from Lonza), EX-CELL302 medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMedTM iPSC culture medium (REPROCELL Inc.) and mixtures thereof, etc., but are not limited to these.

[0082] As shown in the following examples, the proliferation effect of pancreatic endoderm cells was observed in various types of ROCK inhibitors. That is, in the proliferation of pancreatic endoderm cells, it is important to inhibit the function of Rho-kinase (ROCK), and any ROCK inhibitor can be applied to the preparation method of the present invention as long as it can inhibit this function. As ROCK inhibitors, for example, Y-27632 (for example, refer to Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil / HA1077 (for example, refer to Uenata et al., Nature 389: 990-994 (1997)), SR3677 (for example, refer to Feng Y et al., J Med Chem. 51: 6642-6645 (2008)), GSK269962 (for example, refer to Stavenger RA et al., J Med Chem.50:2-5 (2007) or WO2005 / 037197), GSK429286A, H1152 (for example, see Sasaki et al., Pharmacol.Ther.93:225-232 (2002)), Wf-536 (for example, see Nakajima et al., Cancer Chemother Pharmacol.52(4):319-324 (2003)), Thiazovivin and salts or derivatives thereof. Other ROCK inhibitors include antisense nucleic acids, RNA interference-inducing nucleic acids (for example, siRNA), dominant negative variants and expression vectors thereof against ROCK. In addition, as ROCK inhibitors, other well-known low molecular weight compounds and their salts or derivatives can also be used (for example, refer to 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 them, 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.

[0083] Examples of the salt of the compound 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; inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, hydroiodides, nitrates, and phosphates; and acid addition salts such as organic acid salts such as citrates, oxalates, acetates, formates, propionates, benzoates, trifluoroacetates, maleates, tartrates, methanesulfonates, benzenesulfonates, and p-toluenesulfonates.

[0084] When Y-27632 or a salt thereof (e.g., Y-27632 dihydrochloride, etc.) is used as a ROCK inhibitor, the concentration in the culture medium is usually 1 μM or more (e.g., 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM or more), and 500 μM or less (400 μM, 300 μM, 200 μM, 150 μM, 100 μM or less). In addition, when Y-27632 is used, the concentration in the culture 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.

[0085] When GSK269962 or a salt thereof (eg, GSK269962 hydrochloride) 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, and more preferably 2 μM to 10 μM. In one embodiment, it is 2 μM.

[0086] 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, and more preferably 2 μM to 10 μM. In one embodiment, it is 2 μM.

[0087] When fasudil or a salt thereof (eg, fasudil hydrochloride) is used as a 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. In one embodiment, it is 50 μM.

[0088] When H1152 or a salt thereof (eg, H1152 dihydrochloride) 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, and more preferably 2 μM to 10 μM. In one embodiment, it is 2 μM.

[0089] 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, and more preferably 2 μM to 10 μM. In one embodiment, it is 2 μM or 10 μM.

[0090] KGF is a protein called keratinocyte growth factor, and is sometimes referred to as FGF-7. KGF can be commercially available from R&D systems, for example. The concentration of KGF in the culture 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).

[0091] EGF is a protein called epidermal growth factor or Epidermal Growth Factor. EGF can be used, for example, EGF commercially available from R&D systems. The concentration of EGF in the culture 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).

[0092] Nicotinamide may also be included in the culture medium. The concentration of nicotinamide in the culture medium is generally 0.1 mM to 200 mM, preferably 1 mM to 100 mM, and more preferably 5 mM to 50 mM (eg, 10 mM).

[0093] If necessary, the culture medium may contain culture medium additives other than those mentioned above, for example, one or more fetal bovine serum (FBS), horse serum and other sera, knockout serum replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-mercaptoglycerol and other serum substitutes, and may also contain one or more lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, proliferation factors (growth factors), low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, selenic acid, progesterone and putrescine and other substances.

[0094] The culture in the preparation method of the present invention can be cultured under feeder-free conditions and / or under exogenous conditions in all or part of the time period. From the viewpoint of clinical use, the preparation method of the present invention is preferably carried out under feeder-free conditions and exogenous conditions in all time periods. In this specification, "feeder-free" means a culture medium or culture condition that does not contain other cell types (i.e., feeder cells) used to prepare the culture conditions for the cultured target cells to achieve auxiliary effects. In addition, "exogenous" means a culture medium or culture condition that does not contain a component of a biological source different from the biological species of the cultured target cells.

[0095] The culture in the preparation method of the present invention can be any one of suspension culture and adherent culture as long as the desired cell proliferation can be made, preferably adherent culture. In this specification, "suspension culture" means the culture carried out under the condition that the cell or the aggregate of the cell is maintained as suspended in the culture fluid and exists, that is, the culture under the condition that a firm cell-matrix combination (cell-substratum junction) is not formed between the aggregate of the cell or the cell and the culture container. In addition, in this specification, "adherent culture" refers to the culture of the condition that a firm cell-matrix combination is formed between the aggregate of the cell or the cell and the culture equipment etc.

[0096] Examples of culture vessels used for adherent culture include those whose surfaces have been artificially treated for the purpose of improving cell adhesion (e.g., coating treatment with basement membrane modulators, fibronectin, laminin or its fragments, entactin, collagen, gelatin, Synthemax, vitronectin or other extracellular matrices, or polymers such as polylysine and polyornithine, or surface processing such as positive charge treatment). Among them, culture vessels coated with laminin or its fragments are preferred.

[0097] Examples of laminin or its fragment used in the present invention include a fragment comprising laminin-111 and its E8 region, a fragment comprising laminin-211 and its E8 region (e.g., iMatrix-211), a fragment comprising laminin-121 or its E8 region, a fragment comprising laminin-221 or its E8 region, a fragment comprising laminin-332 or its E8 region, a fragment comprising laminin-3A11 or its E8 region, a fragment comprising laminin-411 or its E8 region (e.g., iMatrix-4 ... A fragment containing laminin-421 or its E8 region, a fragment containing laminin-511 or its E8 region (for example: iMatrix-511, iMatrix-511silk), a fragment containing laminin-521 or its E8 region, a fragment containing laminin-213 or its E8 region, a fragment containing laminin-423 or its E8 region, a fragment containing laminin-523 or its E8 region, a fragment containing laminin-212 / 222 or its E8 region, a fragment containing laminin-522 or its E8 region, etc.

[0098] The culture container used when carrying out suspension culture is not particularly limited as long as it is a culture container that can carry out "suspension culture", and can be appropriately determined by those skilled in the art. As such a culture container, for example, flasks, flasks for tissue culture, dishes, petri dishes, dishes for tissue culture, porous dishes, microplates, microporous plates, micropores, multiplates, multi-well plates, chamber slides, culture dishes, tubes, trays, culture bags, roller bottles can be listed. Further, as a container for suspension culture, a bioreactor is illustrated. In order to carry out suspension culture, these culture containers are preferably cell non-adhesive. As a cell non-adhesive culture container, the surface of the culture container can be used for improving the adhesion with the cell without artificial treatment (for example: the coating treatment carried out by the extracellular matrix, etc.) of the culture container, etc.

[0099] The culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C. The culture is carried out in an atmosphere of air containing CO 2 , and the CO 2 concentration is preferably about 2 to 5%.

[0100] In the preparation method of the present invention, since the target cells are obtained in a long period of time, the culture period is not particularly limited, and is usually 2 days or more (for example: 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 20 days or more), 100 days or less (for example: 90 days, 80 days, 70 days, 60 days or less). In one embodiment, in the preparation method of the present invention, since the pancreatic endoderm cells can also be proliferated for more than 100 days, the culture period can exceed 100 days (for example, 110 days, 120 days or more).

[0101] The cell culture density is not particularly limited as long as the cells can proliferate. It is usually 1.0×10 2 ~1.0×10 7 cells / cm 2 , preferably 1.0×10 3 ~1.0×10 6 cells / cm 2 , more preferably 1.0×10 4 ~1.0×10 5 cells / cm 2 .

[0102] The pancreatic endoderm cells used in the preparation method of the present invention can use cells isolated from an organism or commercially available cell strains, but are preferably cells derived from pluripotent stem cells. In addition, in one embodiment, the pancreatic endoderm cells used as starting cells in the preparation method of the present invention are cells derived from patients with hereditary pancreatic diseases. Pancreatic diseases exemplify acute pancreatitis, chronic pancreatitis, type 1 diabetes, type 2 diabetes, pancreatic tumors, Langerhans island tumors, etc. Among pancreatic diseases, diseases caused by gene abnormalities are hereditary pancreatic diseases. As hereditary pancreatic diseases, specifically hereditary pancreatitis, familial pancreatic tumors, cystic fibrosis, etc. can be cited, but are not limited to these. The pancreatic endoderm cells derived from patients with hereditary pancreatic diseases can use cells isolated from patients, preferably somatic cells derived from patients are initialized to establish iPS cells, and pancreatic endoderm cells produced by differentiation induction of the iPS cells by methods known per se. Pancreatic endoderm cells derived from patients with hereditary pancreatic diseases or cells such as β-like cells differentiated from such cells can be used as pancreatic disease models reflecting the pathology of the disease and are therefore suitable for, for example, screening for therapeutic or preventive drugs for pancreatic diseases.

[0103] "Pluripotent stem cells" refer to tissues or cells with various forms or functions of an organism, and have the ability to differentiate into cells of any system of the three germ layers (endoderm, mesoderm, ectoderm). As the pluripotent stem cells used in the present invention, for example, artificial pluripotent stem cells (induced pluripotent stem cells: iPS cells), embryonic stem cells (embryonic stem cells: ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transplantation (nuclear transfer embryonic stem cells: ntES cells), pluripotent germline stem cells (multipotent germline stem cells) ("mGS cells"), embryonic germ stem cells (EG cells), etc., preferably iPS cells (more preferably human iPS cells). In the case where the above-mentioned pluripotent stem cells are ES cells or any cells derived from human embryos, the cells may be cells made by destroying the embryo, or cells made without destroying the embryo, preferably cells made without destroying the embryo.

[0104] ES cells are stem cells established from the inner cell mass of early embryos (e.g., blastocysts) of mammals such as humans or mice, and have pluripotency and the ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and ES cell lines were subsequently established in primates such as humans and monkeys (JA Thompson et al., (1998), Science 282: 1145-1147; JA Thompson et al., (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thompson et al., (1996), Biol. Reprod., 55: 254-259; A. Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165). ES cells can be established by removing the inner cell mass from the blastocyst of the fertilized egg of the target animal and culturing the inner cell mass on a feeder layer of fibroblasts. Alternatively, ES cells can be established using only a single blastomere of an embryo in the cleavage stage before the blastocyst stage (Chung Y. et al., (2008), Cell Stem Cell 2: 113-117), or using an embryo that has arrested development (Zhang X. et al., (2006), Stem Cells 24: 2669-2676.).

[0105] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology, and have almost the same characteristics 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). That is, ES cells established from the inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with a somatic cell nucleus are nt ES (nuclear transfer ES) cells. In order to produce nt ES cells, a combination of nuclear transplantation technology (Cibelli JB et al., (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (described above) is used (Wakayama Kiyoka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Supplement), pp. 47-52). In nuclear transplantation, somatic cell nuclei can be injected into mammalian enucleated unfertilized eggs and cultured for several hours for initialization.

[0106] As the ES cell strain used in the present invention, if it is a mouse ES cell, for example, various mouse ES cell strains established by inGenious targeting laboratory, RIKEN (Rikagaku Rikagakuen), etc. can be used, and if it is a human ES cell strain, for example, various human ES cell strains established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Developmental Medicine, Cellartis, etc. can be used. Specifically, for example, as human ES cell strains, CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains, etc., sold by ESI Bio, H1 strains, H9 strains, etc., sold by WiCellResearch, KhES-1 strains, KhES-2 strains, KhES-3 strains, KhES-4 strains, KhES-5 strains, SSES1 strains, SSES2 strains, SSES3 strains, etc., sold by RIKEN, etc. can be cited.

[0107] iPS cells are cells obtained by introducing specific factors (nuclear reprogramming factors) into mammalian somatic cells or undifferentiated stem cells for reprogramming. Currently, there are various types of iPS cells. In addition to 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 screening for Nanog expression as an index after the introduction of the above four factors (Okita, K., Ichisaka, T. and Yamanaka, S. (2007). Nature 448, 313-317.); iPSCs established by a method without c-Myc (Nakagawa M, Yamanaka S., et al., Nature Biotechnology, (2008) 26, 101-106); iPSCs established by introducing 6 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. In addition, artificial pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, prepared by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920); artificial pluripotent stem cells prepared by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146); artificial pluripotent stem cells prepared by Sakurada et al. (Japanese Patent Laid-Open No. 2008-307007), etc. can also be used.

[0108] In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, 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. Any of the artificial pluripotent stem cells known in the art described in Japanese Unexamined Patent Application Publication No. 7,795-797) or patent gazettes (e.g., Japanese Unexamined Patent Application Publication No. 2008-307007, Japanese Unexamined Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852).

[0109] As artificial pluripotent stem cell strains, various iPSC strains established by NIH, RIKEN, Kyoto University, etc. can be used. For example, in the case of human iPSC strains, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, etc. of RIKEN, 253G1 strain, 253G4 strain, 1201C1 strain, 1205D1 strain, 1210B2 strain, 1383D2 strain, 1383D6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 585A1 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1231A3 strain, FfI-01s04 strain, etc. of Kyoto University can be cited, and 585A1 strain is preferred.

[0110] mGS cells are pluripotent stem cells derived from the testis and are cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various series of cells, and have the property of being able to produce chimeric mice if transplanted into mouse blastocysts (Kanatsu-Shinohara M. et al., (2003) Biol. Reprod., 69: 612-616; Shinohara K. et al., (2004), Cell, 119: 1001-1012). They can self-replicate in a culture medium containing a neurotrophic factor derived from a glial cell line (glial cell line-derived neurotrophic factor (GDNF), glial cell-derived neurotrophic factor), and by repeatedly passaged under the same culture conditions as ES cells, germ stem cells can be obtained (Takebayashi Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Supplement), pp. 41-46, Yodosha (Tokyo, Japan)).

[0111] EG cells are cells established from primordial germ cells in the embryonic period and have the same pluripotency as ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Matsui Y. et al., (1992), Cell, 70: 841-847; JL Resnick et al., (1992), Nature, 359: 550-551).

[0112] The source species of pluripotent stem cells are also not particularly limited, and can be, for example, rodents such as rats, mice, hamsters, guinea pigs, lagomorpha such as rabbits, ungulates such as pigs, cattle, goats, sheep, carnivora such as dogs and cats, primates such as humans, monkeys, macaques, marmosets, gorillas, chimpanzees, etc. The preferred source species is human.

[0113] Differentiation induction of pluripotent stem cells into pancreatic endoderm cells can be performed by known methods such as the methods 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 induction can be performed, for example, by methods including the following:

[0114] A) The process of inducing pluripotent stem cells to differentiate into embryonic endoderm cells,

[0115] B) The process of inducing embryonic endoderm cells to differentiate into primitive intestinal tube cells.

[0116] C) The process of inducing the differentiation of primitive intestinal tube cells into posterior foregut cells.

[0117] D) The process of inducing the differentiation of posterior foregut cells into pancreatic endoderm cells.

[0118] The basal medium or medium additive used in steps A) to D) can use the same basal medium or medium additive as the basal medium or medium additive used in the preparation method of the present invention. In addition, the culture temperature in steps A) to D) is usually about 30 to 40°C, preferably about 37°C, and the culture is carried out in an atmosphere of air containing CO2, and the CO2 concentration is preferably about 2 to 5%. In addition, steps A) to D) are preferably carried out by adherent culture. The definition or method of adherent culture is as described above.

[0119] The differentiation into embryonic endoderm cells in step A) can be performed, 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 generally 2 to 8 days.

[0120] The concentration of activin A used in step A) in the culture medium is, for example, 5 to 1000 ng / mL, preferably 20 to 500 ng / mL, and more preferably 50 to 150 ng / mL.

[0121] As the GSK3β inhibitor used in step A), for example, CHIR98014, CHIR99021, TDZD-8, SB216763, TWS-119, kenpaullone, 1-azakenpaullone, SB216763, SB415286, AR-AO144-18, CT99021, CT20026, etc. can be mentioned. Among them, CHIR99021 is preferred. When CHIR99021 is used, the concentration in the culture medium is usually 0.5 to 5 μM, preferably 1 to 4 μM.

[0122] The ROCK inhibitor used in step A) can be the same as the ROCK inhibitor used in the production method of the present invention. When Y-27632 is used as the ROCK inhibitor, the concentration in the culture medium is usually 1 to 20 μM, preferably 5 to 15 μM.

[0123] Insulin may be further added to the culture medium. The concentration of insulin in the culture medium is generally 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 culture medium may be the concentration of insulin contained in the added B-27 supplement, but is not limited thereto.

[0124] The differentiation into primitive intestinal tube cells in step B) can be performed, for example, by culturing the embryonic endoderm cells obtained in step A) in a medium containing growth factors. The culture period is usually 2 to 8 days.

[0125] As the growth factor used in step B), EGF, KGF, FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is further preferred. The concentration of the growth factor in the culture medium can be appropriately set according to the type of growth factor used, and is generally about 0.1nM to 1000μM, preferably about 0.1nM to 100μM. In the case of EGF, its concentration is about 5 to 2000ng / ml (i.e., about 0.8 to 320nM), preferably about 5 to 1000ng / ml (i.e., about 0.8 to 160nM), and more preferably about 10 to 1000ng / ml (i.e., about 1.6 to 160nM). 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.

[0126] Differentiation into posterior foregut cells in step C) can be performed, for example, by culturing the primitive gut cells obtained in step B) in a medium containing growth factors, retinoic acid receptor agonists such as retinoic acid derivatives, Hedgehog signaling inhibitors, and BMP inhibitors. The culture period is usually 1 to 5 days.

[0127] The type and concentration of the growth factor used in step C) are the same as those described in step B).

[0128] As the retinoic acid receptor agonist used in step C), for example, retinoic acid (all-trans-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexene-1-yl)-2,4,6,8-nonatetraenoic acid (CasNo: 302-79-4)), retinoic acid salts, retinoic acid precursors, retinoic acid derivatives, etc. Examples of retinoic acid salts include sodium retinoic acid, potassium retinoic acid, calcium retinoic acid, etc. Examples of retinoic acid precursors include β-carotene, retinol esters, retinol, retinal, etc. Retinoic acid derivatives refer to artificially modified retinoic acid that retains the functions of natural retinoic acid, and examples thereof include 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)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-naphthyl)-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 them, TTNPB is preferred. When TTNPB is used, the concentration in the culture medium is generally 1 to 50 nM, preferably 5 to 15 nM.

[0129] As the Hedgehog pathway inhibitor used in step C), for example, cyclopamine, jervine, 3-keto-N-(aminoethyl-aminoacetyl-dihydro-cinnamoyl) (KAAD)-cyclopamine, CUR-61414, SANT-1, SANT-2, SANT-3, SANT-4, IPI-926, IPI-269609, GDC-0449 and NVP-LDE-225 can be mentioned. Among them, SANT-1 is preferred. When SANT-1 is used, the concentration in the culture medium is generally 100 to 500 nM, preferably 50 to 150 nM.

[0130] Examples of the BMP inhibitor used in step C) include protein 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 them, LDN-193189 is preferred. When LDN-193189 is used, the concentration in the culture medium is generally 10 to 1000 nM, preferably 100 to 300 nM.

[0131] 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 growth factors and BMP inhibitors. The medium may also contain non-muscle myosin II inhibitors, TGFβ inhibitors, nicotinamide, etc. The culture period is usually 2 to 10 days.

[0132] When performing step D), the posterior foregut cells obtained in step C) can be treated with 0.25% trypsin-EDTA and dispersed by pipetting according to previous reports (Toyoda et al., Stem Cell Research (2015) 14, 185-197), and the 0.25% trypsin-EDTA can be centrifuged and suspended.

[0133] The types and concentrations of the growth factors and BMP inhibitors used in step D) are the same as those described in step B) and step C).

[0134] As the non-muscle myosin II inhibitor used in step D), for example, 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, wortmannin, etc. can be cited. Among them, Blebbistatin is preferred. When Blebbistatin is used as a non-muscle myosin II inhibitor, the concentration in the culture medium is 1 μM to 200 μM, preferably 10 μM to 100 μM.

[0135] The TGFβ inhibitor used in step D) is a substance that inhibits the sequential signal transmission from the binding of TGFβ to the receptor to SMAD. It is not particularly limited as long as it is a substance that inhibits the binding to the ALK family as a receptor or a substance that inhibits the phosphorylation of SMAD caused by the ALK family. Examples thereof include Lefty-1, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276, A-83-01 (WO2009146408), ALK5 inhibitor II (ALK5 inhibitor II) (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine (2-[3-(6-methylpyridin-2-yl)-1H-pyrazol-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 their derivatives, etc. Preferably, it may be ALK5 inhibitor II. When ALK5 inhibitor II is used as a TGFβ inhibitor, the concentration in the culture medium is generally 0.1μM to 50μM, and more preferably 1μM to 20μM.

[0136] The concentration of nicotinamide used in step D) in the medium is usually 1 mM to 100 mM, preferably 5 mM to 50 mM.

[0137] The first day of culturing in step D) is carried out in the presence of a ROCK inhibitor, and then the culture may be carried out in a medium without a ROCK inhibitor. The ROCK inhibitor may be the same as the ROCK inhibitor used in the preparation method of the present invention. When Y-27632 is used as a ROCK inhibitor, the concentration in the medium is generally 1 to 20 μM, preferably 5 to 15 μM.

[0138] As shown in the following examples, it is shown that by culturing pancreatic endoderm cells in the presence of specific low molecular weight compounds or proteins (hereinafter sometimes referred to as "expansion culture promoting factors"), the proliferation efficiency of the cells is improved. Therefore, the preparation method of the present invention may include a step of culturing pancreatic endoderm cells in a culture medium containing an expansion culture promoting factor. As such an expansion culture promoting factor, for example, TGFβ inhibitors, retinoic acid receptor agonists, proliferation factors (for example: NGF, β-cellurin, TGFα, PDGFAA, LIF, IGF-1, FGF9, FGF10, etc.), bone morphogenetic proteins (BMP) (for example: BMP4, BMP7, etc.), etc., among which TGFβ inhibitors and retinoic acid receptor agonists are preferred. As more specific expansion culture promoting factors, Figure 7 The expansion culture promoting factor described in . The expansion culture promoting factor can be used alone or in combination. In one embodiment of the present invention, the culture medium in the preparation method of the present invention contains a TGFβ inhibitor and / or a retinoic acid receptor agonist, but preferably contains both a TGFβ inhibitor and a retinoic acid receptor agonist.

[0139] As a TGFβ inhibitor, the same TGFβ inhibitor as that used in step D) can be used. As TGFβ inhibitors, ALK5 inhibitor II, SB431542, A-83-01, LY2109761, and DMH1 are preferred, and ALK5 inhibitor II is more preferred. When ALK5 inhibitor II is used as a TGFβ inhibitor, the concentration in the culture medium is generally 0.1 μM to 50 μM, and more preferably 1 μM to 20 μM. In one embodiment, it is 10 μM.

[0140] As the retinoic acid receptor agonist, the same retinoic acid receptor agonist as that used in step C) can be used. As the retinoic acid receptor agonist, retinoic acid and TTNPB are preferred, and retinoic acid is more preferred. When retinoic acid or a salt thereof is used as the retinoic acid receptor agonist, the concentration in the culture medium is usually 100nM to 10μM, and more preferably 500nM to 5μM. In one embodiment, it is 1μM.

[0141] In other embodiments 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 at least the ability to differentiate into β-like cells and express at least PDX1 and NKX6.1. Pancreatic endoderm cells can further express one or more gene markers such as SOX9 and GATA4.

[0142] In this specification, "expression" of various gene markers such as PDX1 or the marker is "positive", unless otherwise specified, at least with the meaning of "production of mRNA encoded in the gene", preferably with the meaning of further including "production of protein encoded in mRNA". Therefore, in the case where the production of mRNA encoded in the gene is detected by at least quantitative RT-PCR, the gene can be said to be expressed. On the other hand, in the case where the production of mRNA encoded in the gene is not detected by quantitative RT-PCR (i.e., below the detection limit), or at background levels, it can be said that the gene is not expressed or is negative.

[0143] 2. Pancreatic endoderm cell expansion culture kit

[0144] Further, the present invention provides a kit for expanding and culturing pancreatic endoderm cells comprising a ROCK inhibitor and KGF and / or EGF (hereinafter sometimes referred to as "the kit for expanding and culturing of the present invention"). The "kit for expanding and culturing pancreatic endoderm cells" may also be renamed as "kit for pancreatic endoderm cell proliferation" or "kit for producing pancreatic endoderm cells". The kit for expanding and culturing of the present invention preferably comprises nicotinamide. In addition, the kit for expanding and culturing of the present invention also preferably comprises a TGFβ inhibitor and / or a retinoic acid receptor agonist.

[0145] In addition, the kit for expanded culture of the present invention may include at least one of a basal culture medium, a culture medium additive, a culture container, pancreatic endoderm cells and their precursor cells (e.g., pluripotent stem cells, embryonic endoderm cells, primitive intestinal tube cells, posterior foregut cells, etc.). The definitions or specific examples of each constituent substance such as a ROCK inhibitor, a TGFβ inhibitor, and a retinoic acid receptor agonist contained in the kit for expanded culture of the present invention are all cited from the contents described in the above-mentioned "1. Method for preparing pancreatic endoderm cells".

[0146] 3. Preparation of β-like cells

[0147] As described above, the pancreatic endoderm cells of the present invention have at least the ability to differentiate into β-like cells. Therefore, in other embodiments, a method for manufacturing β-like cells or their precursor cells (hereinafter sometimes referred to as "the method for manufacturing β-like cells of the present invention") including a process for inducing the differentiation of the pancreatic endoderm cells of the present invention into β-like cells or their precursor cells is provided, as well as β-like cells or their precursor cells (hereinafter also referred to as "the method for manufacturing β-like cells of the present invention") obtained in the method. As precursor cells of β-like cells, for example, endocrine cells expressing NGN3, immature β cells expressing insulin and NKX6.1, etc. can be cited. From the perspective of clinical use, the method for manufacturing β-like cells of the present invention is preferably carried out without a feeder layer and without exogenous conditions throughout the process.

[0148] Differentiation induction of pancreatic endoderm cells into β-like cells or their precursor cells can be performed by known methods such as those described in Patent Document 1, Non-Patent Documents 1 and 2. Specifically, differentiation induction can be performed, for example, by methods including the following:

[0149] Step E) a step of inducing differentiation of the pancreatic endoderm cells of the present invention into endocrine precursor cells, and

[0150] Step F) A step of inducing differentiation of endocrine precursor cells into β-like cells.

[0151] The basal medium or medium additive used in steps E) and F) can be the same basal medium or medium additive as that used in the production method of the present invention. In addition, the culture temperature in steps E) and F) is usually about 30 to 40°C, preferably about 37°C, and the culture is carried out in an atmosphere of air containing CO2, and the CO2 concentration is preferably about 2 to 5%. In addition, steps E) and F) are preferably carried out by suspension culture. The definition or method of suspension culture, etc., refer to the contents described in the above-mentioned "1. Production method of pancreatic endoderm cells".

[0152] 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 culture medium containing a γ-secretase inhibitor and a TGFβ inhibitor. The culture medium may contain thyroid hormones, growth factors, Hedgehog pathway inhibitors, retinoic acid derivatives, BMP inhibitors, etc. The culture period is generally 1 to 5 days.

[0153] 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 the above steps C) and D).

[0154] Examples of the γ-secretase inhibitor used in step E) include RO4929097, DAPT (GSI-IX), semagacestat (LY450139), dibenzazepine (dibenzazepine) (YO-01027), etc. Among them, RO4929097 is preferred. When RO4929097 is used, the concentration in the culture medium is usually 0.1 to 10 μM, preferably 0.5 to 5 μM.

[0155] Examples of the thyroid hormone used in step E) include triiodothyronine (T3) and thyroxine (T4). Among them, triiodothyronine is preferred. When triiodothyronine is used, the concentration in the culture medium is generally 0.1 to 10 μM, preferably 0.5 to 5 μM.

[0156] Differentiation into β-like cells in step F) can be performed, for example, by culturing the endocrine progenitor cells obtained in step E) in a medium obtained by removing the γ-secretase inhibitor from the medium used in step E). The culture period is usually 4 to 10 days.

[0157] For other culture conditions or methods, additives to the culture medium, specific examples of culture vessels, surface processing of culture vessels, etc., refer to the contents described in the above-mentioned "1. Method for producing pancreatic endoderm cells".

[0158] In addition, it is also possible to manufacture pancreatic exocrine cells by pancreatic endoderm cells of the present invention by known methods. As this method, for example, the method described in International Publication 2014 / 104403 can be cited, specifically, it is also possible to manufacture by the process of cultivating pancreatic endoderm cells of the present invention in a culture fluid with the addition of a histone deacetylase inhibitor and / or a ligand protein of Notch signal and a protein kinase C activator. The specific histone deacetylase inhibitor, the ligand protein of Notch signal, the kind of protein kinase C activator or the manufacturing method all cite the content of International Publication 2014 / 104403.

[0159] 4. Cell transplantation therapy

[0160] The pancreatic endoderm cells of the present invention and the β-like cells of the present invention (sometimes the term "cells of the present invention" is used as their general term) can be differentiated and induced into islet-like cells by transplantation into mammalian organisms. Therefore, since the cells of the present invention are suitable for cell transplantation therapy, in other embodiments of the present invention, a cell transplantation therapy agent containing the cells of the present invention (hereinafter sometimes referred to as "cell transplantation therapy agent of the present invention") is provided. In addition, the present invention also includes a method for treating pancreatic diseases, wherein 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.) as a treatment object. As pancreatic diseases that become treatment objects, for example, acute pancreatitis, chronic pancreatitis, type 1 diabetes, type 2 diabetes, pancreatic tumors, Langerhans islet tumors, etc. are exemplified.

[0161] The cells or cell transplantation therapy agents of the present invention can be transplanted into the body of a patient in need thereof and used. Transplantation is preferably performed in a region of the body where the cells can be fixed at a certain position, such as subcutaneously, intraperitoneally, in the peritoneal epithelium, omentum, adipose tissue, muscle tissue, or under the capsule of organs such as the pancreas and kidneys. Subcutaneous transplantation with low invasiveness is preferred. The transplanted cells only need to be given a therapeutically effective amount, which can vary according to factors such as the age, weight, size of the transplant site, severity of the disease, etc. of the transplanted subject, and is not particularly limited. For example, it can be set to 10×10 4cells~10×10 11 About cells.

[0162] When the cells of the present invention are used for 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 individual of the transplant target from the viewpoint of not causing rejection. Here, "substantially the same" means that the HLA genotype is consistent to the extent that the immune response to the transplanted cells can be suppressed by immunosuppressants, such as somatic cells with the same HLA type of three loci of HLA-A, HLA-B and HLA-DR or four loci of HLA-C. In the case where sufficient cells cannot be obtained for reasons such as age or physical constitution, they can also be transplanted in a state of being embedded in a capsule or porous container such as polyethylene glycol or silicone to avoid rejection.

[0163] The cells of the present invention are mixed with a pharmaceutically acceptable carrier according to conventional means and prepared as parenteral preparations such as injections, suspensions, and drips. Therefore, in one embodiment, a method for preparing a cell transplantation therapy agent including a step of preparing the cells of the present invention is also provided. This method may also include a step of preparing the cells of the present invention. Further, it may also include a step of preserving the cells of the present invention.

[0164] As pharmaceutically acceptable carriers that can be included in the parenteral preparation, for example, aqueous liquids for injection such as physiological saline, isotonic solutions (isotonic solutions) containing glucose or other auxiliary drugs (such as D-sorbitol, D-mannitol, sodium chloride, etc.) can be cited. The cell transplantation therapy agent of the present invention can be combined with, for example, a buffer (such as phosphate buffer, sodium acetate buffer), an analgesic (such as benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (such as human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc.

[0165] The cell transplantation therapy agent of the present invention can also be provided in a state of being cryopreserved under conditions commonly used for cell cryopreservation, and thawed for use. In this case, serum or its substitute, organic solvent (such as DMSO) etc. may be further included. In this case, the concentration of serum or its substitute is not particularly limited, and 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, and may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0166] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0167] Example

[0168] <Materials and Methods>

[0169] Screening of aging-related reagents

[0170] After differentiation to pancreatic endoderm cells by staged differentiation induction method, 2 × 10 5 / wells were re-seeded. The following culture medium was added to these cultured cells and cultured for 1 week. The culture medium of the control group was a modified MEM supplemented with 0.5×B-27 supplement, 100U / ml penicillin / streptomycin, 100ng / ml KGF / FGF7, 50ng / ml EGF and 10mM nicotinamide. In the intervention group, Y-27632, terrenic acid, soybean yellow, PD98059, metformin, BPTES, ABT263, ARV825 or 17-DMAG were added to the above culture medium, and cultured for 1 week. After fixation with 4% PFA, β-gal staining and NKX6.1 antibody immunostaining were performed, and fluorescent staining images were obtained using BZ-800 (Keyence). Using the BZ-H4CM / macro cell counting function of the same microscope, the number of NKX6.1-positive cells, the NKX6.1-positive ratio, and the β-gal-positive ratio were calculated. An overview of this step is shown in Figure 2-A and 3-A .

[0171] Expanded culture to promote reagent screening

[0172] The cells on day 21 of the expansion culture were cultured for 6 days using a medium in which various growth factors or small molecule compounds were added to the expansion culture medium containing Y-27632. Other conditions were the same as those for the aging-related agent screening.

[0173] PDX1 + / NKX6.1 + Pancreatic endoderm cell expansion culture

[0174] 1. PDX1 + / NKX6.1 + Production of pancreatic endoderm cells

[0175] PDX1 was produced from iPS cells (585A1 strain) or ES cells (KhES-3 strain) in 6-well plates using the method of Toyoda et al. (Toyoda T, et al., Stem Cell Reports 2017). + / NKX6.1 + Pancreatic endoderm cells. Briefly, 1 × 10 6The undifferentiated cells were seeded in 100 μl / well and cultured for 4 days in S1 medium (RPMI supplemented with 1×B-27 supplement, 100 U / ml penicillin / streptomycin, 100 ng / ml activin A and CHIR99021 (Day 1: 3 μM, Days 2-3: 1 μM, Day 4: 0 μM)), 5 days in S2 medium (modified MEM supplemented with 0.5×B-27 supplement, 100 U / ml penicillin / streptomycin and 50 ng / ml KGF / FGF7), and 2 days in S3 medium (modified MEM supplemented with 0.5×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 plated again in S3 medium at 1.5 × 10 cells / mL in a 6-well plate coated with iMatrix-511silk. 6 On the next day, the culture medium was changed to S4 medium (modified MEM supplemented with 0.5×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.

[0176] 2. PDX1 + / NKX6.1 + Pancreatic endoderm cell expansion culture

[0177] (1) After washing the cells prepared in 1 above with PBS (-), add 1 ml of 0.25% trypsin-EDTA and incubate at 37°C, 5% CO2 for 5 minutes. (2) Dissociate the adherent cells by pipetting and add them to a 50 ml centrifuge tube. (3) Add 4 ml of DMEM (Double's modified Eagle medium) / 10% FBS / PS (penicillin-streptomycin) to the centrifuge tube. (4) Centrifuge at 400G for 3 minutes and remove the supernatant. (5) Suspend in modified MEM (containing 10 μM Y-27632, 0.5×B-27 supplement and 100U / ml penicillin / streptomycin) and count the number of cells. (6) When seeding in a 6-well plate, recover the required amount of suspension to make 1×10 6 10 cells / well, centrifuge at 400G for 3 minutes (2×10 cells / well in a 24-well plate) 5cells / well). (7) After removing the supernatant, resuspend in modified MEM (containing 100ng / ml KGF, 50ng / ml EGF, 10mM nicotinamide, 50μM Y-27632, 0.5×B-27 supplement and 100U / ml penicillin / streptomycin), adjust to 2ml / well suspension, and inoculate cells. 1 hour before inoculation, add a turbid solution of 10μli Matrix-511silk and 1.5ml PBS(-) to each well of the 6-well plate for cell inoculation, incubate at 37°C, 5% CO2 for 1 hour, and pre-coat.

[0178] (8) The next day (Day 1), after washing with modified MEM, the medium is replaced with modified MEM (5 ml / well) 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. (9) Three days later (Day 4), after washing with modified MEM, the medium is replaced with the same medium as step (7) (5 ml / well). (10) Three days later (Day 7), the above steps (1) to (7) are performed to carry out the next subculture (expansion culture). From the next day, steps (8), (9) and (10) are repeated, thereby repeating the expansion and subculture. One subculture (expansion culture) is equivalent to 7 days (the above operation). The outline of this step 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 the culture medium: 10 μM) and retinoic acid (Sigma; R2625; CAS: 302-79-4)) (concentration in the culture medium: 1 μM), the ALK inhibitor and RA or DMSO (concentration in the culture medium: 0.1% (v / v)) were added to the culture medium of the above steps (7) to (9).

[0179] Differentiation into β-like cells

[0180] After expansion, the method of Kimura et al. (Non-patent Document 1) was partially modified to induce differentiation into β-like cells. Briefly, pancreatic endoderm cells were detached from a 6-well plate using trypsin and cultured at 3 × 10 5 Cells / ml were suspended in S4 medium. 4The cells were seeded in a v-bottom 96-well plate at 100 μl / well. After incubation at 37°C and 5% CO2 for 1 day, the medium was changed to S5 medium (modified MEM supplemented with 0.5×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 beta-cells) and cultured for 1 week. After that, the medium was exchanged for S6 medium (modified MEM supplemented with 0.5×B-27 supplement, 100 U / ml penicillin / streptomycin, 10 μM ALK5 inhibitor II and 1 μM T3) and cultured for another week.

[0181] Evaluation Method

[0182] 1. Immunostaining

[0183] The expanded cultured cells were washed twice with PBS(-) and fixed with 4% PFA at 4°C for 20 minutes. Blocked with blocking solution (PBS(-) containing 5% donkey serum and 0.4% Triton X-100) at room temperature for 30 minutes. Incubated overnight at 4°C with the primary antibody solution diluted with the blocking solution. After washing the primary antibody solution, incubated with the fluorescent secondary antibody solution diluted with the blocking solution at room temperature for one hour. Immunofluorescence staining images were obtained using BZ-710 or BZ-800 (Keyence).

[0184] 2. Flow Cytometry Analysis

[0185] The expanded cultured cells were dissociated with 0.25% trypsin-EDTA and fixed using the Cytofix / Cytoperm kit (BD Biosciences) according to the experimental protocol. Blocked with a permeabilization solution containing 2% donkey serum. Incubated overnight at 4°C using a primary antibody solution diluted in blocking solution. After washing the primary antibody solution, incubated for one hour at room temperature using a fluorescent secondary antibody solution diluted in blocking solution. Analyzed the stained cells using FACSAriaII (BD Biosciences).

[0186] 3. Quantitative Evaluation of Nuclear Morphology

[0187] The cells on the plastic bottom plate fixed with 4% PFA were stained with Hoechst for nuclear staining. The cell morphology evaluation values ​​including eccentricity (roundness) were obtained using the MeasureObjectSizeShape function of the CellProfiller software for the fluorescent staining images acquired using BZ-800 (Keyence).

[0188] Statistical analysis

[0189] Mann-Whitney U test was used as a nonparametric comparison method between 2 groups. In comparative analysis of more than 3 groups, one-way ANOVA was used. In the case of significant difference in ANOVA, Dunnet's method was used for multiple comparisons of 1 control group and other treatment groups. P value less than 0.05 was defined as significant difference.

[0190] The information of reagents and the like used in the Examples are as follows.

[0191] [Table 1-1]

[0192]

[0193]

[0194] [Table 1-2]

[0195] Niacinamide STEMCELL Technologies 7154 Hoechst 33342 Thermo Fisher Scientific H3570 ALK5 inhibitors Fujifilm Wako Chemicals 018-23023 Triiodothyronine Sigma-Aldrich 64245 RO4929097 Selleck Chemicals S1575 Recombinant human beta-cell protein R&D RSD-261-CE Terrenic acid Santa Cruz sc-200655 Soybean Sigma-Aldrich D7802 PD 98059 Sigma-Aldrich P215 Metformin Pfizer 4987-114-12280-3 BPTES Sigma-Aldrich SML0601 ARV825 Medchem Express HY-16954 ABT263 Medchem Express HY-10087 17-DMAG Selleck Chemicals S1142 FBS biosera FB-1285 / 500

[0196] Example 1: Search for agents that can proliferate pancreatic endoderm cells

[0197] Among the aging-related agents, agents that can proliferate pancreatic endoderm cells derived from human iPS cells were explored. Y-27632, terrenic acid, soybean zein, PD98059, metformin, BPTES, BPTES, ABT263, ARV825, and 17-DMAG were selected as aging-related agents, and NKX6.1-containing cells were cultured in a medium containing each aging-related agent. + S4d4 cells of pancreatic endoderm cells were cultured to measure (1) the number of NKX6.1-positive cells or (2) the ratio of NKX6.1-positive cells and βgal-positive cells. The results are shown in Figure 2-B~2-D .according to Figure 2-B , showing that other aging-related agents did not significantly increase pancreatic endoderm cells, on the other hand, Y-27632 significantly increased target cells, and as a concentration of Y-27632, 50 μM was most suitable for proliferation. Figure 2-C , showing that even when the proportion of pancreatic endoderm cells was used as the outcome, Figure 2-B Similarly, it was observed that Y-27632 50 μM had the greatest effect. Figure 2-D , showing that Y-27632 also has a reducing effect on the proportion of βgal-positive cells (senescent cells). On the other hand, other aging-related agents also reduce the proportion of βgal-positive cells, but when used with Figure 2-BTaken together, it is speculated that the decrease in the proportion of βgal-positive cells caused by BPTES or ABT263 is the result of toxicity.

[0198] Example 2: Verification of the Proliferation Effect of ROCK Inhibitors Other Than Y-27632 on Pancreatic Endoderm Cells

[0199] According to Example 1, the proliferation effect of pancreatic endoderm cells caused by Y-27632, a ROCK inhibitor, was observed. Therefore, it was verified whether the same effect was observed on human iPS cell-derived PDX1 cells with other ROCK inhibitors. + / NKX6.1 + Proliferation effect of pancreatic endoderm cells. The experiment was carried out in the same manner as in Example 1. The results are shown in Figure 3-B and 3-C .according to Figure 3-B and 3-C , the ROCK inhibitors other than Y-27632 (GSK269962, GSK429286A, Fasudil hydrochloride, H1152, and Thiazovivin) also showed the same effect on the proliferation of pancreatic endoderm cells as Y-27632.

[0200] The above strongly suggests that ROCK inhibitors exert a broad pancreatic endoderm cell proliferation effect regardless of the type.

[0201] Example 3: Validation of the expansion culture of pancreatic endoderm cells by Y-27632 (50 μM)

[0202] Using Y-27632, we tested whether pancreatic endoderm cells derived from human iPS cells can be expanded and cultured. The results are shown in Figure 4-B~4-H .according to Figure 4-B By using Y-27632 (50 μM) for expansion culture, it was confirmed that the total cell number increased to 1×10 within 60 days. 5 times more. Figure 4-C , by using Y-27632 (50 μM) for expansion culture, it was confirmed that PDX1 + / NKX6.1 + The number of pancreatic endoderm cells increased to 1×10 5 times more. Figure 4-D , immunostaining images of cells after expansion with Y-27632 (50 μM) confirmed that the cells maintained the expression of PDX1 and NKX6.1, and also maintained the expression of Ki67. Figure 4-E By using Y-27632 (50 μM) for expansion culture, it was confirmed that the proportion of pancreatic endoderm cells or Ki67 positive ratio was maintained. Figure 4-F, it was confirmed that the proliferation ability (= Ki67 positive ratio) of pancreatic endoderm cells was also maintained by using Y-27632 (50 μM) for expansion culture. Figure 4-G , confirmed that even pancreatic endoderm cells that have been expanded twice have the ability to differentiate into β-like cells. Figure 4-H It was confirmed that even when pancreatic endoderm cells derived from ES cells (KhES-3 strain) were used, the pancreatic endoderm cells could be expanded and cultured in the same manner. However, it was confirmed that expansion and culture would be difficult without using Y-27632.

[0203] The above results show that pancreatic endoderm cells can be highly efficiently expanded and cultured for at least 60 days by using Y-27632 (50 μM), and that the expanded pancreatic endoderm cells maintain the ability to differentiate into β-like cells.

[0204] Example 4: Verification of the expansion culture of pancreatic endoderm cells by Y-27632 at varying concentrations

[0205] In Example 3, it was confirmed that pancreatic endoderm cells can be expanded by using 50 μM Y-27632, so the effect on expansion culture was verified when the concentration was reduced to 10 μM. The results are shown in Figure 5-A to 5-C .according to Figure 5-A It was confirmed that even when Y-27632 (10 μM) was used, PDX1 + / NKX6.1 + The number of pancreatic endoderm cells and total cells increased to 1×10 4 On the other hand, according to Figure 5-B and Figure 5-C , showing that in the case of Y-27632 (10 μM), in the cells of KhES-3 strain-derived pancreatic endoderm cells that were passaged 5 times (5 weeks), the cell morphology changed (became an elongated oval shape).

[0206] From the above, it was shown that Y-27632 can be used in culture at a concentration of at least 10 μM in the medium, and that expansion culture can be performed, but from the viewpoint of cell quality, it is preferably used at 50 μM.

[0207] Example 5: Verification of the mechanism by which Y-27632 enables the expansion of pancreatic endoderm cells

[0208] The mechanism by which Y-27632 enables the expansion of pancreatic endoderm cells was verified. In the third passage of the expansion culture, the group not given Y-27632 showed an increase in α-SMA positive cells compared to the group given Y-27632 at a concentration of 50 μM ( Figure 6). That is, by administering Y-27632 to cells, inhibition of fibrosis or epithelial-mesenchymal transition was observed.

[0209] The above results suggest that the main mechanism by which Y-27632 enables the expansion of pancreatic endoderm cells is not due to anti-apoptosis but due to the inhibition of cell aging and the accompanying fibrosis or epithelial-mesenchymal transition.

[0210] Example 6: Exploration of agents that promote proliferation of pancreatic endoderm cells

[0211] In the expansion culture medium using Y-27632, the efficiency of induction into NKX6.1-positive cells and β-like cells tended to decrease gradually with repeated induction. Therefore, screening was carried out using proteins such as growth factors and low molecular weight compounds to try to improve the expansion culture method.

[0212] After three passages of pancreatic endoderm cells using a medium supplemented with Y-27632 (50 μM), the cells were cultured at 2.0×10 5 The cells were seeded in 24-well plates coated with iMatrix at a number of cells / well. Each screening agent was added to a medium containing Y-27632 (50 μM) and cultured. After 6 days, the proportion of NKX6.1-positive cells was studied by cell immunostaining. As a result, ALK5 inhibitors (ALK5i) and retinoic acid receptor agonists were identified as candidate factors for improving expansion culture ( Figure 7 ).

[0213] Example 7: Validation of ALK5 inhibitor (ALK5i) and retinoic acid receptor agonist

[0214] By adding the two candidate factors to the expansion culture method using Y-27632 (50 μM), it was tested whether long-term expansion culture could be performed while maintaining the NKX6.1 positive ratio.

[0215] The pancreatic endoderm cells were subcultured repeatedly every week and the number of cells was counted. At the same time, immunostaining was performed each time to study the NKX6.1 positive ratio, and the cumulative NKX6.1 increase ratio was calculated from these values. This calculation method was performed under two conditions: "DMSO group (Y-27632+DMSO)" and "ALK5i+RA group (Y-27632+ALK5i+RA)". As a result, compared with the Y-27632 alone group, the proliferation efficiency was improved while maintaining the NKX6.1 positive ratio by adding ALK5i and RA ( Figure 8 ).

[0216] Example 8: Verification of β-like cell induction efficiency

[0217] In order to confirm the function of the expanded pancreatic endoderm cells, the efficiency of induction of β-like cells after expansion was investigated.

[0218] The differentiation induction of pancreatic endoderm cells that were repeatedly passaged 5 times by the expansion culture method using "Y-27632+DMSO" or "Y-27632+ALK5i+RA" into β-like cells was promoted. On this basis, the efficiency of cell clusters induced into β-like cells and induced into β-like cells by pancreatic endoderm cells that were not passaged was compared. As a result, the efficiency of induction into β-like cells in the improved expansion culture method ("Y-27632+ALK5i+RA") was high compared to the expansion culture method before the improvement ("Y-27632+DMSO"), and was equivalent to the induction efficiency of pancreatic endoderm cells before the expansion culture was implemented ( Fig. 9 ).

[0219] Industrial Applicability

[0220] The method of the present invention, or pancreatic endocrine cells or pancreatic exocrine cells, including pancreatic endoderm cells produced by the present invention or β-like cells derived from the cells, can be applied to: the development of cell therapy for pancreatic diseases (especially type 1 and type 2 diabetes), the application in drug development screening systems for pancreatic diseases, the preparation of pancreatic disease models using iPS cells derived from patients with hereditary pancreatic diseases, etc.

[0221] This application is based on Japanese Patent Application No. 2022-153013 (filing date: September 26, 2022) filed in Japan, the contents of which are incorporated herein in their entirety.

Claims

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.

2. The method according to claim 1, wherein the culturing is carried out for more than 2 days.

3. The method of claim 1 or 2, wherein the ROCK inhibitor is selected from the group consisting of Y-27632, GSK269962, GSK429286A, Fasudil hydrochloride, H1152 and Thiazovivin.

4. The method of claim 1 or 2, wherein the ROCK inhibitor is Y-27632.

5. The method according to any one of claims 1 to 4, wherein the culture medium contains a TGFβ inhibitor and / or a retinoic acid receptor agonist. 6 . The method of claim 5 , wherein the TGFβ inhibitor is 2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine.

7. The method of claim 5 or 6, wherein the retinoic acid receptor agonist is retinoic acid.

8. The method according to any one of claims 1 to 7, wherein the pancreatic endoderm cells are derived from pluripotent stem cells. 9 . The method according to claim 1 , wherein the pancreatic endoderm cells are cells derived from a patient with a hereditary pancreatic disease.

10. The method according to any one of claims 1 to 9, wherein the culture is culture under feeder-free conditions.

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

12. A kit for expanding and culturing pancreatic endoderm cells, 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 precursor cells thereof, comprising the step of inducing the pancreatic endoderm cells according to claim 11 to differentiate into β-like cells or precursor cells thereof.

15. A cell transplantation therapy agent comprising the pancreatic endoderm cell of claim 11 or a cell produced by the method of claim 14.

16. The agent according to claim 15, which is used for treating diabetes.

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