Method for producing pancreatic β cells

Abstract

The present invention provides a method for producing pancreatic β cells, the method comprising a step for inducing differentiation of pancreatic endocrine progenitor cells into pancreatic β cells in the presence of an endoplasmic reticulum stress reducing drug. Moreover, the present invention also provides: pancreatic β cells obtained by said method; and a drug for treating or preventing diabetes, the drug comprising said pancreatic β cells.

Description

Method for producing pancreatic beta cells

[0001] The present invention relates to a method for producing pancreatic β cells, pancreatic β cells produced by the method, and uses of the pancreatic β cells.

[0002] Diabetes mellitus is a disease caused by insufficient or abnormal insulin secretion from pancreatic beta cells in the pancreas. Because adult pancreatic islet cells have little self-renewal capacity, transplantation of pancreatic beta cells is necessary to restore insulin secretion in diabetic patients. However, a shortage of donors for islet beta cell transplantation is a serious problem. One potential solution to this problem is the use of pluripotent stem cells, such as human induced pluripotent stem cells (iPS cells).

[0003] Many researchers have spent over 20 years attempting to generate pancreatic β cells from human iPS cells and use them in the treatment of diabetes. However, to the inventors' knowledge, pancreatic β cells generated using previously reported differentiation protocols have not yet acquired sufficient functionality (i.e., glucose-responsive insulin secretion) comparable to that of adult cells. Specifically, the in vitro insulin secretion capacity of isolated human adult islets has been reported to be approximately 1-4% / protein content under low glucose conditions and approximately 2-8% under high glucose conditions (Non-Patent Document 1). On the other hand, pancreatic β cells generated by differentiation induction from iPS cells or ES cells have been reported to have functionality of approximately 0.2-1% / protein content under low glucose conditions and approximately 0.5-2% under high glucose conditions (Non-Patent Documents 2 and 3).

[0004] Lorza-Gil E. et al., Sci Rep., 10(1):16497 (2020)Davis JC et al., Cell Rep., 31(6):107623 (2020)Liang S. et al., Cell Rep Methods, 3(5):100466 (2023)

[0005] Therefore, an object of the present invention is to provide a method for producing pancreatic β cells having higher insulin secretion ability than pancreatic β cells produced by conventional methods, and to provide pancreatic β cells obtained by said method.

[0006] While studying the mechanism of diabetes development associated with insulin gene mutations, the present inventors induced differentiation of pancreatic β cells from pluripotent stem cells harboring a mutation in the insulin gene. In this study, they found that cells with a mutation in the insulin gene exhibited increased endoplasmic reticulum (ER) stress as differentiation progressed, contributing to the dysfunction of the resulting pancreatic β cells (specifically, low insulin secretion capacity). The occurrence of ER stress during normal β cell differentiation in animal embryos has not been reported, and the present inventors also confirmed that no ER stress marker-positive cells were detected during normal pancreatic β cell differentiation in mouse embryos (data not shown).

[0007] Therefore, the ER stress observed during the differentiation of pluripotent stem cells carrying the insulin gene mutation into pancreatic β cells was thought to be due to the insulin gene mutation. However, in a series of experiments, the inventors noticed that even in negative control pluripotent stem cells (parental lines without insulin gene mutations), a very small number (less than 5%) of ER stress marker-positive cells were detected during the differentiation process into pancreatic β cells. Initially, they did not pay any attention to this, assuming it was a cellular stress response to the artificial in vitro environment. However, as a control for investigating the relationship between the gene mutation and ER stress, they added an ER stress-reducing drug to the negative control differentiation process. They found that the efficiency of differentiation into pancreatic β cells increased approximately 1.2-fold and the glucose-responsive insulin secretion efficiency of pancreatic β cells increased approximately 6-fold compared to the control group. Because the occurrence of ER stress during differentiation induction was extremely minor, it was completely unexpected that the addition of an ER stress-reducing drug would produce such high-quality pancreatic β cells with such high efficiency. Based on these findings, the present inventors have conducted further research and have completed the present invention.

[0008] That is, the present invention provides the following: [1] A method for producing pancreatic β cells, comprising a step of inducing differentiation of pancreatic endocrine precursor cells into pancreatic β cells in the presence of an endoplasmic reticulum stress-reducing agent. [2] The method described in [1], comprising a step of culturing pancreatic endocrine precursor cells under suspension culture conditions. [3-1] The method described in [1] or [2], comprising a three-dimensional culture step. [3-2] The method described in any one of [1] to [3-1], comprising a shaking culture step. [3-3] The method described in any one of [1] to [3-2], wherein the step of inducing differentiation of pancreatic endocrine precursor cells into pancreatic β cells comprises culturing pancreatic endocrine precursor cells in the presence of an endoplasmic reticulum stress-reducing agent for 5 to 9 days (e.g., 7 days). [4-1] The method described in any one of [1] to [3-3], wherein at least one of the endoplasmic reticulum stress-reducing agents is selected from the group consisting of 4-phenylbutyric acid, azoramide, an ATPase inhibitor of VCP, and a berberrubine acetic acid adduct. [4-2] The method according to any one of [1] to [4-1], wherein at least one of the endoplasmic reticulum stress-reducing agents is 4-phenylbutyric acid or azoramide. [5] The method according to any one of [1] to [4-2], wherein at least one of the endoplasmic reticulum stress-reducing agents is 4-phenylbutyric acid. [6] The method according to any one of [1] to [5], wherein two or more endoplasmic reticulum stress-reducing agents are used. [7] The method according to any one of [1] to [6], wherein the step of inducing differentiation into pancreatic β cells comprises a step of culturing pancreatic endocrine precursor cells in the presence of zinc sulfate. [8] The method according to any one of [1] to [7], wherein the pancreatic β cells are glucose-responsive insulin-secreting cells. [9-1] The method according to any one of [1] to [8], wherein the pancreatic β cells have a mutation in the insulin gene. [9-2] The method according to [9-1], wherein the mutation in the insulin gene is present in only one allele of the cells. [10-1] The method according to any one of [1] to [9-2], wherein the pancreatic endocrine precursor cells are obtained by inducing the differentiation of pancreatic endoderm cells in the presence of an endoplasmic reticulum stress-reducing agent. [10-2] The method according to any one of [1] to [9-2], further comprising, prior to the step of inducing the differentiation of pancreatic endocrine precursor cells into pancreatic β cells, a step of inducing the differentiation of pancreatic endoderm cells into pancreatic endocrine precursor cells in the presence of an endoplasmic reticulum stress-reducing agent.[10-3] The method according to [10-2], wherein the step of inducing differentiation of pancreatic endoderm cells into pancreatic endocrine precursor cells comprises culturing pancreatic endoderm cells for 5 to 9 days (e.g., 7 days) in the presence of an endoplasmic reticulum stress-reducing agent. [10-4] The method according to any one of [10-1] to [10-3], wherein at least one of the endoplasmic reticulum stress-reducing agents is selected from the group consisting of 4-phenylbutyric acid, azoramide, an ATPase inhibitor of VCP, and a berberrubine acetic acid adduct. [10-5] The method according to any one of [10-1] to [10-4], wherein at least one of the endoplasmic reticulum stress-reducing agents is 4-phenylbutyric acid or azoramide (preferably, 4-phenylbutyric acid). [11-1] The method according to any one of [10-1] to [10-5], wherein the pancreatic endoderm cells are obtained by inducing differentiation of posterior foregut endoderm cells in the presence of an endoplasmic reticulum stress-reducing agent. [11-2] The method according to any one of [10-2] to [10-5], comprising a step of inducing the differentiation of posterior foregut endoderm cells into pancreatic endoderm cells in the presence of an endoplasmic reticulum stress-reducing agent before the step of inducing the differentiation of pancreatic endoderm cells into pancreatic endocrine precursor cells. [11-3] The method according to [11-2], wherein the step of inducing the differentiation of posterior foregut endoderm cells into pancreatic endoderm cells comprises a step of culturing the posterior foregut endoderm cells in the presence of an endoplasmic reticulum stress-reducing agent for 5 to 9 days (e.g., 7 days). [11-4] A method for producing pancreatic β cells, comprising: (1) a step of inducing the differentiation of posterior foregut endoderm cells into pancreatic endoderm cells in the presence of an endoplasmic reticulum stress-reducing agent, (2) a step of inducing the differentiation of pancreatic endoderm cells into pancreatic endocrine precursor cells in the presence of an endoplasmic reticulum stress-reducing agent, and (3) a step of inducing the differentiation of pancreatic endocrine precursor cells into pancreatic β cells in the presence of an endoplasmic reticulum stress-reducing agent. [11-5] The method according to any one of [11-1] to [11-4], wherein at least one of the endoplasmic reticulum stress-reducing agents is selected from the group consisting of 4-phenylbutyric acid, azoramide, an ATPase inhibitor of VCP, and a berberrubine acetic acid adduct. [11-6] The method according to any one of [11-1] to [11-5], wherein at least one of the endoplasmic reticulum stress-reducing agents is 4-phenylbutyric acid or azoramide (preferably, 4-phenylbutyric acid).[12-1] The method according to any one of [11-1] to [11-6], wherein the posterior foregut endoderm cells are obtained by inducing differentiation of archenterocytes in the presence of an endoplasmic reticulum stress-reducing drug. [12-2] The method according to any one of [11-2] to [11-6], comprising a step of inducing differentiation of archenterocytes into posterior foregut endoderm cells in the presence of an endoplasmic reticulum stress-reducing drug prior to the step of inducing differentiation of posterior foregut endoderm cells into pancreatic endoderm cells. [12-3] The method according to [12-2], wherein the step of inducing differentiation of archenterocytes into posterior foregut endoderm cells comprises a step of culturing the archenterocytes in the presence of an endoplasmic reticulum stress-reducing drug for 1 to 5 days (e.g., 3 days). [12-4] A method for producing pancreatic β cells, comprising: (1) inducing differentiation of gastrula cells into posterior foregut endoderm cells in the presence of an endoplasmic reticulum stress-reducing agent, (2) inducing differentiation of the posterior foregut endoderm cells into pancreatic endoderm cells in the presence of an endoplasmic reticulum stress-reducing agent, (3) inducing differentiation of the pancreatic endoderm cells into pancreatic endocrine precursor cells in the presence of an endoplasmic reticulum stress-reducing agent, and (4) inducing differentiation of the pancreatic endocrine precursor cells into pancreatic β cells in the presence of an endoplasmic reticulum stress-reducing agent. [12-5] The method according to any one of [12-1] to [12-4], wherein at least one of the endoplasmic reticulum stress-reducing agents is selected from the group consisting of 4-phenylbutyric acid, azolamide, an ATPase inhibitor of VCP, and a berberrubine acetic acid adduct. [12-6] The method according to any one of [12-1] to [12-5], wherein at least one of the endoplasmic reticulum stress-reducing agents is 4-phenylbutyric acid or azoramide (preferably 4-phenylbutyric acid). [12-7] The method according to any one of [12-1] to [12-6], wherein the archenterocytes are obtainable by inducing the differentiation of definitive endoderm cells. [12-8] The method according to [12-7], wherein the definitive endoderm cells are obtainable by inducing the differentiation of pluripotent stem cells. [13-1] The method according to any one of [1] to [12-7], wherein the pancreatic β cells are derived from pluripotent stem cells. [13-2] The method according to any one of [1] to [13-1], wherein the pancreatic β cells are of human origin. [14-1] A method for producing pancreatic β cells, comprising a step of culturing pancreatic β cells in the presence of an endoplasmic reticulum stress-reducing agent.[14-2] The method according to [14-1], wherein at least one of the endoplasmic reticulum stress-reducing agents is selected from the group consisting of 4-phenylbutyric acid, azoramide, an ATPase inhibitor of VCP, and a berberrubine acetic acid adduct. [14-3] The method according to [14-2], wherein at least one of the endoplasmic reticulum stress-reducing agents is 4-phenylbutyric acid or azoramide (preferably, 4-phenylbutyric acid).

[15] The method according to any one of [14-1] to [14-3], wherein the starting pancreatic β cells are pancreatic β cells obtained by the method according to any one of [1] to [13-2] or pancreatic β cells constituting pancreatic islets in vivo.

[16] The method according to any one of [14-1] to

[15] , wherein the culture period is 30 days or longer.

[17] Pancreatic β cells obtained by the method according to any one of [1] to

[16] .

[18] A therapeutic or preventive agent for diabetes, comprising the pancreatic β cells according to

[17] . [19-1] A method for screening for a therapeutic or preventive agent for diabetes, comprising: (1) culturing the pancreatic β cells of

[17] in the presence or absence of a test substance; (2) measuring the insulin-producing ability of the pancreatic β cells in the presence or absence of the test substance; and (3) selecting the test substance as a candidate for a therapeutic or preventive agent for diabetes when the insulin-producing ability of the pancreatic β cells is higher in the presence of the test substance compared to in the absence of the test substance. [19-2] The method of [19-1], wherein the pancreatic β cells have a mutation in the insulin gene. [19-3] The method of [19-2], wherein the mutation in the insulin gene is present in only one allele of the cells.

[20] A method for treating or preventing diabetes, comprising transplanting an effective amount of the pancreatic β cells of

[17] into a mammal.

[21] The pancreatic β cells of

[17] for use in treating or preventing diabetes.

[22] Use of the pancreatic β cells of

[17] in the manufacture of a therapeutic or preventive agent for diabetes.

[0009] The present invention makes it possible to produce pancreatic β cells with higher insulin secretion capacity than pancreatic β cells produced by conventional methods. Furthermore, by adding a step of treating conventional pancreatic β cell induction methods with an endoplasmic reticulum stress-reducing drug for a certain period of time, it becomes possible to produce pancreatic β cells with even better insulin secretion capacity. Because the cells produced in this way can have properties closer to those of in vivo pancreatic β cells, they are expected to be useful in transplantation therapy into the pancreas, for example.

[0010] This example shows an outline of the differentiation induction protocol for insulin-producing cells. Cells were induced to differentiate over a 30-day period using either rotational culture or three-dimensional (3D) culture. The percentage of endoplasmic reticulum (ER) stressed (BIP)-positive cells among insulin-positive cells was approximately 5% on day 30 of differentiation induction from pluripotent stem cells to pancreatic beta cells. n = 3, error bars: standard error, ns: not significant, **: P < 0.01, ***: P < 0.001 (one-way ANOVA, Tukey's test). Improvement in the efficiency of pancreatic beta cell differentiation induction by the addition of PBA. When differentiation into pancreatic beta cells was induced using an ER stress-reducing drug (4-phenylbutyric acid; PBA), the differentiation efficiency was approximately 1.2-fold higher on day 30 compared to the control. n = 3, error bars: standard error, ****: P < 0.0001 (two-way ANOVA). Improvement in insulin secretion efficiency in pancreatic beta cells by the addition of PBA. When ER stress-reducing agents (PBAs) were used to induce differentiation into pancreatic beta cells, insulin secretion efficiency improved at 30 days compared to controls, and in a glucose tolerance test, it was approximately sixfold higher. n = 4, error bars: standard error, ns: not significant, *: P < 0.05 (two-way ANOVA). Another ER stress-reducing agent (azoramide) improved insulin cell differentiation induction efficiency (the percentage of INS-positive cells among DAPI-positive cells). When azoramide was used to induce differentiation into pancreatic beta cells in plate culture, no ER-stressed cells (BIP-positive cells) were observed at 32 days compared to controls, and the number of insulin-secreting cells increased approximately 1.4-fold. In 3D culture, the addition of PBAs reduced the percentage of endoplasmic reticulum (ER)-stressed (BIP-positive) cells among insulin-positive cells. Without the addition of ER stress-reducing agents, the percentage of BIP-positive cells was approximately 6.8% at 30 days after differentiation of pluripotent stem cells into pancreatic beta cells. On the other hand, when PBA was added from Stage 3 to Stage 6, the percentage of BIP-positive cells was 0%. n=3, error bars: standard error, ***: P < 0.001 (unpaired t test). INS indicates insulin. Same below. Improved efficiency of pancreatic beta cell differentiation induction by adding PBA in 3D culture.When cells were induced to differentiate into pancreatic beta cells using the ER stress-reducing drug (4-phenylbutyric acid; PBA), differentiation efficiency was approximately 1.8-fold higher at 30 days compared to the control. n = 3, error bars: standard error, ***: P < 0.001 (unpaired t test). Comparison of rotational culture and 3D culture. The addition of PBA increased the efficiency of pancreatic beta cell differentiation induction by approximately 1.2-fold at 30 days after differentiation induction in rotational culture, but by approximately 1.8-fold in 3D culture. n = 11 (rotational culture), 5 (3D culture). Error bars: standard error, ***: P < 0.001, ****: P < 0.0001 (unpaired t test). Improvement of insulin secretion efficiency in pancreatic beta cells by the addition of PBA in 3D culture. When cells were induced to differentiate into pancreatic beta cells using the ER stress-reducing drug (PBA), glucose-responsive insulin secretion efficiency was approximately 1.8-fold higher at 30 days after differentiation induction compared to the control. Insulin secretion was significantly improved in both the PBA-treated and control groups in 3D culture compared to rotational culture (Figure 4). n = 3, error bars: standard error, ns: not significant, *: P < 0.05, **: P < 0.01 (RM two-way ANOVA). The efficiency of insulin cell differentiation induction in 3D culture was improved by other ER stress reducers (azoramide), ATPase inhibitors (KUS121), and Mitol inducers (berberrubin acetate adduct; denoted as Mitol in the graph). No ER-stressed cells (BIP-positive cells) were observed with the addition of azoramide, KUS121, or berberrubine acetate adduct. Left: Graph based on the results in Figure 10. The efficiency of insulin cell differentiation induction at 30 days after differentiation induction increased approximately 1.2-fold, 1.3-fold, and 1.4-fold, respectively. Furthermore, the combined use of PBA with KUS121 or berberrubine acetate adduct increased the percentage of insulin-producing cells by approximately 1.8-fold, respectively (n=3-6, error bars: standard error, ****: P < 0.0001 by ordinary one-way ANOVA).Right panel: Insulin secretion increased approximately 1.2-fold (Azoramide), approximately 1.2-fold (KUS121), approximately 1.1-fold (Berberrubin acetate adduct), approximately 1.4-fold (KUS121 + PBA), and approximately 1.4-fold (Berberrubin acetate adduct + PBA). n = 3-9; error bars indicate standard error; ****: P < 0.0001, ***: P < 0.001, **: P < 0.01, *: P < 0.05 (mixed-effect model, Fisher's LSD test). Verification of the timing and duration of PBA administration in inducing differentiation of pluripotent stem cells into pancreatic beta cells. Analysis was performed on day 30 after the initiation of pancreatic beta cell differentiation induction. No BIP-positive cells were observed when cells were cultured in the presence of PBA during stages 3-6 (days 7-30) or stages 4-6 (days 10-30). Left: Graph based on the results of Figure 12. When cells were cultured in the presence of PBA during Stages 3-6 (Day 7-Day 30) or Stages 4-6 (Day 10-Day 30), differentiation induction efficiency was significantly improved compared to the control. n = 3, error bars: standard error, ****: P < 0.0001, ***: P < 0.001, ns: not significant (ordinary one-way ANOVA). Right: Addition of PBA during the same period also significantly improved insulin secretion. n = 3, error bars: standard error, ****: P < 0.0001, *: P < 0.05, ns: not significant (mixed-effect model, Fisher's LSD test). Evaluation of ER stress after extending Stage 6 for one month. Culture in the absence of PBA increased BIP-positive cells (i.e., increased ER stress). This was accompanied by a decrease in both the percentage of insulin-producing cells and insulin secretion. On the other hand, when cultured in the presence of PBA, no ER stress was observed, and the efficiency of insulin cell differentiation induction and insulin secretion ability were maintained even when Stage 6 was extended for one month. Upper left graph: n=3, error bars: standard error, ****: P < 0.0001, **: P < 0.01, ns: not significant (by ordinary one-way ANOVA).Upper right graph: n=3, error bars: standard error, ****: P < 0.0001, **: P < 0.01, ns: not significant (ordinary one-way ANOVA). Lower graph: n=3, error bars: standard error, ns: not significant, *: P < 0.05, **: P < 0.01, ****: P < 0.0001 (RM two-way ANOVA). Increased insulin secretion in pancreatic islets by PBA addition. When pancreatic islets isolated from human donors were cultured in the presence of PBA for four days after isolation, ER stress was alleviated and the proportion of insulin cells increased. At the same time, glucose-responsive insulin secretion was also improved by approximately 1.5-fold. GCG stands for glucagon. Left graph: n=3, error bars: standard error, ****: P < 0.0001 (unpaired t-test). Center graph: n=3, error bars: standard error, ***: P < 0.001 (by unpaired t test). Right graph: n=6, error bars: standard error, ns: not significant, *: P < 0.05, **: P < 0.01, ****: P < 0.0001 (by RM two-way ANOVA).

[0011] 1. Method for Producing Pancreatic β Cells The present invention provides a method for producing pancreatic β cells from pancreatic endocrine precursor cells. Specifically, the present invention provides a method for producing pancreatic β cells (hereinafter sometimes referred to as the "production method of the present invention"), which comprises a step of inducing the differentiation of pancreatic endocrine precursor cells into pancreatic β cells in the presence of an endoplasmic reticulum stress-reducing agent (in other words, in a medium containing an endoplasmic reticulum stress-reducing agent) (hereinafter also referred to as the "pancreatic β cell differentiation induction step").

[0012] As used herein, "pancreatic endocrine progenitor cells" refer to cells that have the ability to differentiate into pancreatic β cells and express the neurogenin 3 (NGN3) gene. Furthermore, "pancreatic β cells" are also called "insulin-producing cells" and refer to cells that produce and secrete insulin. Typically, pancreatic β cells express the NKX6-1 (NK6 Homeobox 1) gene and the insulin gene (NKK6-1 + / Insulin +) cells. In a preferred embodiment, the pancreatic β cells obtained by the production method of the present invention are glucose-responsive insulin-secreting cells. As used herein, "glucose-responsive insulin-secreting cells" refers to cells whose insulin secretion is promoted by the presence of glucose, and preferably refers to cells whose insulin secretion level varies depending on the glucose concentration. At least when the insulin concentration secreted by the cells after 1 hour of culture is higher when the glucose concentration in the medium is 28.0 mM (high glucose concentration) compared to when the glucose concentration is 2.8 mM (low glucose concentration), it can be said that the insulin secretion level of the cells varies depending on the glucose concentration.

[0013] In this specification, unless otherwise specified, "cells" includes "cell populations." Furthermore, unless otherwise specified, "cells" refers to those obtained by cell culture. A cell population may be composed of one type of cell, or may be composed of two or more types of cells. Furthermore, unless otherwise specified, "cell populations" also include "cell aggregates" (also called "cell clumps").

[0014] Thus, the term "pancreatic endocrine precursor cells" encompasses cell populations containing pancreatic endocrine precursor cells and cell aggregates containing pancreatic endocrine precursor cells (also referred to as "pancreatic endocrine precursor cell masses"). Similarly, the term "pancreatic β cells" encompasses cell populations containing pancreatic β cells and cell aggregates containing pancreatic β cells (also referred to as "pancreatic β cell masses"). In one embodiment of the present invention, pancreatic endocrine precursor cells are cultured in the form of cell aggregates. In another embodiment of the present invention, pancreatic β cells are in the form of cell aggregates. The proportion of pancreatic endocrine precursor cells or pancreatic β cells contained in a cell population or cell aggregate (number of pancreatic endocrine precursor cells or number of pancreatic β cells / total number of cells) is not particularly limited, but is preferably 20% or more, and may be 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. The same applies to other cell types.

[0015] As used herein, unless otherwise specified, the terms "expressing" or "positively expressing" a gene are used to mean at least "the production of mRNA encoded by the gene," but preferably also "the production of a protein encoded by the mRNA."

[0016] As used herein, "endoplasmic reticulum stress-reducing drugs" (hereinafter also referred to as "ER stress-reducing drugs"), also known as "endoplasmic reticulum stress-improving drugs," refer to substances that can reduce (including "improve"; the same applies below) stress on the endoplasmic reticulum of cells. Substances that have such effects include those that induce the reversion of misfolded proteins in the endoplasmic reticulum to their normal structure, those that act directly or indirectly on the endoplasmic reticulum to reduce stress, those that alleviate factors that cause endoplasmic reticulum stress, such as a decrease or depletion of intracellular ATP concentration and oxidative stress, and those that alleviate stress responses caused by ER stress (e.g., mitochondrial dysfunction, inflammatory responses, apoptosis, etc.). Examples of ER stress-reducing drugs include chemical chaperones that contribute to the formation and stabilization of protein higher-order structures, substances that inhibit downstream signals of endoplasmic reticulum stress sensors, and valosin-containing protein (VCP) ATPase inhibitors. Three ER stress sensor pathways are known: the PERK (PKR-like endoplasmic reticulum kinase) pathway, the IRE1 (Inositol requiring 1) pathway, and the ATF6 (Activating transcription factor 6) pathway (Hetz, C., 2012, Nature Rev. Mol. Cell Biol., Vol. 13, pp. 89-102). Therefore, the ER stress-reducing drug used in the present invention may inhibit signal transduction in any of these pathways.

[0017] Specifically, chemical chaperones used as ER stress-reducing drugs in the present invention include, for example, 4-phenylbutyric acid (PBA), tauroursodeoxycholic acid, and trehalose, as well as BIX (BiP inducer X), which induces chaperone proteins. Substances that inhibit downstream signaling of ER stress sensors include, for example, Mitol (which functions through ubiquitination of IRE1α) or Mitol derivatives, salubrinal, guanabenz, GSK2606414, GSK2656157, ISRIB, STF-083010, MKC-3946, toyocamycin, nelfinavir, sunitinib, 4μ8C (7-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-8-carboxaldehyde), azoramide, and arctigenin. In addition, the ER stress-reducing drug may be a natural compound contained in plants, such as astragaloside IV, baicalein, berberine, crosin, elatoside C, ginsenoside Rb1, honokiol, icariin, mangiferin, notoginsenoside R1, or pterostilbene.

[0018] An example of a Mitol inducer is a berberrubine acetic acid adduct. The berberrubine acetic acid adduct is a compound having the structure shown in formula (I) or (II) below, as disclosed in WO 2025 / 053283. The berberrubine acetic acid adduct can be synthesized based on the descriptions in, for example, WO 2025 / 053283, Sato M. et al., bioRxiv. (2025); doi: 10.1101 / 2025.05.01.651794, and the like. Among these, compounds having a structure in which n is 4 in formula (I) or (II) below are preferred.

[0019]

[0020]

[0021] [In formula (I) or (II), n is an integer of 1 to 10 (preferably 4 to 7)]

[0022] A VCP ATPase inhibitor refers to a compound that inhibits at least the ATPase activity of VCP, preferably specifically inhibiting ATPase activity. Examples of such VCP ATPase inhibitors include KUS121 (sodium (E)-4-amino-3-((6-(4-fluoro-2-methylphenyl)pyridin-3-yl)diazenyl)naphthalene-1-sulfonate; CAS number: 1357164-52-3), KUS31, KUS69, KUS94, and KUS187. These compounds have been shown to specifically inhibit VCP ATPase activity, suppress intracellular ATP loss under ER stress, and alleviate ER stress-induced cell death (Ikeda HO et al., Sci Rep. 4:5970 (2014); Nakano N. et al., Heliyon. 2(4):e00096 (2016)). The structural formulas of these compounds are shown below with reference to Figure 1a in Ikeda HO et al., Sci Rep. 4:5970 (2014). Note that the document also discloses a compound (KUS11) other than the above compounds.

[0023]

[0024] Among these, preferred ER stress-reducing agents are PBA, azoramide, an ATPase inhibitor of VCP (particularly KUS121), and berberrubine acetic acid adduct (particularly a compound having a structure in which n is 4 in the above formula (I) or (II)), with PBA being particularly preferred. Multiple types of ER stress-reducing agents may be used in combination (for example, a combination of PBA and another ER stress-reducing agent, etc.).

[0025] When PBA is used as the ER stress-reducing drug, its concentration in the medium is typically 10 μM to 100 mM, preferably 200 μM to 10 mM, and more preferably 1 mM to 5 mM (in one embodiment, 2.5 mM). When azoramide is used as the ER stress-reducing drug, its concentration in the medium is typically 0.1 μM to 1 mM, preferably 1 μM to 100 μM, and more preferably 5 μM to 20 μM (in one embodiment, 10 μM). When KUS121 is used as the ER stress-reducing drug, its concentration in the medium is typically 0.5 μM to 5 mM, preferably 5 μM to 500 μM, and more preferably 20 μM to 100 μM (in one embodiment, 50 μM). When a berberrubine acetic acid adduct is used as the ER stress-reducing agent, its concentration in the medium is typically 0.1 μM to 1 mM, preferably 1 μM to 100 μM, and more preferably 5 μM to 20 μM (10 μM in one embodiment). When an ER stress-reducing agent other than these is used, the concentration of the ER stress-reducing agent in the medium is selected appropriately.

[0026] In the production method of the present invention, the culture may be either suspension culture (also referred to as three-dimensional culture) or adherent culture (also referred to as plate culture), but suspension culture is preferred from the viewpoint of insulin secretion ability. Furthermore, suspension culture and adherent culture may be switched at any time. As used herein, "suspension culture" refers to culture performed 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.

[0027] When the production method of the present invention is carried out using suspension culture, if the pancreatic endocrine precursor cells are provided in a state adherent to a culture vessel, the production method of the present invention can be carried out by exchanging the medium containing the pancreatic endocrine precursor cells for a medium containing a pancreatic β cell induction promoter and culturing under suspension culture conditions. Alternatively, if the pancreatic endocrine precursor cells are provided in a suspended state in a medium, the medium containing the pancreatic endocrine precursor cells can be exchanged for a medium containing a pancreatic β cell induction promoter and culturing under suspension culture conditions.

[0028] When pancreatic endocrine precursor cells are provided as cell aggregates, they may be substantially separated (or dissociated) by any method to separate them into single cells, and then cultured. Examples of the separation method include mechanical separation and separation using a separation solution having protease activity and collagenase activity (e.g., Accutase, a solution containing trypsin and collagenase). TM , Accumax TM (Nacalai Tesque)) or separation using a separation solution having only collagenase activity. The separated or dissociated cells can be further isolated using methods such as flow cytometry or mass cytometry using surface antigens as an indicator, magnetic cell separation, or affinity columns on which the desired antigens are immobilized.

[0029] Incubators used for suspension culture include, but are not limited to, 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. However, for shaking culture (also referred to as "agitation culture"), as described below, sealed incubators are preferred. Examples of such incubators include tissue culture flasks, culture bags, and roller bottles. Furthermore, to enable culture under non-adhesive conditions, the incubator is preferably non-cell-adhesive. Examples of non-cell-adhesive incubators include those whose surfaces have not been artificially treated to improve cell adhesion (e.g., coated with an extracellular matrix, etc.) or those whose surfaces have been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA)).

[0030] Alternatively, a culture vessel can be used that has fine wells on the cell culture surface, does not have a flat surface, and has a low-adhesion coating of a protein or the like. By using such a culture vessel, a large number of cell aggregates (e.g., about 700 cells) can be easily produced in a single culture vessel. Examples of such a culture vessel include EZSPHERE (registered trademark).

[0031] Shaking culture of pancreatic endocrine precursor cells can induce pancreatic β cells with superior insulin-producing ability compared to cultures without shaking. Without being bound by any theory, it is speculated that the physical shaking of the medium during shaking culture allows for the supply of oxygen and nutrients and the efficient exchange of excreted waste products, thereby inducing pancreatic β cells with superior insulin-producing ability. Therefore, the shaking culture used in the present invention is not limited as long as it can physically shake the medium. Shaking culture can be performed, for example, by placing the culture vessel in which the pancreatic endocrine precursor cells are cultured on a shaker, rotator, or the like; this method is also called rotation culture. Shaking culture can also be performed by placing the pancreatic endocrine precursor cells in an environment where a stirrer, impeller, or the like is rotating.

[0032] Shaking culture may be performed throughout the entire suspension culture period in the pancreatic β-cell differentiation induction step, or only for a portion of the period; however, it is preferable to perform the culture throughout the entire period. Furthermore, parameters such as the speed of shaking culture can be appropriately determined by those skilled in the art. For example, when shaking culture is performed using a wave-type 3D shaker (e.g., Mini-Shaker 3D, manufactured by Biosan, etc.), the shaking speed can be set within a range of, for example, 5 to 60 rpm. When shaking culture is performed using a reciprocating shaker (e.g., NS-LR, manufactured by AS ONE, etc.), the shaking speed can be set within a range of, for example, 15 to 60 rpm. When shaking culture is performed using a seesaw shaker (e.g., NS-S, manufactured by AS ONE, etc.), the shaking speed can be set within a range of, for example, 5 to 50 rpm. Alternatively, for example, a spinner flask (e.g., 3152, manufactured by Corning, etc.) can be placed on a magnetic stirrer and culture can be performed at a rotation speed that does not cause the cell aggregates to settle visually. Culture can also be performed using a three-dimensional rotary suspension culture device (e.g., CellPet CUBE, manufactured by J-Tech Corporation; Clinostar, manufactured by Cellvivo, etc.). When performing shaking culture using a three-dimensional rotary suspension culture device, the rotation speed range can be set, for example, between 15 and 60 rpm. In addition, shaking culture may involve rotation perpendicular to the direction of gravity (horizontal rotation) or parallel to the direction of gravity (vertical rotation), with horizontal rotation being preferred.

[0033] In the examples described below, it was shown that three-dimensional (3D) culture using EZSPHERE (registered trademark) improved the efficiency of differentiation into pancreatic β cells and insulin secretion ability. Therefore, the culture method used in the present invention is preferably 3D culture. As used herein, 3D culture refers to culture in an environment in which cells can form a spatial three-dimensional structure. 3D culture reproduces intercellular interactions and relationships with the extracellular matrix in a manner closer to a physiological environment, which can be advantageous in forming tissue-like structures, maintaining cell function, inducing differentiation, etc. 3D culture may be performed throughout the entire period of the production method of the present invention or only for a portion of the period, but it is preferably performed throughout the entire period.

[0034] 3D culture can be broadly classified into anchorage-dependent and anchorage-independent 3D culture. Anchorage-dependent 3D culture refers to a method of culturing cells three-dimensionally using scaffold materials such as extracellular matrices (e.g., Matrigel, hydrogel, collagen gel, laminin, etc.). Anchorage-independent 3D culture refers to a method of suspending or aggregating cells on a low-adhesion culture surface or special container to form cell aggregates such as spheroids. Examples of anchorage-independent 3D culture include culture using a low-adhesion U-bottom culture vessel or a culture vessel that does not have a flat culture surface and has a low-adhesion coating with a protein or other material (e.g., EZSPHERE®, etc.). EZSPHERE® is a low-adhesion culture vessel with multiple microwells. Using this cell vessel, cells seeded in each well naturally gather due to gravity, efficiently forming uniformly sized cell aggregates. The 3D culture may be static culture or the above-mentioned shaking culture, but static culture is preferred from the viewpoint of avoiding cell stress due to shaking. Therefore, in a preferred embodiment, the 3D culture is static culture using a culture vessel that does not have a flat culture surface and whose culture surface is low-adhesion coated with a protein or the like.

[0035] On the other hand, adherent culture can be typically performed by culturing cells using a culture vessel coated with, for example, Matrigel (BD Biosciences), Synthemax (Corning), collagen, gelatin, heparan sulfate proteoglycan, entactin, or a combination thereof.

[0036] Alternatively, from the viewpoint of xeno-free, laminin or a fragment thereof is also preferred. Examples of laminin or a fragment thereof include laminin-111 or a fragment thereof comprising its E8 region, laminin-211 or a fragment thereof comprising its E8 region (e.g., iMatrix-211), laminin-121 or a fragment thereof comprising its E8 region, laminin-221 or a fragment thereof comprising its E8 region, laminin-332 or a fragment thereof comprising its E8 region, laminin-3A11 or a fragment thereof comprising its E8 region, laminin-411 or a fragment thereof comprising its E8 region (e.g., iMatrix-411), laminin-421 or a fragment thereof comprising its E8 region, and laminin-511 or a fragment thereof comprising 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] Examples of pancreatic β-cell induction promoters used in the present invention include those described in Non-Patent Documents 2 and 3. Specific examples include zinc salts (e.g., zinc sulfate, zinc chloride, zinc sulfide, etc.), inhibitors of ALK5 and CDK8 / 19 (e.g., ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine (Cas No. 446859-33-2)), and combinations of ALK5 inhibitors (e.g., SB431542, SB525334, IN1130, EW-7197, etc.) with CD8 / 19 inhibitors (e.g., Senexin B, TR06096159, etc.)). These compounds may be used in combination. When zinc sulfate is used as the pancreatic β cell induction promoter, its concentration in the medium is typically 0.1 μM to 1 mM, preferably 1 μM to 100 μM, and more preferably 5 μM to 20 μM (10 μM in one embodiment). When a pancreatic β cell induction promoter other than zinc sulfate is used, the concentration of the pancreatic β cell induction promoter in the medium is selected appropriately.

[0038] The duration of the pancreatic β cell differentiation induction step is not particularly limited, and examples include 3 days or more, 4 days or more, 5 days or more, 6 days or more, and 7 days or more. Typically, it is 3 to 11 days, preferably 4 to 10 days, and more preferably 5 to 9 days (7 days in one embodiment). The period during which cells are cultured in the presence of an ER stress-reducing drug (hereinafter also referred to as "contacting cells with an ER stress-reducing drug") may be only a portion of the pancreatic β cell differentiation induction step, but preferably is the entire duration of the step.

[0039] The pancreatic endocrine precursor cells used in the present invention can be obtained by known methods. Examples include isolation from biological tissue (e.g., pancreas, etc.) using known techniques, differentiation-inducing methods for pancreatic endoderm cells, and obtaining them from companies such as ATCC. Pancreatic endocrine precursor cells can be isolated from biological tissue using, for example, methods using flow cytometry or mass cytometry with surface antigens as an indicator, magnetic cell separation, affinity columns on which the desired antigens are immobilized, etc.

[0040] Pancreatic endocrine precursor cells can also be obtained, for example, from pluripotent stem cells through a multistep differentiation induction process. "Pluripotent stem cells" refer to stem cells that can differentiate into various tissues and cells with 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 (nuclear transfer embryonic stem cells (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 a human embryo, the cells may be cells produced by destroying an embryo or cells produced without destroying an embryo, but from an ethical point of view, cells produced without destroying an embryo are preferred.

[0041] 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).

[0042] 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.

[0043] 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.

[0044] Various iPSC lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, etc. are available as induced pluripotent stem cell lines. Examples of 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 Ff-I01s04 lines. Other cell lines include the ChiPSC12 line.

[0045] The induced pluripotent stem cells used in the present invention may be cells derived from a patient with diabetes caused by a genetic mutation. Cells induced to differentiate from pluripotent stem cells derived from a patient with diabetes caused by a genetic mutation can serve as disease models that reflect the pathology of the disease, making them suitable for screening therapeutic or preventive drugs for the disease. Alternatively, pluripotent stem cells derived from a patient with diabetes caused by a genetic mutation can be genetically repaired by genome editing using a CRISPR-Cas system or other methods, and then differentiated into the desired cells, making them suitable for use as a therapeutic agent for the disease. In one embodiment, the induced pluripotent stem cells used in the present invention or cells (e.g., pancreatic β cells) obtained by differentiation induction from such cells have a mutation in the insulin gene (e.g., a mutation in only one allele of the insulin gene in the cells). Pancreatic β cells with such a genetic mutation can reflect the pathology of diabetes and are therefore suitable for screening therapeutic or preventive drugs for diabetes.

[0046] The species of origin of pluripotent stem cells and other cells (e.g., pancreatic β cells) is not particularly limited, and may be, for example, cells from 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 of origin is human.

[0047] Differentiation of pluripotent stem cells into pancreatic endocrine progenitor cells can be induced by known methods such as those described in Toyoda T, et al. Stem Cell Reports 2017, WO2020 / 059892 (US2021 / 0353686), Kimura et al., Cell Chemical Biology 2020 Dec 17;27(12):1561-1572.e7, Konagaya et al., Scientific Reports. 2019 Jan 24;9(1):640, WO2017 / 047797 (US2018 / 0327719), and non-patent literature 2 and 3. Specifically, differentiation can be induced by a method comprising, 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 archenterocytes, C) a step of inducing differentiation from archenterocytes into posterior foregut endoderm cells, D) a step of inducing differentiation from posterior foregut endoderm cells into pancreatic endoderm cells, and E) a step of inducing differentiation from pancreatic endoderm cells into pancreatic endocrine precursor cells. Thus, the production method of the present invention may comprise at least one of the above steps A) to E).

[0048] 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 at least one (preferably all) of a ROCK inhibitor, a GSK3β inhibitor, and a PI3K inhibitor. The medium may further contain insulin. The culture period is typically 2 to 8 days (in one embodiment, 4 days). In one embodiment, the ROCK inhibitor, GSK3β inhibitor, and PI3K inhibitor are contained in the medium only for a portion of the culture period (e.g., only on day 1). Herein, definitive endoderm cells are cells that differentiate into cells that form tissues of organs such as the digestive tract, lung, thyroid, pancreas, and liver during mammalian development. As used herein, "definitive endoderm cells" refer to cells that express at least one selected from the group consisting of SOX17, FOXA2, and CXCR4, preferably cells that express at least SOX17.

[0049] The concentration of activin A in the medium used in step A) is typically 5 to 1000 ng / mL, preferably 20 to 500 ng / mL, more preferably 50 to 150 ng / mL.

[0050] Examples of the ROCK inhibitor used in step A) 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 (e.g., see Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (e.g., 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 against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof. Among these, Y-27632 is preferred. These may be used in combination. When Y-27632 is used as a ROCK inhibitor, the concentration in the medium is typically 1 to 20 μM, preferably 5 to 15 μM.

[0051] 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. These inhibitors may be used in combination with multiple types. When using CHIR99021, the concentration in the medium is typically 0.5 to 5 μM, preferably 1 to 4 μM. Examples of PI3K inhibitors used in step A) include PI-103, ZSTK474, NVP-BEZ235, LY294002, and wortmannin. Among these, wortmannin is preferred. These inhibitors may be used in combination with multiple types. When wortmannin is used, the concentration in the medium is typically 5 to 1000 nM, preferably 20 to 500 nM, more preferably 50 to 150 nM.

[0052] The differentiation into archenterocytes in step B) can be carried out, for example, by culturing the definitive endoderm cells obtained in step A) in a medium containing a growth factor. The medium may further contain a GSK3β inhibitor and / or a BMP inhibitor. The culture period is typically 1 to 4 days (in one embodiment, 2 days). As used herein, "archenterocytes" refer to cells that express at least one of HNF1β and FOXA2, preferably cells that express at least HNF1β, and more preferably cells that express both.

[0053] The growth factors used in step B) are preferably EGF, KGF, or FGF10, more preferably EGF or KGF, and even more preferably KGF. These may be used in combination. The concentration of the growth factor in the medium is determined appropriately depending on the type of growth factor used. For example, when KGF is used as the growth factor, the concentration in the medium is typically 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably 50 ng / mL. In the case of EGF, the concentration in the medium is typically 5 to 2000 ng / mL, preferably 5 to 1000 ng / mL, and more preferably 10 to 1000 ng / mL. In the case of FGF10, the concentration in the medium is typically 5 to 2000 ng / mL, preferably about 10 to 1000 ng / mL, and more preferably 10 to 1000 ng / mL.

[0054] The GSK3β inhibitor used in step B) can be the same as the GSK3β inhibitor used in step A). ​​When CHIR99021 is used as the GSK3β inhibitor, its concentration in the medium is typically 0.1 to 1 μM, preferably 0.1 to 0.5 μM. Examples of BMP inhibitors used in step B 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. These inhibitors may be used in combination. When LDN-193189 is used, the concentration in the medium is typically 1 to 1000 nM, preferably 10 to 100 nM, more preferably 20 to 70 nM.

[0055] The differentiation into posterior foregut endoderm cells in step C) can be carried out, for example, by culturing the archenterocytes obtained in step B) in a medium containing a growth factor. The medium may further contain at least one (preferably all) of a retinoic acid receptor agonist such as retinoic acid, a Hedgehog pathway inhibitor, and a BMP inhibitor. As used herein, "posterior foregut endoderm cells" refer to cells that have the ability to differentiate into pancreatic endoderm cells and express the PDX1 gene and / or the HNF4α gene.

[0056] The types of growth factors and BMP inhibitors used in step C), their concentrations in the medium, and the like are the same as those described in step B). KGF is a preferred growth factor used in step C). LDN-193189 is a preferred BMP inhibitor used in step C).

[0057] 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, retinoic acid or a salt thereof is preferred. These may be used in combination. When retinoic acid or a salt thereof is used, the concentration in the medium is typically 0.1 to 100 μM, preferably 0.5 to 10 μM.

[0058] 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. These inhibitors may be used in combination. When SANT-1 is used, its concentration in the medium is typically 0.01 μM to 1 μM, preferably 0.1 μM to 0.5 μM.

[0059] The culture period in step C) is typically 1 to 5 days (3 days in one embodiment). Furthermore, step C) preferably includes a step of culturing in the presence of an ER stress-reducing drug. The period during which the ER stress-reducing drug is brought into contact with the cells may be only a part of the period of step C), but is preferably the entire period of the step. The type of ER stress-reducing drug used in step C), its concentration in the medium, and the like are as described in the pancreatic β cell differentiation induction step.

[0060] The differentiation into pancreatic endoderm cells in step D) can be carried out, for example, by culturing the posterior foregut endoderm cells obtained in step C) in a medium containing a growth factor and a BMP inhibitor. The medium may also contain a retinoic acid receptor agonist, a non-muscle myosin II inhibitor, nicotinamide, etc. In one embodiment, the medium used in step D) contains a growth factor, a BMP inhibitor, a retinoic acid receptor agonist, and nicotinamide. As used herein, "pancreatic endoderm cells" refer to cells that have the ability to differentiate into pancreatic endocrine precursor cells and express the PDX1 (pancreatic and duodenal homeobox 1) gene, and typically also express the NKX6.1 gene.

[0061] The types and concentrations in the medium of the growth factor, BMP inhibitor, and retinoic acid receptor agonist used in step D) are described in steps B) and C) by reference. EGF is preferred as the growth factor used in step D). LDN-193189 is preferred as the BMP inhibitor used in step D). Retinoic acid or a salt thereof is preferred as the retinoic acid receptor agonist used in step D). When retinoic acid or a salt thereof is used as the retinoic acid receptor agonist, the concentration in the medium is typically 1 nM to 10 μM, preferably 10 nM to 1 μM, and more preferably 50 nM to 200 nM. The concentration of nicotinamide in the medium used in step D) is typically 1 mM to 100 mM, preferably 5 mM to 50 mM.

[0062] 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. These inhibitors may be used in combination.

[0063] The culture period in step D) is typically 5 to 9 days (7 days in one embodiment). Furthermore, step D) preferably includes a step of culturing in the presence of an ER stress-reducing drug. The period during which the ER stress-reducing drug is brought into contact with the cells may be only a part of the period of step D), but is preferably the entire period of the step. The type of ER stress-reducing drug used in step D), its concentration in the medium, and the like are as described in the pancreatic β cell differentiation induction step.

[0064] The differentiation into pancreatic endocrine precursor cells in step E) can be carried out, for example, by culturing the pancreatic endoderm cells obtained in step D) in a medium containing an inhibitor of ALK5 and CDK8 / 19, and a Notch signal inhibitor. The medium may also contain at least one (preferably all) of a retinoic acid receptor agonist, a Hedgehog pathway inhibitor, and thyroid hormones. The medium may also contain an actin polymerization inhibitor (e.g., only on day 1 of step E).

[0065] The types of ALK5 and CDK8 / 19 inhibitors, retinoic acid receptor agonists, and hedgehog pathway inhibitors used in step E) are as described in the pancreatic β cell differentiation induction step and step C). The ALK5 and CDK8 / 19 inhibitor used in step E) is preferably ALK5 inhibitor II. The retinoic acid receptor agonist used in step E) is preferably retinoic acid or a salt thereof. The hedgehog pathway inhibitor used in step E) is preferably SANT-1. When ALK5 inhibitor II is used as the ALK5 and CDK8 / 19 inhibitor, the concentration in the medium is typically 1 μM to 10 mM, preferably 10 μM to 1000 μM, and more preferably 50 μM to 200 μM. When retinoic acid or a salt thereof is used as the retinoic acid receptor agonist, its concentration in the medium is typically 1 nM to 10 μM, preferably 10 nM to 1 μM, and more preferably 50 nM to 200 nM. When SANT-1 is used as the hedgehog pathway inhibitor, its concentration in the medium is typically 0.01 μM to 1 μM, and preferably 0.1 μM to 0.5 μM.

[0066] Examples of Notch signal inhibitors include DAPT (CAS No. 208255-80-5), γ-Secretase Inhibitor XXI (CAS No. 209986-17-4), γ-Secretase Inhibitor I (CAS No. 133407-83-7), Dibenzazepine (CAS No. 209984-56-5), LY411575 (CAS No. 209984-57-6), and RO4929097 (CAS No. 847925-91-1). Among these, γ-Secretase Inhibitor XXI is preferred. These inhibitors may also be used in combination. When using γ-Secretase Inhibitor XXI as a Notch signal inhibitor, its concentration in the medium is typically 0.1 to 5 μM, preferably 0.5 to 2 μM.

[0067] As used herein, thyroid hormones include thyroid hormone (also called triiodothyronine (T3)) and T4, as well as substances similar to T3 or that mimic the action of T3. Substances similar to T3 or that mimic the action of T3 include, for example, thyroid hormone receptor agonist compounds, specifically, for example, DITPA (also called 3,5-diiodothyropropionic acid or DITPA), thyroid hormone receptor subtype β (TRβ)-selective agonists, and thyroid hormone receptor subtype α1 (TRα1)-selective agonists. Among these, T3 is preferred as the thyroid hormone used in step E). These may also be used in combination. When T3 is used as the thyroid hormone, the concentration in the medium is typically 0.1 to 5 μM, preferably 0.5 to 2 μM.

[0068] Examples of actin polymerization inhibitors used in step E) include latrunculin A, latrunculin B, and chaetoglobosin A, with latrunculin A being preferred. These may be used in combination. When latrunculin A is used as the actin polymerization inhibitor, its concentration in the medium is typically 0.1 to 5 μM, preferably 0.5 to 2 μM.

[0069] The culture period in step E) is typically 5 to 9 days (7 days in one embodiment). Furthermore, step E) preferably includes a step of culturing in the presence of an ER stress-reducing drug. The period during which the ER stress-reducing drug is brought into contact with the cells may be only a part of the period of step E), but is preferably the entire period of the step. The type of ER stress-reducing drug used in step E), its concentration in the medium, and the like are as described in the pancreatic β cell differentiation induction step.

[0070] In the production method of the present invention, it is preferable to carry out the culture period in steps D) and E) entirely in the presence of an ER stress-reducing drug, in which case the culture will be carried out in the presence of an ER stress-reducing drug during the period in which posterior foregut endoderm cells are induced to pancreatic β cells. Furthermore, it is more preferable to carry out the culture period in steps C) to E) in the presence of an ER stress-reducing drug, in which case the culture will be carried out in the presence of an ER stress-reducing drug during the period in which archenterocytes are induced to pancreatic β cells.

[0071] The starting cells used in the above steps A) to E) may be obtained by isolation from biological tissues using known techniques, may be obtained by a method of inducing differentiation of each precursor cell, or may be obtained from a company such as ATCC.

[0072] As shown in the Examples below, it has been shown that ER stress-reducing agents not only have a high efficiency of inducing differentiation into pancreatic β cells but also have an effect of proliferating pancreatic β cells. Therefore, the production method of the present invention may include a step of culturing pancreatic β cells obtained in the step of inducing pancreatic β cell differentiation in the presence of an ER stress-reducing agent (hereinafter also referred to as "step F").

[0073] In another aspect, the present invention also provides a method for producing pancreatic β cells (hereinafter also referred to as the "expansion culture method of the present invention"), which comprises culturing pancreatic β cells in the presence of an endoplasmic reticulum stress-reducing agent. The starting pancreatic β cells used in this method may be pancreatic β cells obtained by the production method of the present invention, or may be pancreatic β cells that constitute pancreatic islets in a living organism (particularly, a human). The "production of pancreatic β cells" can also be interpreted as a "method for concentrating pancreatic β cells" or a "method for improving the insulin secretion ability of pancreatic β cells."

[0074] The culture period for step F) and the expansion culture method of the present invention can be appropriately set based on the desired cell number, etc., but is typically 1 day or more, preferably 10 days or more, and more preferably 30 days or more. The culture period for step F) or the expansion culture method of the present invention is preferably 180 days or less, preferably 90 days or less, and more preferably 60 days or less. Step F) preferably includes a step of culturing in the presence of an ER stress-reducing drug. The period during which the ER stress-reducing drug is contacted with the cells may be only a portion of the period of step F) or the expansion culture method of the present invention, but is preferably the entire period of the step. The type of ER stress-reducing drug used in step F) or the expansion culture of the present invention, the concentration in the medium, each compound, and the like are described in the pancreatic β cell differentiation induction step and step E).

[0075] Furthermore, although the culture in steps A) to F) or the expansion culture method of the present invention may be adherent culture, it is preferable that all steps be performed by suspension culture. The definitions and methods of adherent culture and suspension culture are as described above. Furthermore, when performing suspension culture, shaking culture may also be performed, but static culture is preferred.

[0076] When performing the above steps A) to F) or the expansion culture method of the present invention, if the starting cells are provided as cell clusters, they may be cultured as single cells by substantially separating or dissociating them by any method, or they may be cultured in the state of cell clusters. Examples of the separation method include mechanical separation such as pipetting, or separation using a separation solution having protease activity and collagenase activity (for example, Accutase, a solution containing trypsin and collagenase). TM, Accumax TM (Nacalai Tesque) or separation using a separation solution having only collagenase activity.

[0077] The medium used in the present invention can typically be prepared by adding various differentiation-inducing factors, such as pancreatic β cell induction-promoting substances (e.g., substances listed as "Differentiation inducers" in FIG. 1), to a basal medium. Examples of such basal media include MCDB 131 medium, IMDM, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof (e.g., DMEM / F12 medium, etc.). In one embodiment, MCDB 131 medium is used in the pancreatic β cell differentiation induction step. In one embodiment, RPMI 1640 medium is used in the above step A). ​​In one embodiment, MCDB 131 medium is used in the above step B). In one embodiment, DMEM / F12 medium is used in the above step C). In one embodiment, DMEM / F12 medium is used in the above step D). In one embodiment, MCDB 131 medium is used in the above steps E) and F) and the expansion culture method of the present invention.

[0078] These basal media may contain serum (e.g., human serum, fetal bovine serum (FBS), horse serum, goat serum, rabbit serum, etc.) or may be serum-free. In the case of serum-free media, they may optionally contain one or more serum substitutes, such as albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), ITS supplement (e.g., ITS-G, ITS-A, ITS-X, all Fujifilm Wako Pure Chemical Industries), fatty acids, insulin, sodium selenite, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The medium may also contain one or more substances such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins (e.g., nicotinamide, ascorbic acid), glucose, heparin, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, glucagon, hydrocortisone, epidermal growth factor (EGF), dexamethasone, and the like.

[0079] The culture temperature in each step of the production method of the present invention or the expansion culture method of the present invention is typically 30 to 40°C, preferably 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration preferably of 2 to 5%.

[0080] The seeding density of cells in each step of the production method of the present invention or in the expansion culture method of the present invention 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.

[0081] In each step of the production method of the present invention or the expansion culture method of the present invention, cells may be cultured under feeder-free conditions and / or xeno-free conditions. In the production method of the present invention or the expansion culture method of the present invention, all steps may be performed under feeder-free and xeno-free conditions. 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.

[0082] Each substance used in the present invention may be synthesized by a known method, or a commercially available product may be used. Furthermore, when the substance is a protein or peptide, the target protein or peptide may be obtained from a cell that expresses it. Cells that express the target protein or peptide can be produced by inserting DNA encoding the target protein or peptide into a known expression vector and then introducing the resulting expression vector into an appropriate host cell.

[0083] When the substance used in the present invention is a protein or peptide such as activin A, its origin is not particularly limited, but is preferably mammalian (e.g., human, mouse, rat, monkey, bovine, equine, porcine, canine, etc.), with human-derived proteins being particularly preferred. In one embodiment, the protein or peptide used in the present invention is of human origin or an orthologue of a human protein or peptide in another mammalian species. Furthermore, the protein or peptide used in the production method of the present invention includes not only wild-type proteins but also variants thereof having similar functions (e.g., promoting differentiation of target cells). Examples of variants include proteins or peptides with high identity (e.g., 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of a specific wild-type protein or peptide.

[0084] When the substance used in the present invention is a compound, not only the free form but also its pharmacologically acceptable salts are encompassed by the compound. The pharmacologically acceptable salts vary depending on the type of compound, but examples include inorganic base salts such as alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salts, and ammonium salts, as well as base addition salts such as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine, as well as inorganic acid salts such as mesylate, hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, and acid addition salts such as organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate.

[0085] When the compound used in the present invention has isomers such as optical isomers, stereoisomers, positional isomers, and rotational isomers, either one of the isomers or a mixture thereof is encompassed by the compound. These isomers can be obtained as a single product by known synthesis methods, separation methods (e.g., concentration, solvent extraction, column chromatography, recrystallization, etc.), optical resolution methods (e.g., fractional recrystallization, chiral column method, diastereomer method, etc.), etc. The compound used in the present invention may be crystalline, and both a single crystalline form and a crystalline mixture are encompassed by the compound. Crystals can be produced by crystallization using known crystallization methods. The compound used in the present invention may be a solvate (e.g., hydrate, etc.) or a non-solvate (e.g., non-hydrate, etc.), and both are encompassed by the compound. Furthermore, the compound used in the present invention may contain isotopes (e.g., 3 H, 14 C. 35 S, 125 Also included are compounds labeled with iodine, i.e., iodine-1, iodine-2, iodine-3, iodine-4, iodine-5, iodine-6, iodine-7, iodine-8, iodine-9, iodine-10, iodine-11, iodine-12, iodine-13, iodine-14, iodine-15,

[0086] The production method or expansion culture method of the present invention may include a step of recovering the target cells obtained in each step. The recovered cells may be cryopreserved using a cell cryopreservation solution. Furthermore, each of the obtained cells may be counted using a cell counter, or may be labeled with an antibody against a cell surface marker and then selected or purified by flow cytometry, mass cytometry, magnetic cell separation, or the like.

[0087] 2. Uses of Pancreatic β Cells In another aspect of the present invention, pancreatic β cells (hereinafter, sometimes referred to as "pancreatic β cells of the present invention") obtained by the production method or expansion culture method of the present invention ("obtained" can be read as "obtained" as appropriate) are also provided. The pancreatic β cells of the present invention may be in the form of a pancreatic β cell mass, or may be single cells isolated by flow cytometry, mass cytometry, magnetic cell separation, or the like.

[0088] Because the pancreatic β cells of the present invention can be used for the treatment and prevention of diabetes, in another aspect, a therapeutic or preventive agent for diabetes (hereinafter, sometimes referred to as the "medicine of the present invention") comprising the pancreatic β cells of the present invention is provided. The present invention also encompasses a method for treating or preventing diabetes, in which an effective amount of the pancreatic β cells of the present invention is transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) to be treated or prevented. Unless otherwise specified, the therapeutic or preventive agent (or therapeutic or preventive method) for diabetes herein also encompasses a medicament (or method) that can both treat and prevent the disease.

[0089] Examples of diabetes to be treated or prevented include type 1 diabetes, type 2 diabetes, borderline diabetes, secondary diabetes, gestational diabetes, and pancreatic diabetes. Furthermore, the cause of the diabetes may be unclear, may be due to a genetic abnormality, may be due to another disease, or may be due to other causes. Examples of genes involved in the genetic abnormality include the insulin gene, hepatocyte nuclear transcription factor (HNF) 4α gene, glucokinase gene, HNF1α gene, insulin promoter factor (IPF) 1 gene, HNF1β gene, neuroD1 gene, insulin receptor gene, and mitochondrial gene. The pancreatic β cells of the present invention exert a therapeutic or preventive effect against diabetes when transplanted into the pancreas, liver, peritoneal cavity, subcutaneous tissue, etc.

[0090] The amount of pancreatic β cells to be transplanted should be a therapeutically or prophylactically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of diabetes of the transplant recipient. For example, the number of cells may be, but is not limited to, 10 × 10 4 Cell ~10×10 11 More specifically, methods for transplanting the pancreatic β cells of the present invention into a patient include, for example, a method in which the obtained pancreatic β cells are suspended in physiological saline or the like and directly transplanted into the pancreas, liver, abdominal cavity, subcutaneous tissue, or the like of the patient, and a method in which the pancreatic β cells are three-dimensionally cultured on a scaffold composed of Matrigel or the like and the obtained pancreatic β cell mass is transplanted.

[0091] When the pancreatic β cells of the present invention are used for transplantation, it is desirable to use pancreatic β 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 prevent 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 pancreatic β cells of the present invention are prepared as parenteral preparations such as injections, suspensions, and infusions by mixing with a pharmaceutically acceptable carrier according to conventional methods. Accordingly, in one embodiment, a method for producing a therapeutic or preventive agent for diabetes, comprising the step of formulating the pancreatic β cells of the present invention, is also provided. Such a method may also comprise the step of preparing the pancreatic β cells of the present invention. Furthermore, the method may also comprise the step of preserving the pancreatic β 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 auxiliary agents (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), etc. The pancreatic β cells of the present invention may be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0094] The pancreatic β cells of the present invention are provided in a cryopreserved state under conditions typically used for cryopreserving cells, and can be thawed immediately before use. In this case, serum or a serum substitute, an organic solvent (e.g., DMSO), or the like may further be contained. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be 1-30% (v / v) or 5-20% (v / v). The concentration of the organic solvent is not particularly limited, but may be 0-50% (v / v) or 5-20% (v / v).

[0095] The pancreatic β cells of the present invention can also be used in a method for screening candidate drugs that are useful for treating or preventing diabetes. Thus, in yet another aspect of the present invention, there is provided a method for screening for therapeutic or preventive drugs for diabetes, comprising the steps of: (1) culturing the pancreatic β cells of the present invention in the presence or absence of a test substance; (2) measuring the insulin-producing ability of the pancreatic β cells in the presence or absence of the test substance; and (3) selecting the test substance as a candidate drug for treating or preventing diabetes when the insulin-producing ability of the pancreatic β cells is higher in the presence of the test substance than in the absence of the test substance. Examples of such diabetes include the same types of diabetes as those targeted for treatment or prevention by the pharmaceutical of the present invention.

[0096] In the above step (1), the period for culturing the pancreatic β cells of the present invention in the presence of a test substance (in other words, contacting the pancreatic β cells of the present invention with the test substance) is not particularly limited, but is typically 1 minute to 5 days, preferably 1 hour to 1 day. Furthermore, "in the absence of a test substance" may also be, for example, in the presence of a substance other than the test substance that is known to have no therapeutic or preventive effect on diabetes. Furthermore, the cells cultured in the absence of the test substance as a control in step (1) may be different cells prepared by the same method as the cells cultured in the presence of the test substance, or the same cells prior to culture with the test substance, or other cells that allow the effect of the test substance to be evaluated. In the present invention, cells having a mutation in a gene associated with the onset of diabetes, such as the insulin gene, may also be used.

[0097] The insulin-producing ability in step (2) can be measured, for example, by measuring the amount of insulin mRNA or protein in cells or in the medium using known methods (e.g., RT-qPCR, Western blotting, ELISA, etc.). Alternatively, the amount of C-peptide or the expression level of the reporter gene, which correlates with the amount of insulin production, may be used. The expression level of the reporter gene can be measured by a method appropriate for the type of reporter gene. For example, if the reporter gene is a luciferase gene, it can be measured based on luminescence intensity. If the reporter gene is a fluorescent protein gene, it can be measured based on fluorescence intensity. If the reporter gene is a chromogenic enzyme gene, it can be measured based on color intensity. Alternatively, reporter gene expression can be detected or measured using a fluorescence imaging system or a luminescence imaging system.

[0098] Test substances used in the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low-molecular-weight compounds, and natural compounds. Test substances may also be existing or candidate components of pharmaceuticals, nutritional foods, etc. Furthermore, by simultaneously contacting two or more test substances with cells, interactions, synergistic effects, etc. between the test substances can be verified.

[0099] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0100] Example 1: Induction of differentiation from human induced pluripotent stem cells into insulin-producing cells An outline of the differentiation induction method is shown in Figure 1. The detailed method is described below. In Examples 1 and 2, suspension culture and rotational culture (150 rpm) were performed throughout the entire period. Furthermore, as shown in Example 4 below, insulin-producing cells could be efficiently induced by an ER stress-reducing drug by seeding the same number of cells onto a cell aggregate formation plate (EZSPHERE (registered trademark) 6-well plate, IWAKI #4810-900) and performing static culture instead of rotational culture.

[0101] Human iPS cell line ChiPSC12 (Takara Bio, Cellartis #Y00280) was used. iPS cell culture (maintenance of undifferentiated state and passage) was performed using the Cellartis DEF-CS 500 Culture System (Takara Bio #Y30010) according to the manufacturer's instructions. The outline of the culture procedure is as follows: iPS cell culture was initiated on a culture plate coated with Cellartis DEF-CS 500 coat 1 (Takara Bio #Y30012) using iPS cell medium (Cellartis DEF-CS medium, Takara Bio #Y30010) at 37°C under 5% CO2 (Day 0). Hereafter, the time point or cells X days after the start of culture will sometimes be referred to as "Day X." Medium changes were performed daily starting the day after passaging, and the cells were passaged every 3–4 days.

[0102] Before initiating differentiation, we first prepared spheroids from undifferentiated iPS cells. Using TrypLE (Gibco #A1285901), undifferentiated iPS cell colonies were detached from the culture plate and dissociated into single cells. The iPS cells were dispersed in iPS cell medium and plated at 2.5 × 10 cells per well in a low-attachment 6-well plate (Costar 6-well flat bottom ultra-low attachment, Corning #3471). 6 The cells were seeded at a density of 1 / 3 and cultured at 37°C, 5% CO2 with rotation (150 rpm) for 1 day. All subsequent cultures were performed under the same gas, temperature, and rotation speed settings.

[0103] <Induction of differentiation of undifferentiated iPS cells into definitive endoderm cells (Stage 1) after spheroid formation.> Rotating culture was performed in RPMI 1640 with GlutaMAX medium (Gibco, #61870-010) containing various differentiation inducers. The medium was supplemented with 1% penicillin-streptomycin solution (FujiFilm #168-2319) and 2% B-27 supplement (Gibco #17604044). The differentiation inducers used were activin A (100 ng / mL, Biotech #338-AC-01M) and GSK3β inhibitors CHIR99021 (3 μM, FujiFilm CultureSure #034-23103), Y-27632 (10 μM, FujiFilm CultureSure #036-24023), and wortmannin (100 nM, Sigma #W3144). After 24 hours of culture, the medium was replaced with RPMI 1640 with GlutaMAX medium containing activin A (100 ng / mL) and 1% B-27 vitamin A-free supplement. The same medium was replaced daily and the cells were cultured for an additional 3 days.

[0104] <Induction of Differentiation into Gastrula Cells (Stage 2)> MCDB131 medium (Sigma-Aldrich #M8537) containing various differentiation inducers was changed daily for a total of 2 days. The medium was supplemented with 1% penicillin-streptomycin solution, GlutaMAX (2 mM, Gibco #35050061), 1% B-27 Vitamin A-free supplement, sodium bicarbonate (1.5 g / L, Gibco #250800094), and vitamin C (0.25 μM, Sigma-Aldrich #A4544). Keratinocyte Growth Factor (KGF) (50 ng / mL, R&D #251-KG), LDN-193189 (50 nM, Sigma-Aldrich #SML0559), and CHIR99021 (0.25 μM) were used as differentiation inducers.

[0105] <Induction of differentiation into posterior foregut endoderm cells (Stage 3)> DMEM / F12 with GlutaMAX medium (Gibco #10565018) containing various differentiation-inducing factors was used. The medium was supplemented with 1% penicillin-streptomycin solution, 1% B-27 vitamin A-free supplement (Gibco #12587010), and vitamin C (0.25 μM). The differentiation inducers used were keratinocyte growth factor (KGF) (50 ng / mL), retinoic acid (2 μM, Sigma-Aldrich #R2625), LDN-193189 (50 nM), and SANT-1 (0.25 μM, Sigma-Aldrich #S4572), as well as 4-phenylbutyric acid (PBA) (2.5 mM, Sigma-Aldrich #P21005) or azoramide (10 μM, Sigma-Aldrich #SML1560). The medium was changed daily for a total of 3 days. Negative controls were cultured without PBA or azoramide.

[0106] <Induction of Differentiation into PDX1-Positive Pancreatic Endoderm Cells (Stage 4)> Next, the cells were cultured for 7 days in DMEM / F12 with GlutaMAX medium containing various differentiation inducers. The medium was supplemented with 1% penicillin-streptomycin solution, 1% B-27 vitamin A-free supplement, and vitamin C (0.25 μM). The differentiation inducers used were epidermal growth factor (EGF) (100 ng / mL, R&D #236-EG), LDN-193189 (50 nM), retinoic acid (100 nM), nicotinamide (10 mM, Wako #145-01205), PBA (2.5 mM), or azoramide (10 μM). Negative controls were treated without PBA or azoramide. Samples after 16 days (Stage 4) of culture are designated "Day 16."

[0107] <Induction of differentiation into pancreatic endocrine progenitor cells (Stage 5)> Cells were cultured in MCDB131 medium containing various differentiation-inducing factors, including 1% penicillin-streptomycin solution, GlutaMAX (2 mM), bovine serum albumin (BSA) (2%, Sigma-Aldrich #A9418-50G), sodium bicarbonate (1.17 g / L), vitamin C (0.25 μM), insulin transferrin selenium ethanolamine (ITS-X) supplement (0.5%, Gibco #51500056), heparin (10 μg / ml, Sigma-Aldrich #H3149), and glucose (3.6 g / L, Gibco #A2494001). The differentiation inducers used were ALK5 inhibitor II (10 μM, Wako #012-23021), γ-secretaase inhibitor XXI (1 μM, Sigma-Aldrich #565789), retinoic acid (100 nM), SANT-1 (0.25 μM), 3,3',5-triiodo-L-thyronine sodium salt (T3) (1 μM, Sigma-Aldrich #T6397), and latrunculin A (1 μM, Tocris #3973), as well as PBA (2.5 mM) or azoramide (10 μM). Negative controls were omit- ed PBA or azoramide. After 24 hours, the medium was changed to one lacking latrunculin A from the differentiation inducers listed above. The medium was then changed every 48 hours for a total of 7 days. Samples cultured for 23 days (Stage 5) are labeled "Day 23."

[0108] <Final Differentiation Induction into NKX6.1+ / Insulin+ Cells (Stage 6)> MCDB131 medium containing various differentiation inducers was supplemented with the following additives and cultured for 7 days with medium changes every 48 hours: zinc sulfate heptahydrate (10 μM, Sigma-Aldrich #Z0251), 1% penicillin-streptomycin solution, GlutaMAX (2 mM), BSA (2%), sodium bicarbonate (1.17 g / L), vitamin C (0.25 μM), insulin transferrin selenium ethanolamine (ITS-X) supplement (0.5%, Gibco, 51500056), heparin (10 μg / ml), glucose (3.6 g / L), MEM nonessential amino acids (5%, Gibco #11140050), and PBA (2.5 mM) or azoramide (10 μM). Neither PBA nor Azoramide was added to the negative control group. Samples after 30 days of culture (Stage 6) are labeled "Day 30."

[0109] Example 2: Verification of the induction efficiency and insulin secretion ability of insulin-producing cells. Cultured samples were collected on Day 16, Day 23, and Day 30 and immunostained using an insulin antibody / BIP (ER stress marker) antibody. The results are shown in Figure 2. Insulin-positive cells began to appear on Day 9 after the start of differentiation induction, and although very few, insulin-positive and BIP-positive cells were observed. The number of both positive cells gradually increased thereafter, but even on Day 30, they accounted for less than 5% of the total. Therefore, it was found that very slight ER stress occurred in insulin-positive cells over the culture period, and that this gradually accumulated. No obvious apoptosis was observed during this process.

[0110] The results of counting the number of insulin-positive cells are shown in Figure 3. The addition of PBA, an ER stress-reducing drug, improved the efficiency of insulin cell differentiation induction, and on day 30, 1.2 times more insulin-positive cells were observed than in the control group.

[0111] Next, a glucose-loaded insulin secretion test was performed using the day 30 samples (Figure 4). The samples were cultured for 1 hour in a medium containing low glucose (2.8 mM), then transferred to a medium containing high glucose (28 mM) for another hour, and the culture medium was collected. PBA was not added to the culture medium during the secretion test. To prevent false positives due to the detection of insulin contained in the differentiation-inducing culture medium, C-peptide concentration was measured instead of insulin using ELISA (human C-Peptide Ultrasensitive ELISA kit, Mercodia #10-1141-01). Furthermore, by measuring C-peptide (insulin) contained within pancreatic β cells after the secretion test, we evaluated the percentage of C-peptide (insulin) actually secreted by the cells.

[0112] In response to low glucose stimulation, the amount of C-peptide (insulin) secreted in the PBA-added group increased approximately eight-fold compared to the control group. Furthermore, in response to high glucose stimulation, the amount of C-peptide (insulin) secreted in the PBA-added group increased to approximately six-fold compared to the control group. In the control group, there was no significant difference in C-peptide (insulin) secretion between low and high glucose stimulation, but in the PBA-added group, C-peptide (insulin) secretion also increased significantly and markedly in response to an increase in glucose concentration.

[0113] Therefore, it was revealed that using an endoplasmic reticulum stress-reducing drug (e.g., PBA) during the process of inducing differentiation of wild-type pluripotent stem cells (without mutations in the insulin gene) into pancreatic beta cells in culture not only increases differentiation efficiency, but also results in pancreatic beta cells with high glucose-responsive insulin secretion ability comparable to that of adult pancreatic beta cells.

[0114] Example 3: Verification of the effects of ER stress-reducing drugs other than PBA We investigated whether the efficiency of insulin cell differentiation induction could be improved by using another ER stress-reducing drug (azoramide). Plate culture was performed from Stage 1 to Stage 5, and the same procedure as in Example 1 was used, except that azoramide was added instead of PBA. As a result, ER stress (BIP positive) in insulin cells disappeared, as with the use of PBA, and the number of insulin cells increased by approximately 1.4-fold on Day 32 of differentiation induction (Figure 5).

[0115] Example 4: Induction of Differentiation from Human Induced Pluripotent Stem Cells to Insulin-Producing Cells by Three-Dimensional (3D) Culture. The rotational culture of Example 1 was entirely replaced with 3D culture, and the other conditions were the same to induce differentiation of human iPS cells into insulin-producing cells. 3D culture was performed by seeding equal numbers of cells onto a cell aggregate formation plate (EZSPHERE® 6-well plate, IWAKI #4810-900) and statically culturing the cells throughout the entire period. Hereinafter, unless otherwise specified, 3D culture refers to static culture using EZSPHERE.

[0116] 3D culture, like rotational culture, significantly suppressed the generation of ER-stressed cells (Figure 6), and was able to induce pancreatic beta cells more efficiently than rotational culture (Figure 7). Figure 8 shows the results of comparing the proportion of pancreatic beta cells in rotational culture and 3D culture. Figure 8 shows that on day 30 of differentiation induction, the efficiency of PBA-induced differentiation into pancreatic beta cells was approximately 1.2-fold in rotational culture, but increased to approximately 1.8-fold in 3D culture.

[0117] Example 5: Verification of insulin secretion ability by 3D culture The insulin secretion ability of the pancreatic β cells obtained in Example 4 was evaluated using the same method as in Example 2. When differentiation into pancreatic β cells was induced using an ER stress-reducing agent (PBA), insulin secretion efficiency improved compared to the control on day 30 of differentiation induction, and in the case of a glucose tolerance test, it was improved by approximately 1.8-fold (Figure 9). In 3D culture, insulin secretion was significantly improved not only in the PBA-treated group but also in the control group, so it is presumed that the PBA effect resulted in a lower improvement in insulin secretion ability compared to rotational culture.

[0118] Example 6: Verification of the Effects of ER Stress-Reducing Agents Other Than PBA in 3D Culture We investigated whether the use of ER stress-reducing agents other than PBA, or a combination of these agents, in 3D culture would similarly increase the differentiation efficiency into pancreatic β cells and insulin secretion capacity. Specifically, azoramide, the ATPase inhibitor KUS121, or the Mitol inducer berberrubine acetate adduct were used as ER stress-reducing agents other than PBA. One or two of these compounds were added to the culture medium from the start of Stage 3 to the end of Stage 6. The concentrations of each compound in the culture medium were as follows: PBA: 2.5 mM; Azoramide: 10 μM; KUS121: 50 μM; Berberrubine acetate adduct: 10 μM; PBA (2.5 mM) + KUS121 (50 μM); PBA (2.5 mM) + berberrubine acetate adduct (10 μM).

[0119] The structural formula of the specific berberrubine acetic acid adduct used in this example is shown below with reference to Figure 2a in Sato M. et al., Sato M. et al., bioRxiv. (2025); doi: 10.1101 / 2025.05.01.651794.

[0120]

[0121] No ER-stressed cells (BIP-positive cells) were observed with the addition of PBA, KUS121, or berberrubine acetate adduct (Fig. 10). Furthermore, the number of insulin cells at 30 days after differentiation induction increased by approximately 1.2-fold, 1.3-fold, and 1.4-fold, respectively (Fig. 11). Furthermore, the combined use of PBA with KUS121 or berberrubine acetate adduct increased the efficiency of insulin cell differentiation induction by approximately 1.8-fold (Fig. 11). Furthermore, the insulin secretory capacity increased by approximately 1.2-fold (azoramide), 1.2-fold (KUS121), 1.1-fold (berberrubine acetate adduct), 1.4-fold (KUS121 + PBA), and 1.4-fold (berberrubine acetate adduct + PBA), respectively (Fig. 11). These results demonstrate that while all ER stress-reducing drugs improve insulin cell differentiation induction and insulin secretory capacity, the combination with PBA is even more effective.

[0122] Example 7: Effect of PBA Administration on Differentiation Induction from Pluripotent Stem Cells to Pancreatic β Cells Analysis was performed 30 days after the start of differentiation induction into pancreatic β cells by 3D culture. When cells were cultured in the presence of PBA during Stages 3-6 (Day 7-Day 30) or Stages 4-6 (Day 10-Day 30), no BIP-positive cells were observed, and the differentiation induction efficiency and insulin secretion capacity were significantly improved compared to the control (Figures 12 and 13). On the other hand, when cells were cultured in the presence of PBA during Stages 3-4 (Day 7-Day 17) or Stages 3-5 (Day 7-Day 23), the generation of BIP-positive cells was not suppressed, and no significant differences were observed in the differentiation induction efficiency and insulin secretion capacity compared to the control (Figures 12 and 13).

[0123] Example 8: Verification of the effect of PBA on pancreatic β cells Pancreatic β cells obtained at Stage 6 (Day 30) were cultured for an additional 30 days in the presence of PBA, and the degree of reduction in ER stress was evaluated. For the additional 30 days, pancreatic β cells were cultured under the same culture conditions as Stage 6. When pancreatic β cells were cultured in the absence of PBA, ER stress increased, and the proportion of insulin cells and insulin secretion capacity decreased accordingly. On the other hand, when cultured in the presence of PBA, no ER stress was observed even when Stage 6 was extended for one month, and the efficiency of insulin cell differentiation induction and insulin secretion capacity were maintained.

[0124] Pancreatic islets isolated from brain-dead donors lose insulin secretion and become less functional over time when maintained under conventional culture conditions. Therefore, islet transplantation requires transplantation into diabetic patients as soon as possible after islet isolation from the donor. Therefore, we also evaluated the effect of PBA on pancreatic β cells, which comprise the islets in vivo. Human islets were obtained from the Integrated Islet Distribution Program (IIDP) in the United States. Isolated islets were cultured in human islet culture medium (25 mM HEPES, 1x GlutaMAX (Gibco), 1% Penicillin-Streptomycin, 10% FBS-containing CMRL (Gibco) Medium) supplemented with 10 mM PBA at 37°C under 5% CO2 for 4 days. ER stress was significantly reduced in β cells from PBA-treated islets compared to the control (untreated) group (Figure 15). Furthermore, the PBA group showed a slight increase in β cell number compared to the control group (Figure 15). Furthermore, the glucose-responsive insulin secretion capacity of the beta cells in the PBA group was improved by 1.5 times compared to the control group. This effect is equivalent to the PBA effect on iPS cell-derived insulin cells (1.8 times). These results are useful findings that point to the possibility of application to human islet transplantation.

[0125] These results demonstrate that ER stress-reducing drugs not only have a high efficiency of inducing differentiation into pancreatic beta cells, but also have a proliferation effect on pancreatic beta cells.

[0126] The present invention makes it possible to produce pancreatic β cells from pluripotent stem cells (in a preferred embodiment, with a higher induction efficiency than conventional methods) that have functionality similar to that of adult pancreatic islets (i.e., glucose-responsive insulin secretion ability). These pancreatic β cells can be used as a source of pancreatic islets for transplantation into diabetic patients and are also expected to contribute to the development of diabetes treatment drugs.

[0127] This application is based on patent application No. 2024-104475 filed in Japan (filing date: June 27, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing pancreatic beta cells, comprising the step of inducing differentiation of pancreatic endocrine precursor cells into pancreatic beta cells in the presence of an endoplasmic reticulum stress-reducing drug.

2. The method according to claim 1, which comprises culturing pancreatic endocrine precursor cells under suspension culture conditions.

3. The method according to claim 1 or 2, which comprises a three-dimensional culture step.

4. The method according to any one of claims 1 to 3, wherein at least one of the endoplasmic reticulum stress-reducing agents is selected from the group consisting of 4-phenylbutyric acid, azoramide, an ATPase inhibitor of VCP, and a berberrubine acetic acid adduct.

5. The method according to any one of claims 1 to 4, wherein at least one of the endoplasmic reticulum stress reducing agents is 4-phenylbutyric acid.

6. The method according to any one of claims 1 to 5, wherein two or more endoplasmic reticulum stress-reducing drugs are used.

7. The method according to any one of claims 1 to 6, wherein the step of inducing differentiation into pancreatic β cells comprises a step of culturing pancreatic endocrine precursor cells in the presence of zinc sulfate.

8. The method according to any one of claims 1 to 7, wherein the pancreatic β cells are glucose-responsive insulin-secreting cells.

9. The method according to any one of claims 1 to 8, wherein the pancreatic β cells have a mutation in the insulin gene.

10. A method according to any one of claims 1 to 9, wherein the pancreatic endocrine precursor cells are obtained by inducing differentiation of pancreatic endoderm cells in the presence of an endoplasmic reticulum stress-reducing drug.

11. The method according to claim 10, wherein the pancreatic endoderm cells are obtained by inducing differentiation of posterior foregut endoderm cells in the presence of an endoplasmic reticulum stress-reducing agent.

12. The method according to claim 11, wherein the posterior foregut endoderm cells are obtained by inducing differentiation of archenterocytes in the presence of an endoplasmic reticulum stress-reducing agent.

13. The method of any one of claims 1 to 12, wherein the pancreatic beta cells are derived from pluripotent stem cells.

14. A method for producing pancreatic beta cells, comprising a step of culturing pancreatic beta cells in the presence of an endoplasmic reticulum stress-reducing drug.

15. The method according to claim 14, wherein the starting pancreatic beta cells are pancreatic beta cells obtained by the method according to any one of claims 1 to 13 or pancreatic beta cells constituting pancreatic islets in a living organism.

16. The method according to claim 14 or 15, wherein the culture period is 30 days or more.

17. Pancreatic β cells obtained by the method according to any one of claims 1 to 16.

18. A therapeutic or preventive agent for diabetes, comprising the pancreatic beta cells of claim 17.

19. A method for screening for therapeutic or preventive drugs for diabetes, comprising: (1) culturing the pancreatic beta cells described in claim 17 in the presence or absence of a test substance; (2) measuring the insulin production ability of the pancreatic beta cells in the presence or absence of the test substance; and (3) selecting the test substance as a candidate substance for therapeutic or preventive drugs for diabetes when the insulin production ability of the pancreatic beta cells is higher in the presence of the test substance compared to in the absence of the test substance.

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