Proliferation inhibitor

Abstract

The purpose of the present invention is to provide a technique for removing non-endocrine and non-target cells that co-exist with differentiation-induced insulin-secreting cells. The present invention relates to a method for producing an insulin-producing cell population, the method comprising a step for treating, with a PLK inhibitor, an insulin-producing cell population that is differentiation-induced from pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to a method for removing non - endocrine off - target cells present in an insulin - producing cell population or pancreatic β - cell population obtained by inducing differentiation from pluripotent stem cells. [Background of the Invention]

[0002] Research is underway to induce differentiation of pluripotent stem cells such as iPS cells and ES cells into insulin - secreting cells such as insulin - producing cells and pancreatic β - cells for application in the treatment of diabetes.

[0003] To date, various methods have been developed and reported for inducing differentiation of pluripotent stem cells into an insulin - secreting cell population (Non - Patent Document 1). However, the insulin - secreting cell population obtained by differentiation induction contains non - endocrine cells together with the target insulin - secreting cells. Therefore, a method for more efficiently obtaining the target insulin - secreting cells has been eagerly desired for promoting their use in diabetes treatment.

Prior Art Documents

Non - Patent Documents

[0004]

Non - Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The inventors of the present invention have found that, among cell populations induced to differentiate from pluripotent stem cells into insulin-producing cells or pancreatic β cells, there are non-endocrine cells (hereinafter sometimes referred to as "CHGA-negative cells") characterized by being negative for chromogranin A (hereinafter referred to as "CHGA") separately from these insulin-secreting cells (insulin-producing cells and pancreatic β cells). They have also found that CHGA-negative cells include highly proliferative cells (CHGA-negative and Ki67-positive cells) characterized by being positive for the Ki67 marker.

[0006] When attempting to apply induced-differentiated insulin-secreting cells to the treatment of diabetes and the like, it is extremely important to strictly control cells other than insulin-secreting cells from the viewpoint of safety. In addition, the contamination and survival of highly proliferative cells may have an adverse effect on the recipient and affect the long-term engraftment of the transplanted insulin-secreting cells, which is not preferable.

[0007] Therefore, an object of the present invention is to provide a method for removing CHGA-negative cells coexisting with induced-differentiated insulin-secreting cells.

Means for Solving the Problems

[0008] As a result of intensive studies to solve the above problems, the inventors of the present invention have found that by treating a pancreatic progenitor cell population, preferably an endocrine progenitor cell population, more preferably an insulin-producing cell population, or a cell population at a subsequent differentiation stage obtained by inducing differentiation from pluripotent stem cells with a PLK inhibitor, the proliferation of CHGA-negative cells can be suppressed, and it is possible to obtain a pancreatic progenitor cell population, an endocrine progenitor cell population, an insulin-producing cell population, or a cell population at a subsequent differentiation stage with a reduced content of these cells.

[0009] The present invention is based on these novel findings and includes the following inventions. [1] A method for producing an insulin-producing cell population, comprising a step of treating an insulin-producing cell population induced to differentiate from pluripotent stem cells with a PLK inhibitor. [1-1] A method for producing an insulin-producing cell population, comprising the step of treating an endocrine progenitor cell population induced to differentiate from pluripotent stem cells with a PLK inhibitor. [1-2] A method for producing an insulin-producing cell population, comprising the step of treating a pancreatic progenitor cell population induced to differentiate from pluripotent stem cells with a PLK inhibitor. [2] The production method according to any one of [1] to [1-2], wherein the produced cell population contains CHGA-negative cells at a ratio of 20% or less. [3] The production method according to any one of [1] to [2], wherein the produced cell population contains CHGA-negative and Ki67-positive cells at a ratio of 3% or less. [4] The production method according to any one of [1] to [3], wherein the produced cell population contains insulin-positive and NKX6.1-positive cells at a ratio of 15% or more. [5] The production method according to any one of [1] to [4], further comprising the step of differentiating the cell population treated with the PLK inhibitor. [6] The production method according to any one of [1] to [5], wherein the treatment is performed with a PLK inhibitor at 3 μM or less. [7] A method for suppressing the proliferation of CHGA-negative cells present in an insulin-producing cell population induced to differentiate from pluripotent stem cells, comprising: treating the cell population with a PLK inhibitor. [7-1] A method for suppressing the proliferation of CHGA-negative cells present in an endocrine progenitor cell population induced to differentiate from pluripotent stem cells, comprising: treating the cell population with a PLK inhibitor. [7-2] A method for suppressing the proliferation of CHGA-negative cells present in a pancreatic progenitor cell population induced to differentiate from pluripotent stem cells, comprising: treating the cell population with a PLK inhibitor. [8] The method according to [7], wherein the insulin-producing cell population is treated with a PLK inhibitor at 3 μM or less. [9] The method according to [7] or [8], wherein the CHGA-negative cells present in the insulin-producing cell population are reduced to a ratio of 20% or less.

[10] A method according to any one of [7] to [9], which reduces the proportion of CHGA-negative and Ki67-positive cells present in a cell population induced to differentiate from pluripotent stem cells to 3% or less.

[11] A method according to any one of [7] to

[10] , which increases the proportion of insulin-positive and NKX6.1-positive cells present in a cell population induced to differentiate from pluripotent stem cells to 15% or more.

[12] A cell population of insulin-producing cells induced to differentiate from pluripotent stem cells or a cell population at a subsequent differentiation stage, which is treated with a PLK inhibitor and contains CHGA-negative cells at a proportion of 20% or less.

[13] The cell population of

[12] , which contains CHGA-negative and Ki67-positive cells at a proportion of 3% or less.

[14] The cell population of

[12] or

[13] , which contains insulin-positive and NKX6.1-positive cells at a proportion of 15% or more.

[15] A cell population according to any one of

[12] to

[14] , which is used for transplantation.

[16] A medicament for treating diabetes, which contains a cell population according to any one of

[12] to

[15] .

[17] A method for treating or preventing diabetes, which includes a step of transplanting a cell population of insulin-producing cells induced to differentiate from pluripotent stem cells or a cell population at a subsequent differentiation stage, which is treated with a PLK inhibitor.

[18] A production method according to any one of [1] to [6], wherein the PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor.

[19] A production method according to any one of [1] to [6], wherein the PLK4 inhibitor is a substance with a 50% inhibitory concentration (IC 50 ) of less than 5 nM for PLK4.

[20] A production method according to any one of [1] to [6], wherein the PLK4 inhibitor is (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]ethenyl]-1H-indazol-6-yl]-5'-methoxy-spiro[cyclopropane-1,3'- 3 H]indol]-2'(1'H)-one (CAS No. 1338800-06-8) or a salt thereof.

[21] A method for producing any one of [1] to [6], wherein the PLK1 inhibitor is a substance having an IC50 of less than 5 nM for PLK1. 50

[22] A method for producing any one of [1] to [6], wherein the PLK1 inhibitor is (R)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide (CAS No. 929095-18-1) or a salt thereof.

[23] A method according to any one of [7] to

[11] , wherein the PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor.

[24] A method according to any one of [7] to

[11] , wherein the PLK4 inhibitor is a substance having an IC50 of less than 5 nM for PLK4. 50

[25] A method according to any one of [7] to

[11] , wherein the PLK4 inhibitor is (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]ethenyl]-1H-indazol-6-yl]-5'-methoxy-spiro[cyclopropane-1,3'- 3 H]indol]-2'(1'H)-one (CAS No. 1338800-06-8) or a salt thereof.

[26] A method according to any one of [7] to

[11] , wherein the PLK1 inhibitor is a substance having an IC50 of less than 5 nM for PLK1. 50

[27] A method according to any one of [7] to

[11] , wherein the PLK1 inhibitor is (R)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide (CAS No. 929095-18-1) or a salt thereof.

[28] A cell population according to any one of

[12] to

[15] , wherein the PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor.

[29] A medicament for treating diabetes, comprising the cell population of

[28] . ​​​The method of

[17] , wherein the PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor. The method of [5], wherein the step of differentiating an insulin-producing cell population treated with a PLK inhibitor is performed by transplanting into an animal. Any one of the methods of [7] to

[11] , which reduces the absolute number of CHGA-negative cells present in the cell population. Any one of the methods of [7] to

[11] , which does not reduce the number of cells that are not CHGA-negative cells present in the cell population. Any cell population of

[12] to

[14] that contains CHGA-negative and Ki67-positive cells at a ratio of 2% or less. Any cell population of

[12] to

[14] that contains CHGA-negative and Ki67-positive cells at a ratio of 1% or less. A prodrug containing any cell population of

[12] to

[14] ,

[34] and

[35] .

[37] A method for producing an insulin-producing cell population or a pancreatic β-cell population, comprising: (1) a step of treating an insulin-producing cell population or a pancreatic β-cell population with a PLK inhibitor; and (2) a step of embedding the insulin-producing cell population in a gel containing a biocompatible material. A method comprising the above.

[38] A method for producing an insulin-producing cell population or a pancreatic β-cell population, comprising: (0) a step of increasing the purity of a target cell population to at least 70% or more by a method of purifying the target cell population; (1) a step of treating an insulin-producing cell population or a pancreatic β-cell population with a PLK inhibitor; and (2) a step of differentiating the insulin-producing cell population treated with the PLK inhibitor. A method comprising the above.

[39] The method of

[30] , wherein the PLK4 inhibitor is a substance having an IC 50 of less than 5 nM for 50% inhibition of PLK4.

[40] The method according to

[30] , wherein the PLK4 inhibitor is (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]ethenyl]-1H-indazol-6-yl]-5'-methoxy-spiro[cyclopropane-1,3'- 3 H]indol]-2'(1'H)-one (CAS No. 1338800-06-8) or a salt thereof.

[41] The method according to

[30] , wherein the PLK1 inhibitor is a substance having an IC 50 50% inhibitory concentration (IC50) of less than 5 nM for PLK1.

[42] The method according to

[30] , wherein the PLK1 inhibitor is (R)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide (CAS No. 929095-18-1) or a salt thereof.

[43] The method according to any one of

[17] ,

[30] , and

[39] to

[42] , wherein the cell population contains CHGA-negative cells at a ratio of 20% or less.

[44] The method according to any one of

[17] ,

[30] , and

[39] to

[42] , wherein the cell population contains CHGA-negative and Ki67-positive cells at a ratio of 3% or less.

[45] The method according to any one of

[17] ,

[30] , and

[39] to

[42] , wherein the cell population contains insulin-positive and NKX6.1-positive cells at a ratio of 15% or more.

[46] A cell population induced to differentiate from pluripotent stem cells or a cell population at a subsequent differentiation stage, treated with a PLK inhibitor, for use in a method for treating or preventing diabetes.

[47] The cell population according to

[46] , wherein the PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor.

[48] The cell population according to

[47] , wherein the PLK4 inhibitor is a substance having an IC 50 50% inhibitory concentration (IC50) of less than 5 nM for PLK4.

[49] The cell population of

[47] , wherein the PLK4 inhibitor is (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]ethenyl]-1H-indazol-6-yl]-5’-methoxy-spiro[cyclopropane-1,3’- 3 H]indol]-2’(1’H)-one (CAS No. 1338800-06-8) or a salt thereof.

[50] The cell population of

[47] , wherein the PLK1 inhibitor is a substance with a 50% inhibitory concentration (IC 50 ) of less than 5 nM for PLK1.

[51] The cell population of

[47] , wherein the PLK1 inhibitor is (R)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide (CAS No. 929095-18-1) or a salt thereof.

[52] The cell population of any one of

[46] to

[51] , containing CHGA-negative cells at a ratio of 20% or less.

[53] The cell population of any one of

[46] to

[51] , containing CHGA-negative and Ki67-positive cells at a ratio of 3% or less.

[54] The cell population of any one of

[46] to

[51] , containing CHGA-negative and Ki67-positive cells at a ratio of 2% or less.

[55] The cell population of any one of

[46] to

[51] , containing CHGA-negative and Ki67-positive cells at a ratio of 1% or less.

[56] The cell population of any one of

[46] to

[51] , containing insulin-positive and NKX6.1-positive cells at a ratio of 15% or more.

[57] Use of a cell population producing insulin or a cell population at a subsequent differentiation stage, which is induced to differentiate from pluripotent stem cells and treated with a PLK inhibitor, in the manufacture of a medicament for treating or preventing diabetes.

[58] The use of

[57] , wherein the PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor.

[59] The PLK4 inhibitor, wherein the 50% inhibitory concentration (IC 50Use of a substance with

[60] The PLK4 inhibitor is (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]ethenyl]-1H-indazol-6-yl]-5’-methoxy-spiro[cyclopropane-1,3’- 3 H]indol]-2’(1’H)-one (CAS No. 1338800-06-8) or a salt thereof, for

[58] .

[61] The PLK1 inhibitor has a 50% inhibitory concentration (IC 50 ) of less than 5 nM, for

[58] .

[62] The PLK1 inhibitor is (R)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide (CAS No. 929095-18-1) or a salt thereof, for

[58] .

[63] Use according to any one of

[57] to

[62] , wherein the cell population contains CHGA-negative cells at a ratio of 20% or less.

[64] Use according to any one of

[57] to

[62] , wherein the cell population contains CHGA-negative and Ki67-positive cells at a ratio of 3% or less.

[65] Use according to any one of

[57] to

[62] , wherein the cell population contains CHGA-negative and Ki67-positive cells at a ratio of 2% or less.

[66] Use according to any one of

[57] to

[62] , wherein the cell population contains CHGA-negative and Ki67-positive cells at a ratio of 1% or less.

[67] Use according to any one of

[57] to

[62] , wherein the cell population contains insulin-positive and NKX6.1-positive cells at a ratio of 15% or more. This specification includes the content described in the specification and / or drawings of Japanese Patent Application No. 2019-191876, which is the basis of the priority of this application. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Advantages of the Invention

[0010] According to the present invention, a method for removing CHGA-negative cells coexisting with differentiated insulin-producing cells can be provided.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] 1. Terms Hereinafter, the terms described in this specification will be explained.

[0012] In this specification, "about" indicates a value that varies by plus or minus 25%, 20%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% respectively with respect to a reference value. Preferably, the terms "about" or "approximately" indicate a range of plus or minus 15%, 10%, 5%, or 1% respectively with respect to a reference value.

[0013] In this specification, "comprise(s) or comprising" means indicating the inclusion of elements following that phrase, but not being limited thereto. Therefore, it suggests the inclusion of elements following that phrase, but does not suggest the exclusion of any other arbitrary elements.

[0014] In this specification, "consist(s) of or consisting of" means including all elements following that phrase and being limited thereto. Therefore, the phrase "consist(s) of" indicates that the listed elements are required or essential, and that no other elements substantially exist.

[0015] In this specification, "not using feeder cells" means basically not containing feeder cells and not using a medium preconditioned by culturing feeder cells. Therefore, substances such as growth factors and cytokines secreted from feeder cells are not contained in the medium.

[0016] As used herein, the term "feeder cell" or "feeder" refers to a cell that, when co-cultured with another type of cell, provides an environment that can support and allow the growth of those cells. Feeder cells may be derived from the same species as the cells they support, or from a different species. For example, as a feeder for human cells, human dermal fibroblasts or human embryonic stem cells may be used, or primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts may be used. Feeder cells can be inactivated by, for example, radiation or mitomycin C treatment.

[0017] As used herein, "adhesion" refers to the attachment of cells to a vessel, for example, the attachment of cells to a cell culture dish or flask of sterile plastic (or coated plastic) in the presence of an appropriate medium. Some cells cannot be maintained or grown in culture without adhering to a cell culture vessel. In contrast, non-adherent cells can be maintained and proliferated in culture without attachment to a vessel.

[0018] As used herein, "culture" refers to maintaining, growing, and / or differentiating cells in an in vitro environment. "Culturing" means sustaining, growing, and / or differentiating cells in vitro, for example, in a cell culture dish or flask. Culturing includes two-dimensional culture (planar culture) and three-dimensional culture (suspension culture).

[0019] As used herein, "enrich" and "enrichment" refer to increasing the amount of a specific component in a composition such as a composition of cells, and "enriched", when used to describe a composition of cells, e.g., a cell population, refers to a cell population in which the amount of a specific component is increased as compared to the proportion of such a component in the cell population before enrichment. For example, a composition such as a cell population can be enriched with respect to a target cell type, and thus the proportion of the target cell type is increased as compared to the proportion of target cells present in the cell population before enrichment. The cell population can also be enriched for the target cell type by cell selection and sorting methods known in the art. The cell population can also be enriched by the specific sorting or selection processes described herein. In certain embodiments of the invention, by a method of enriching a target cell population, the cell population is enriched by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% with respect to the target cell population.

[0020] As used herein, "deplete" and "depletion" refer to decreasing the amount of a specific component in a composition such as a composition of cells, and "depleted", when used to describe a composition of cells, e.g., a cell population, refers to a cell population in which the amount of a specific component is decreased as compared to the proportion of such a component in the cell population before depletion. For example, a composition such as a cell population can be depleted with respect to a target cell type, and thus the proportion of the target cell type is decreased as compared to the proportion of target cells present in the cell population before depletion. The cell population can also be depleted for the target cell type by cell selection and sorting methods known in the art. The cell population can also be depleted by the specific sorting or selection processes described herein. In certain embodiments of the invention, by a method of depleting a target cell population, the cell population is decreased (depleted) by at least 50%, 80%, 85%, 90%, 95%, 97%, 98% or 99% with respect to the target cell population.

[0021] As used herein, "purify" and "purification" refer to removing impurities in a composition such as a cell composition to make it pure for a specific component, and "purified", when used to describe a cell composition, e.g., a cell population, refers to a cell population in which the amount of impurities has decreased compared to the proportion of such components in the cell population before purification, and the purity of a specific component has been improved. For example, a composition such as a cell population can be purified with respect to a target cell type, and thus the proportion of the target cell type increases compared to the proportion of target cells present in the cell population before purification. The cell population can also be purified for the target cell type by cell selection and sorting methods known in the art. The cell population can also be purified by the specific sorting or selection processes described herein. In certain embodiments of the present invention, by a method of purifying a target cell population, the purity of the target cell population can be at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99%, or the impurities (including contaminating cells) can be undetectable.

[0022] As used herein, "not reducing the number of cells" means that the number of cells does not significantly decrease due to the implementation of the method of the present invention, meaning that there is no significant difference between the number of cells before the implementation of the method and the number of cells after the implementation of the method. However, a decrease in the number of cells that is not caused by the implementation of the method of the present invention (e.g., natural death of cells that can normally occur in conventionally known cell culture and differentiation processes) can occur. Therefore, "not reducing the number of cells" also includes cases where the reduction rate of cells after implementation compared to before implementation of the method of the present invention is 30% or less, 20% or less, 10% or less, or 5% or less.

[0023] As used herein, "suppressing proliferation" means that the number of cells does not increase significantly due to the implementation of the method of the present invention, i.e., there is no significant increase between the number of cells before the implementation of the method and the number of cells after the implementation of the method. Therefore, "suppressing proliferation" also includes cases where the cell growth rate after the implementation of the method of the present invention is 30% or less, 20% or less, 10% or less, or 5% or less compared to before the implementation of the method.

[0024] As used herein, "marker" means a cell antigen or its gene that is specifically expressed by a predetermined cell type, such as "marker protein", "marker gene", etc. Preferably, the marker is a cell surface marker, in which case, enrichment, isolation, and / or detection of viable cells can be carried out. The marker can be a positive selection marker or a negative selection marker.

[0025] Detection of the marker protein can be performed using an immunological assay with an antibody specific to the marker protein, such as ELISA, immunostaining, flow cytometry. Detection of the marker gene can be performed using nucleic acid amplification methods and / or nucleic acid detection methods known in the art, such as RT-PCR, microarray, biochip, etc. As used herein, "positive" for the marker protein means being detected as positive by flow cytometry, and "negative" means being below the detection limit by flow cytometry. Also, as used herein, "positive" for the marker gene means being detected by RT-PCR, and "negative" means being below the detection limit by RT-PCR.

[0026] As used herein, "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter within a cell.

[0027] As used herein, the term "factor having CDK8 / 19 inhibitory activity" means any substance having inhibitory activity against CDK8 / 19. In contrast to other proteins of the same CDK family, CDK8 is not required for cell proliferation, and inhibition of CDK8 has no significant effect under normal conditions. CDK19 is similar to CDK8, and inhibition of CDK8 is usually accompanied by inhibition of CDK19.

[0028] A "growth factor" is an endogenous protein that promotes the differentiation and / or proliferation of specific cells. Examples of "growth factors" include, for example, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), transferrin, various interleukins (e.g., IL-1 to IL-18), various colony-stimulating factors (e.g., granulocyte / macrophage-colony stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ, etc.) and other cytokines having an effect on stem cells, such as stem cell factor (SCF) and erythropoietin (Epo).

[0029] As used herein, the term "ROCK inhibitor" means a substance that inhibits Rho-kinase (ROCK: Rho-associated, coiled-coil containing protein kinase), and may be a substance that inhibits either ROCK I or ROCK II. The ROCK inhibitor is not particularly limited as long as it has the above-mentioned function. For example, N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (which may also be referred to as Y-27632 in this specification), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine (H-1152), 4β-[(1R)-1-aminoethyl]-N-(4-pyridinyl)benzene-1α-carboxamide (Wf-536), N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4β[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (Y-30141), N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide (GSK269962A), N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A) can be mentioned. The ROCK inhibitor is not limited to these, and antisense oligonucleotides or siRNAs against the mRNA of ROCK, antibodies that bind to ROCK, dominant negative ROCK mutants, etc. can also be used as ROCK inhibitors, and they can be commercially available or synthesized according to known methods.

[0030] As used herein, the term "GSK3β inhibitor" refers to a substance having inhibitory activity against GSK3β (glycogen synthase kinase 3β). GSK3 (glycogen synthase kinase 3) is a type of serine / threonine protein kinase and is involved in many signaling pathways related to glycogen production, apoptosis, and maintenance of stem cells. There are two isoforms of GSK3, namely α and β. The "GSK3β inhibitor" used in the present invention is not particularly limited as long as it has GSK3β inhibitory activity, and may be a substance having both GSK3β inhibitory activity and GSK3α inhibitory activity in combination.

[0031] Examples of GSK3β inhibitors include CHIR98014 (2-[[2-[(5-nitro-6-aminopyridin-2-yl)amino]ethyl]amino]-4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidine), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), TWS-119 (3-[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy]phenol), Kenpaullone, 1-azakenpaullone, SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), and AR-AO144-18, CT99021, CT20026, BIO, BIO-acetoxime, pyridocarbazole-cyclopentadienylruthenium complex, OTDZT, alpha-4-dibromoacetophenone, lithium, and the like. GSK3β is not limited to these, and antisense oligonucleotides or siRNAs against the mRNA of GSK3β, antibodies that bind to GSK3β, dominant negative GSK3β mutants, etc. can also be used as GSK3β inhibitors and can be commercially available or synthesized according to known methods.

[0032] As used herein, "serum substitute" refers to, for example, KnockOut TMSerum replacements include KSR (Thermo Fisher Scientific), StemSure® Serum Replacement (Wako), B-27 supplement, N2-supplement, albumin (e.g., lipid-rich albumin), insulin, transferrin, fatty acids, collagen precursors, trace elements (e.g., zinc, selenium (e.g., sodium selenite)), 2-mercaptoethanol, 3'-thiol glycerol, or mixtures thereof (e.g., ITS-G). Preferred serum replacements are B-27 supplement, KSR, StemSure® Serum Replacement, and ITS-G. When adding a serum replacement to the medium, the concentration in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight. In the present invention, it is preferable to use a "serum replacement" instead of serum.

[0033] 2. Insulin-producing cell population in which the proliferation of CHGA-negative cells is suppressed The present invention relates to an insulin-producing cell population in which the proliferation of CHGA-negative cells is suppressed, and these cell populations can be obtained by treating with a PLK inhibitor. The present invention also relates to a method for suppressing the proliferation of CHGA-negative cells by treating with a PLK inhibitor.

[0034] "CHGA-negative cells" means non-endocrine cells characterized by negative expression of the CHGA marker, which are present in a pancreatic progenitor cell population, preferably an endocrine progenitor cell population, more preferably an insulin-producing cell population or a cell population at a later differentiation stage, induced to differentiate from pluripotent stem cells during the process of differentiating from pluripotent stem cells to pancreatic β cells.

[0035] "CHGA-negative cells" may include cells characterized by the expression of Ki67 as a marker (i.e., CHGA Negative and Ki67-positive cells). "Ki67" is known as a cell cycle-related nuclear protein, and its expression is observed in the G1, S, G2, and M phases of proliferating cells, but not in the G0 phase where cell proliferation has ceased. Therefore, it is also known as a marker for cell proliferation and the cell cycle. For this reason, CHGA Negative and Ki67-positive cells are cells showing high proliferative activity.

[0036] The "insulin-producing cell population" according to the present invention means a cell population containing insulin-producing cells obtained by inducing differentiation from pluripotent stem cells. The "insulin-producing cells" mean cells characterized by the observation of the expression of insulin markers (i.e., insulin-positive cells). The "insulin-producing cells" may express the marker of NK6 homeobox 1 (NKX6.1), and preferably, are cells expressing both the markers of insulin and NKX6.1 (i.e., insulin-positive and NKX6.1-positive cells).

[0037] The "insulin-producing cell population" according to the present invention has a lower content of CHGA-negative cells compared to an insulin-producing cell population obtained by inducing differentiation from pluripotent stem cells according to a conventionally known method, and the content rate (which may be described as "ratio" in this specification) is 40% or less, or 30% or less, preferably 20% or less, more preferably 15% or less, still more preferably 10% or less, for example, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. The lower limit of the content rate is not particularly limited, but is 0% or more, 0.1% or more, or 0.5% or more. The content rate can be represented by using two numerical values respectively selected from the numerical values of the above upper and lower limits. For example, the content rate is 0.5% to 40%, or 0.5% to 30%, preferably 0.5% to 20%, more preferably 0.5% to 15%, still more preferably 0.5% to 10%. On the other hand, the "insulin-producing cell population" according to the present invention is a cell population in which insulin-positive cells, particularly insulin-positive and NKX6.1-positive cells, are enriched, compared to an insulin-producing cell population obtained by inducing differentiation from pluripotent stem cells according to a conventionally known method, and the content rate of insulin-positive and NKX6.1-positive cells is 14% or more, preferably 15% or more, more preferably 20% or more, still more preferably 25% or more, for example, 30% or more, 35% or more, 40% or more, or 45% or more. The upper limit of the content rate is not particularly limited, but is 70% or less, 60% or less, or 50% or less. The content rate can be represented by using two numerical values respectively selected from the numerical values of the above upper and lower limits. For example, the content rate is 14% to 50%, preferably 15% to 50%, more preferably 20% to 50%, still more preferably 25% to 50%.

[0038] The "insulin-producing cell population" according to the present invention can be obtained by treating a pancreatic progenitor cell population, preferably an endocrine progenitor cell population, more preferably an insulin-producing cell population, or a cell population at a subsequent differentiation stage, which is obtained by inducing differentiation from pluripotent stem cells, with a PLK inhibitor. By treating a cell population at a predetermined differentiation stage with a PLK inhibitor, the proliferation of CHGA-negative cells can be suppressed, the cell content thereof can be decreased, and insulin-positive cells, preferably insulin-positive and NKX6.1-positive cells, can be enriched.

[0039] In the process of differentiating from pluripotent stem cells into pancreatic β cells, it is known that cells having different characteristics appear according to the differentiation stage (WO2009 / 012428, WO2016 / 021734). For example, this differentiation stage can be roughly classified into pluripotent stem cells, embryonic endoderm cells, primitive gut tube cells, posterior foregut cells, pancreatic progenitor cells, endocrine progenitor cells, insulin-producing cells, and pancreatic β cells in the order of relatively undifferentiated.

[0040] As used herein, "pluripotency" means the ability to differentiate into tissues and cells having various different forms and functions and to differentiate into cells of any lineage of the three germ layers. "Pluripotency" is distinguished from "totipotency", which can differentiate into all tissues of a living body including the blastocyst, in that it cannot differentiate into the blastocyst and thus does not have the ability to form an individual.

[0041] As used herein, "multipotency" means the ability to differentiate into cells of a plurality of limited lineages. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent but not pluripotent.

[0042] As used herein, the term "pluripotent stem cell" refers to embryonic stem cells (ES cells) and cells having the same pluripotency as ES cells, i.e., cells that potentially have the ability to differentiate into various tissues of a living body (all of endoderm, mesoderm, and ectoderm). Examples of cells having the same pluripotency as ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in this specification). Preferably, in the present invention, the pluripotent stem cells are human pluripotent stem cells.

[0043] As for "ES cells", if they are mouse ES cells, various mouse ES cell lines established by inGenious, RIKEN (RIKEN), etc. can be used. If they are human ES cells, various human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis can be used. For example, as ES cell lines, NIH's CHB-1 to CHB-12 lines, RUES1 line, RUES2 line, HUES1 to HUES28 lines, WiCell Research Institute's H1 line, H9 line, RIKEN's KhES-1 line, KhES-2 line, KhES-3 line, KhES-4 line, KhES-5 line, SSES1 line, SSES2 line, SSES3 line, etc. can be used.

[0044] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells". In addition to iPS cells 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), iPS cells derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131:861-872.), Nanog-iPS cells established by selecting and establishing using the expression of Nanog as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells produced by a method that does not contain c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106)), and iPS cells established by introducing six factors without using viruses (Okita K et al. Nat.Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA.et al., Science (2007) 318:1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ.et al., Nature (2007) 451:141-146), induced pluripotent stem cells produced by Sakurada et al. (Japanese Patent Laid-Open No. 2008-307007), etc. can also be used.

[0045] In addition, any of the known induced pluripotent stem cells in the art described in all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No 7, 795-797) or patents (e.g., JP 2008-307007, JP 2008-283972, US 2008 / 2336610, US 2009 / 047263, WO2007 / 069666, WO2008 / 118220, WO2008 / 124133, WO2008 / 151058, WO2009 / 006930, WO2009 / 006997, WO2009 / 007852) can be used.

[0046] As induced pluripotent cell lines, various iPS cell lines established by NIH, RIKEN, Kyoto University, etc. can be used. For example, in the case of human iPS cell lines, RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, Kyoto University's Ff-WJ-18 strain, Ff-I01s01 strain, Ff-I01s02 strain, Ff-I01s04 strain, Ff-I01s06 strain, Ff-I14s03 strain, Ff-I14s04 strain, QHJI01s01 strain, QHJI01s04 strain, QHJI14s03 strain, QHJI14s04 strain, 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, CDI's MyCell iPS Cells (21525.102.10A) strain, MyCell iPS Cells (21526.101.10A) strain, etc. can be mentioned.

[0047] As used herein, the term "pancreatic progenitor cell population" means a cell population containing pancreatic progenitor cells. As used herein, pancreatic progenitor cells mean cells characterized by the expression of at least one marker of PDX-1, NKX6.1, PTF-1α, GATA4, and SOX9.

[0048] The pancreatic progenitor cell population is a cell population containing pancreatic progenitor cells at a ratio of 30% or more, preferably 40% or more, more preferably 50% or more, still more preferably 60% or more, and even more preferably 70% or more. In addition to pancreatic progenitor cells, the pancreatic progenitor cell population may contain other cells (e.g., endocrine progenitor cells, insulin-producing cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0049] As used herein, the "endocrine progenitor cell population" means a cell population containing endocrine progenitor cells. As used herein, endocrine progenitor cells are cells characterized by the expression of at least one marker of CHGA, NeuroD, and NGN3 and the absence of the expression of markers of pancreatic-related hormone systems (e.g., insulin, etc.). Endocrine progenitor cells may express markers such as PAX-4, NKX2.2, Islet-1, PDX-1, PTF-1α, etc.

[0050] The endocrine progenitor cell population is a cell population containing endocrine progenitor cells at a ratio of 30% or more, preferably 40% or more, more preferably 50% or more, still more preferably 60% or more, and even more preferably 70% or more. In addition to endocrine progenitor cells, the endocrine progenitor cell population may contain other cells (e.g., pancreatic progenitor cells, insulin-producing cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0051] The ratio of a specific cell in a cell population can be determined based on a known method capable of calculating the number of cells, such as flow cytometry.

[0052] "Insulin-producing cells" are as described above, and an "insulin-producing cell population" usually contains insulin-producing cells at a ratio of 5% or more, preferably 10% or more, more preferably 15% or more, still more preferably 20% or more, even more preferably 25% or more, and particularly preferably 30% or more. In addition to insulin-producing cells, the cell population may contain other cells (e.g., endocrine progenitor cells; other pancreatic hormone-producing cells expressing at least one marker of glucagon, somatostatin, and pancreatic polypeptide; Ki67-positive cells, CHGA-negative cells, etc.).

[0053] As used herein, "pancreatic β-cells" mean cells that are more mature than "insulin-producing cells". Specifically, they mean cells that express at least one marker of MAFA, UCN3, and IAPP, which are maturation markers of pancreatic β-cells, or cells characterized by an increased insulin secretion response upon glucose stimulation.

[0054] A "pancreatic β-cell population" is a cell population containing pancreatic β-cells that can be obtained by differentiating and maturing an insulin-producing cell population, preferably by differentiating and maturing it in vivo. In addition to pancreatic β-cells, the cell population may contain other cells (e.g., insulin-producing cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0055] Cell populations at each differentiation stage can be obtained using known methods for inducing the differentiation of pluripotent stem cells into pancreatic β-cells. That is, the cell population of each target can be obtained by utilizing the following differentiation induction steps: Step 1) Induce differentiation from pluripotent stem cells into definitive endoderm cells; Step 2) Induce differentiation from definitive endoderm cells into gut tube cells; Step 3) Induce differentiation from gut tube cells into posterior foregut cells; Step 4) Induce differentiation from posterior foregut cells into pancreatic progenitor cells; Step 5) Induce differentiation from pancreatic progenitor cells into endocrine progenitor cells; Step 6) Induce differentiation from endocrine progenitor cells into insulin-producing cells. The following describes each step, but the induction of differentiation into each cell is not limited to these methods.

[0056] Step 1) Differentiation into definitive endoderm cells Pluripotent stem cells are first differentiated into definitive endoderm cells. Methods for inducing definitive endoderm from pluripotent stem cells are already known, and any of these methods may be used. Preferably, pluripotent stem cells are cultured in a medium containing activin A, more preferably a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, to differentiate into definitive endoderm cells. The number of cells at the start of culture is not particularly limited, and is 22,000 - 150,000 cells / cm 2 , preferably 22,000 - 100,000 cells / cm 2 , more preferably 22,000 - 80,000 cells / cm 2 . The culture period is 1 to 4 days, preferably 1 to 3 days, and particularly preferably 3 days.

[0057] The culture temperature is not particularly limited, but is carried out at 30 - 40°C (for example, 37°C). Also, the carbon dioxide concentration in the culture vessel is, for example, about 5%. The culture may be carried out either in two-dimensional culture or three-dimensional culture.

[0058] As the medium used in this step, basic media used for culturing mammalian cells such as RPMI 1640 medium, MEM medium, iMEM medium, DMEM / F12 medium, Improved MEM Zinc Option medium, Improved MEM / 1%B-27 / Penisilin Streptomycin medium, MCDB131 / 20mM Glucose / NaHCO3 / FAF-BSA / ITS-X / GlutaMAX TM / ascorbic acid / Penisilin Streptomycin medium, etc. can be used.

[0059] The concentration of activin A in the medium is usually 30 - 200 ng / mL, preferably 50 - 150 ng / mL, more preferably 70 - 120 ng / mL, and particularly preferably about 100 ng / mL. In another aspect, activin A can be included in the medium in a low dose, for example, in an amount of 5 to 100 ng / mL, preferably 5 to 50 ng / mL, more preferably 5 to 10 ng / mL. In yet another aspect, the concentration of activin A in the medium is about 0.1 to 100 ng / mL, preferably about 1 to 50 ng / mL, more preferably about 3 to 10 ng / mL.

[0060] The concentration of the GSK3β inhibitor in the medium is appropriately set according to the type of GSK3β inhibitor used. For example, when CHIR99021 is used as the GSK3β inhibitor, the concentration is usually 2 to 5 μM, preferably 2 to 4 μM, particularly preferably about 3 μM.

[0061] The concentration of the ROCK inhibitor in the medium is appropriately set according to the type of ROCK inhibitor used. For example, when Y27632 is used as the ROCK inhibitor, the concentration is usually 5 to 20 μM, preferably 5 to 15 μM, particularly preferably about 10 μM.

[0062] Insulin can be further added to the medium. Insulin can be included in the medium in an amount of 0.01 to 20 μM, preferably 0.1 to 10 μM, more preferably 0.5 to 5 μM. The concentration of insulin in the medium may be the concentration of insulin contained in the added B-27 supplement, but is not limited thereto.

[0063] In a specific aspect, after culturing for 1 day in a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, the medium is further cultured for 2 days while changing the medium every day in a medium containing only activin A. Alternatively, in the presence of a low dose of activin A, pluripotent stem cells can be produced by performing a first culture in a medium containing 0.01 to 20 μM of insulin, and then performing a second culture in a medium not containing insulin.

[0064] Step 2) Differentiation into primitive gut tube cells The embryonic endoderm cells obtained in step 1) are further cultured in a medium containing a growth factor to induce differentiation into primitive streak cells. The culture period is 2 to 8 days, preferably about 4 days.

[0065] The culture temperature is not particularly limited, but is carried out at 30 to 40 °C (for example, 37 °C). Also, the carbon dioxide concentration in the culture vessel is, for example, about 5%. The culture may be carried out either in two-dimensional culture or three-dimensional culture.

[0066] As in step 1), a basal medium used for culturing mammalian cells can be used as the medium. In addition to the growth factor, a serum substitute, vitamins, antibiotics, etc. may be appropriately added to the medium.

[0067] As the growth factor, EGF, KGF, and FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is even more preferred.

[0068] The concentration of the growth factor in the medium is appropriately set depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / mL (that is, about 0.8 to 320 nM), preferably about 5 to 1000 ng / mL (that is, about 0.8 to 160 nM), more preferably about 10 to 1000 ng / mL (that is, about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / mL (that is, about 0.3 to 116 nM), preferably about 10 to 1000 ng / mL (that is, about 0.6 to 58 nM). For example, when KGF is used as the growth factor, the concentration is usually 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL.

[0069] Step 3) Differentiation into posterior foregut cells The primitive streak cells obtained in step 2) are further cultured in a medium containing a growth factor, cyclopamine, noggin, etc. to induce differentiation into posterior foregut cells. The culture period is 1 to 5 days, preferably about 2 days. The culture may be carried out either in two-dimensional culture or three-dimensional culture.

[0070] The culture temperature is not particularly limited, but it is carried out at 30 to 40 °C (for example, 37 °C). Also, the carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0071] As in step 1), a basal medium used for culturing mammalian cells can be used as the medium. In addition to growth factors, serum substitutes, vitamins, antibiotics, etc. may be appropriately added to the medium.

[0072] As the growth factor, EGF, KGF, and FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is even more preferred.

[0073] The concentration of the growth factor in the medium is appropriately set depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / mL (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / mL (i.e., about 0.8 to 160 nM), more preferably about 10 to 1000 ng / mL (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / mL (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / mL (i.e., about 0.6 to 58 nM). For example, when KGF is used as the growth factor, the concentration is usually 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL.

[0074] The concentration of cyclopamine in the medium is not particularly limited, but is usually 0.5 to 1.5 μM, preferably 0.3 to 1.0 μM, and particularly preferably about 0.5 μM.

[0075] The concentration of noggin in the medium is not particularly limited, but is usually 10 to 200 ng / mL, preferably 50 to 150 ng / mL, and particularly preferably about 100 ng / mL.

[0076] Step 4) Differentiation into pancreatic progenitor cells The posterior foregut cells obtained in Step 3) are further cultured in a medium containing a factor having CDK8 / 19 inhibitory activity, preferably in a medium containing a factor having CDK8 / 19 inhibitory activity and a growth factor, to induce differentiation into pancreatic progenitor cells. The culture period is 2 to 10 days, preferably about 5 days. The culture may be performed either in two-dimensional culture or three-dimensional culture.

[0077] In the case of two-dimensional culture, according to the previously reported method (Toyoda et al., Stem cell Research (2015) 14, 185 - 197), the posterior foregut cells obtained in Step 3) are treated with 0.25% trypsin-EDTA and dispersed by pipetting. The obtained dispersion is subjected to centrifugation, and the collected cells are resuspended in a small amount of fresh medium. Then, the cell suspension is reseeded into the fresh medium of Step 4).

[0078] As in Step 1), a basal medium used for culturing mammalian cells can be used as the medium. In addition to growth factors, a serum substitute, vitamins, antibiotics, etc. may be appropriately added to the medium.

[0079] As the factor having CDK8 / 19 inhibitory activity, the various compounds or their salts described above can be used. Depending on the compound or its salt used, the addition amount to the medium is appropriately determined, but is usually about 0.00001 μM to 5 μM, preferably 0.00001 μM to 1 μM. As the concentration of the factor having CDK8 / 19 inhibitory activity in the medium, a concentration that reaches an inhibitory activity of 50% or more against CDK8 / 19 is preferred.

[0080] As the growth factor, EGF, KGF, and FGF10 are preferred, KGF and / or EGF are more preferred, and KGF and EGF are even more preferred.

[0081] The concentration of the growth factor in the medium is appropriately set depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / mL (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / mL (i.e., about 0.8 to 160 nM), more preferably about 10 to 1000 ng / mL (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / mL (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / mL (i.e., about 0.6 to 58 nM). For example, when using KGF and EGF as growth factors, the concentration of EGF is usually 5 to 150 ng / mL, preferably 30 to 100 ng / mL, particularly preferably about 50 ng / mL, and the concentration of KGF is usually 10 to 200 ng / mL, preferably 50 to 150 ng / mL, particularly preferably about 100 ng / mL.

[0082] The first day of the culture in step 4) may be carried out in the presence of a ROCK inhibitor, and thereafter, the culture may be carried out in a medium not containing the ROCK inhibitor.

[0083] In addition, the medium may contain a protein kinase C (PKC) activator. As the PKC activator, PDBu (PKC activator II), TPB (PKC actovator V), etc. are used, but not limited thereto. The concentration of the PKC activator is added at about 0.1 to 100 ng / mL, preferably about 1 to 50 ng / mL, more preferably about 3 to 10 ng / mL.

[0084] In addition, dimethyl sulfoxide and / or activin (1 to 50 ng / mL) may be added to the medium.

[0085] In any of the steps, in addition to the above-described components, a serum substitute (e.g., B-27 supplement, ITS-G) may be added to the medium. Further, if necessary, amino acids, L-glutamine, GlutaMAX (product name), non-essential amino acids, vitamins, nicotinamide, antibiotics (e.g., Antibiotic-Antimycotic (sometimes referred to as AA in this specification), penicillin, streptomycin, or a mixture thereof), antibacterial agents (e.g., amphotericin B), antioxidants, pyruvic acid, buffers, inorganic salts, etc. may be added. When adding an antibiotic to the medium, the concentration in the medium is usually 0.01 to 20% by weight, preferably 0.1 to 10% by weight. The culture may be performed by either two-dimensional culture or three-dimensional culture.

[0086] In the case of two-dimensional culture, cell culture is carried out by adherent culture without using feeder cells. During culture, culture vessels such as dishes, flasks, microplates, cell culture sheets such as OptiCell (product name) (Nunc), etc. are used. The culture vessel is preferably surface-treated to improve the adhesiveness (hydrophilicity) with cells and coated with a cell adhesion substrate such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel (e.g., BD Matrigel (Nippon Becton Dickinson)), vitronectin, etc. As the culture vessel, a culture vessel coated with type I collagen, Matrigel, fibronectin, vitronectin, or poly-D-lysine, etc. is preferred, and a culture vessel coated with Matrigel or poly-D-lysine is more preferred.

[0087] The culture temperature is not particularly limited, but is carried out at 30 to 40 °C (e.g., 37 °C). Also, the carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0088] The pancreatic progenitor cells obtained in step 4) can be further purified using a known surface marker such as glycoprotein 2 (GP2). The above purification can be carried out by a method known per se, for example, using beads immobilized with an anti-GP2 antibody.

[0089] Step 5) Differentiation into endocrine progenitor cells The pancreatic progenitor cells obtained in Step 4) are further cultured in a medium containing a growth factor to induce differentiation into endocrine progenitor cells. The culture may be performed either in two-dimensional culture or three-dimensional culture. In the case of two-dimensional culture, the pancreatic progenitor cells obtained in Step 4) are dispersed by treating with 0.25% trypsin-EDTA and then pipetting, centrifuged, dispersed again by treating with 0.25% trypsin-EDTA and pipetting, the obtained dispersion is subjected to centrifugation, the collected cells are resuspended in a small amount of fresh medium, and the cell suspension is reseeded into the fresh medium of Step 5). The culture period is 2 to 3 days, preferably about 2 days.

[0090] As in Step 1), a basal medium used for culturing mammalian cells can be used as the medium. According to the previously reported (Nature Biotechnology 2014;32:1121-1133), SANT1, retinoic acid, ALK5 inhibitor II, T3, and LDN are added to the medium, and further, a Wnt inhibitor, a ROCK inhibitor, FGF (preferably FGF2), a serum substitute, vitamins, antibiotics, etc. may be added as appropriate.

[0091] The culture is performed in non-adherent culture without using feeder cells. During the culture, a dish, flask, microplate, multi-well plate (Nunc), etc. or a bioreactor is used. The culture vessel is preferably surface-treated to reduce the adhesiveness to cells.

[0092] The culture temperature is not particularly limited, but is performed at 30 to 40 °C (for example, 37 °C). Also, the carbon dioxide concentration in the culture vessel is about 5%, for example.

[0093] Step 6) Differentiation into insulin-producing cells The endocrine progenitor cells obtained in Step 5) are further cultured in a medium containing a growth factor to induce differentiation into insulin-producing cells. The culture period is 10 to 30 days, preferably about 10 to 20 days.

[0094] The medium can be a basal medium used for culturing mammalian cells, similar to that in step 1). According to the previously reported (Nature Biotechnology 2014;32:1121-1133), ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor XX, γ-secretase inhibitor RO, N-cysteine, AXL inhibitor, and ascorbic acid can be added to the medium. Additionally, a Wnt inhibitor, a ROCK inhibitor, FGF (preferably FGF2), a serum substitute, vitamins, antibiotics, etc. can be appropriately added. For example, ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, and ascorbic acid can be added to the medium, or T3, ALK5 inhibitor II, ZnSO4, heparin, N-acetylcysteine, Trolox, and R428 can be added.

[0095] The culture can be performed either in 2D culture or 3D culture. Feeder cells are not used for the culture. In the case of 3D culture, it is carried out in non-adherent culture. During the culture, dishes, flasks, microplates, multi-well plates (Nunc), etc. or bioreactors are used. The culture vessel is preferably surface-treated to reduce its adhesiveness to cells.

[0096] The culture temperature is not particularly limited, but it is carried out at 30 - 40 °C (for example, 37 °C). Also, the carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0097] Differentiation into pancreatic β cells The cells obtained in the above step can be induced to differentiate into pancreatic β cells. The step of differentiating into a pancreatic β cell population can be carried out by transplanting an endocrine progenitor cell population or a cell population at a later differentiation stage, preferably an insulin-producing cell population, into an animal body.

[0098] The "animal" is preferably a mammal, for example, humans, non-human primates, pigs, cows, horses, sheep, goats, llamas, dogs, cats, rabbits, mice, guinea pigs, etc., but preferably humans.

[0099] The transplantation is preferably performed in a living body region where the cell population can be fixed at a certain position. For example, it can be performed under the skin, intraperitoneally, on the peritoneal epithelium, omentum, adipose tissue, muscle tissue, or under the capsule of each organ such as the pancreas and kidney of an animal. The number of cells to be transplanted can vary depending on factors such as the differentiation stage of the cells to be transplanted, the age, weight, size of the transplantation site, and severity of the disease of the transplantation target, and is not particularly limited. For example, it can be about 10×10 4 cells to 10×10 11 cells. The transplanted cell population can be induced to differentiate in the in-vivo environment and differentiate into the target cell population, preferably the pancreatic β-cell population. Thereafter, it may be recovered or left in the living body as it is.

[0100] At the time of transplantation, the cell population may be embedded in a gel containing a biocompatible material and transplanted. For example, a cell population embedded in a gel containing a biocompatible material can be encapsulated in a device such as a capsule, bag, or chamber and transplanted into the living body.

[0101] In the present invention, "embedding" means dispersing and accommodating an endocrine progenitor cell population or a cell population at a subsequent differentiation stage in a gel containing a biocompatible material.

[0102] As used herein, the term "biocompatible material" means any material that, when implanted in a living body and retained for a short or long term, does not induce a significant immune response or harmful biological reactions (e.g., toxic reactions, blood coagulation, etc.). Further, the "biocompatible material" is preferably a biodegradable material. Such materials include polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), polyethylene glycol (PEG), polyhydroxyethyl methacrylate, polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), poly(ethylene-co-vinyl acetate) (PEVA), polyacrylamide, polyethylene oxide, polyethyleneamine, polyhydroxybutyric acid, poly(N-vinylpyrrolidone), polyvinyl alcohol, polypropylene fumarate, polyacrylic acid, poly-e-caprolactone, polymethacrylic acid, polyvinylidene difluoride (PVDF), pectic acid, hyaluronic acid, heparan sulfate, chondroitin sulfate, heparan sulfate proteoglycan, heparin, chitin, chitosan, xanthan, carboxymethyl cellulose, carboxymethyl chitosan, alginate, alginate ester, collagen, cellulose, silk fibroin, keratin, gelatin, fibrin, pullulan, laminin, gellan, silicon, urethane, elastin, etc. and their modified forms, as well as combinations thereof. The surface of the "biocompatible material" may, if necessary, be subjected to surface modifications that enable cell adhesion (e.g., coating with a cell adhesion substrate (such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, matrigel, vitronectin, etc.)) or modification with functional groups known to control cell growth, differentiation, and function (e.g., amino groups, carboxyl groups, hydroxyl groups, methacrylic acid groups, acrylic acid groups, etc.). In certain embodiments, alginate or alginate ester can be preferably used as the "biocompatible material".

[0103] The alginate may be any water-soluble salt, and metal salts, ammonium salts, etc. can be used. For example, sodium alginate, calcium alginate, ammonium alginate, etc. can be preferably used.

[0104] The alginate ester (also referred to as propylene glycol alginate) is a derivative in which propylene glycol is ester-bonded to the carboxyl group of alginic acid.

[0105] The ratio (M / G ratio) of mannuronic acid to guluronic acid contained in the alginate is arbitrary. Generally, when M > G, a gel rich in flexibility can be formed, and when M < G, a strong gel can be formed. In the present invention, those containing guluronic acid at a ratio of 10 - 90%, 20 - 80%, 30 - 70%, or 40 - 60% can be used.

[0106] The preparation of the gel using the alginate or alginate ester can be carried out according to known methods (WO2010 / 032242, WO2011 / 154941), and can be obtained by adding a crosslinking agent to a solution of the alginate or alginate ester to cause gelation.

[0107] The alginate or alginate ester can be included in the solvent in an amount of 0.05 - 10% by weight, preferably 0.1 - 5% by weight, more preferably 0.5 - 3% by weight. The solvent may be any one that can dissolve the alginate or alginate ester, and water, physiological saline, etc. can be used.

[0108] The crosslinking agent may be any substance that can gel a solution of alginate or alginate ester, and is not particularly limited, but polyvalent metal cations can be used. As the polyvalent metal cations, divalent metal cations are preferred, and more preferably calcium ions, strontium ions, and barium ions. The crosslinking agent can be used in the form of a salt, and in the present invention, at least one selected from calcium chloride, strontium chloride, and barium chloride can be used as the crosslinking agent.

[0109] Nanofibers can be included in the gel containing alginate or alginate ester. Nanofibers are natural or synthetic fibers having a diameter in the nanometer range. Examples of natural nanofibers include those containing one or more polysaccharides such as collagen, cellulose, silk fibroin, keratin, gelatin, and chitosan. Examples of synthetic nanofibers include polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), poly(lactide-co-glycolide) (PLGA), poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), and poly(ethylene-co-vinyl acetate) (PEVA). The nanofibers can be included in the gel containing alginate in an amount of less than 1% by weight, for example, 0.9% by weight, 0.8% by weight, 0.7% by weight, 0.6% by weight, 0.5% by weight, or less. The lower limit of the amount of nanofibers included in the gel containing alginate or alginate ester is not particularly limited, but can be 0.05% by weight or more, preferably 0.1% by weight or more.

[0110] The embedding of the cell population into the gel containing alginate or alginate ester can be carried out by any means and is not particularly limited. For example, it can be carried out by mixing the cell population in a solution of alginate or alginate ester and gelling it.

[0111] The cell population is 1×10 4 cells~1×10 9cells / mL, preferably 1×10 7 cells to 1×10 8 It can be included in an amount selected from

[0112] The gelation of a solution of alginate or alginate ester containing a cell population can be carried out by adding a crosslinking agent to the solution. The amount of the crosslinking agent to be added can be an amount selected from 0.1 to 5% by weight, for example, 0.1 to 1% by weight, based on the solution. The gelation can be carried out in a container having a predetermined configuration and / or shape used for cell culture or cell transplantation, or in a mold designed to obtain a gel conforming to the container.

[0113] Alternatively, it may be carried out by forming a gel capsule containing alginic acid according to a known method (WO2010 / 010902). That is, a solution of alginate or alginate ester containing a cell population may be dropped into a solution of a crosslinking agent. Depending on the shape of the nozzle and the dropping method at the time of dropping, the size of the droplets can be adjusted, and thus the size of the gel capsule containing alginic acid can be defined. The dropping method is not particularly limited, and it can be carried out by a method such as an air spray method, an airless spray method, or an electrostatic spray method. The size of the gel capsule containing alginic acid is not particularly limited, but it can be 5 mm or less, 1 mm or less, or 500 μm or less in diameter. The crosslinking agent solution can contain a crosslinking agent in an amount selected from 0.1 to 10% by weight, for example, 0.1 to 5% by weight.

[0114] In the present invention, the "PLK inhibitor" is a substance having inhibitory activity against Polo-like kinase (PLK). PLK is a serine / threonine kinase family conserved in eukaryotes and is a kinase responsible for the progression of the M phase of the cell cycle. Four types, PLK1, PLK2, PLK3, and PLK4, are known. The PLK inhibitor used in the present invention only needs to be able to suppress the growth of CHGA-negative cells, and it may be an inhibitor against any of PLK1, PLK2, PLK3, and PLK4. The inhibitor against each PLK may be an inhibitor against another PLK. The PLK inhibitor used in the present invention only needs to be able to suppress the growth of CHGA-negative cells, and it may be a substance having other activities (such as inhibitory activity) in addition to the PLK inhibitory activity. Preferably, in the present invention, the "PLK inhibitor" is an inhibitor having inhibitory activity against PLK1 or PLK4, and preferably an inhibitor having more selectivity against PLK1 or PLK4. For example, a substance having a 50% inhibitory concentration (IC50) of 1 μM or less, preferably 100 nM or less, more preferably 50 nM or less, still more preferably 10 nM or less, particularly preferably 5 nM or less, and most preferably 3 nM or less against PLK1 or PLK4 can be suitably used. As a method for determining the PLK inhibitory activity, it may be selected from known methods, and examples include a determination method using Serine / Threonine Kinase Assay Kits (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.). In the present invention, conventionally known "PLK inhibitors" can be used and can be found in patent documents or non-patent documents.

[0115] As the PLK inhibitor that can be used in the present invention, (R)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide (CAS No. 929095-18-1) (in this specification, this compound may be described as "CAS929095-18-1"), (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]ethenyl]-1H-indazol-6-yl]-5'-methoxy-spiro[cyclopropane-1,3'- 3H]Indol]-2’(1’H)-one (CAS No. 1338800-06-8) (in this specification, this compound may be referred to as "CAS1338800-06-8"), 2-[[2-Fluoro-4-[[(2-fluoro-3-nitrophenyl)methyl]sulfonyl]phenyl]thio]-5-methoxy-N-(5-methyl-1H-pyrazol-3-yl)-6-(4-morpholinyl)-4-pyrimidineamine (CAS No. 1798871-30-3), 2-[[2-Fluoro-4-[[(2-fluoro-3-nitrophenyl)methyl]sulfonyl]phenyl]thio]-5-methoxy-N-(5-methyl-1H-pyrazol-3-yl)-6-(1-piperidinyl)-4-pyrimidineamine (CAS No. 1798871-31-4), 7-Nitro-5-(trifluoromethyl)-2-benzothiazolecarboxamide-3-oxide (CAS No. 40533-25-3), 5-(5,6-Dimethoxy-1H-benzimidazol-1-yl)-3-[[2-(trifluoromethyl)phenyl]methoxy]-2-thiophenecarboxamide (CAS No. 660868-91-7), 4-[(9-Cyclopentyl-7,7-difluoro-6,7,8,9-tetrahydro-5-methyl-6-oxo-5H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino]-2-fluoro-5-methoxy-N-(1-methyl-4-piperidinyl)benzamide hydrochloride (CAS No. 2108449-45-0), 3-(1,3-Benzodioxol-5-yl)-N-[(1S)-1-phenylethyl]-isoxazolo[5,4-c]pyridin-5-amine (CAS No. 1082739-92-1), (1S,6bR,9aS,11R,11bR)11-(Acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno[4,5,-h]-2-h]-2-benzopyran-3,6,9-trione (CAS No. 19545-26-7), 5-Cyano-7-nitro-2-benzothiazolecarboxamide-3-oxide (CAS No.40647-02-7), 2,3,4,5-tetrahydro-7-hydroxy-1H-benzo[furo[2,3-c]azepin-1-one (CAS No. 521937-07-5), 1-[6-[(3-acetyl-2,4,6-trihydroxy-5-methylphenyl)methyl]-5,7-dihydroxy-2,2-dimethyl-2H-1-benzopyran-8-yl]-3-phenyl-2-propen-1-one (CAS No. 82-08-6), N-[[4-[(6-chloro-3-pyridinyl)methoxy]-3-methoxyphenyl]methyl]-3,4-dimethoxy-benzenethanamine hydrochloride (CAS No. 1052532-15-6), A66 (CAS No. 1166227-08-2), (R)-2-(1-(7-methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)ethylamino)benzoic acid (CAS No. 1173900-33-8), N-[(4-methoxyphenyl)sulfonyl]-N-[2-[(1E)-2-(1-oxide-4-pyridinyl)ethenyl]phenyl]-acetamide (CAS No. 173529-46-9), sodium (E)-2-((2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenyl)amino)acetate (CAS No. 1225497-78-8), 4-((9-cyclopentyl-7,7-difluoro-5-methyl-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino)-2-fluoro-5-methoxy-N-(1-methylpiperidin-4-yl)benzamide (CAS No. 1137868-52-0), 4-((6-chloro-2-methoxyacridin-9-yl)methyl)-2-((4-(2-(dimethylamino)ethyl)piperazin-1-yl)methyl)phenol (CAS No. 2247919-28-2), 2-methyl-5-(1-methylethyl)-1-[O-(2-methylbenzoyl)oxime]-2,5-cyclohexadiene-1,4-dione (CAS No.321688-88-4), 9-cyclopentyl-2-[[2-ethoxy-4-(4-hydroxy-1-piperidinyl)phenyl]amino]-5,7,8,9-tetrahydro-5-methyl-6H-pyrimido[4,5-b][1,4]diazepin-6-one (CAS No. 1228817-38-6), 2,5-cyclohexadiene-1,4-dione, 2-methyl-5-(1-methylethyl)-, 1-oxime (CAS No. 17302-61-3), etc. or salts thereof are exemplified (but not limited thereto). Further, as long as these compounds have PLK inhibitory activity, preferably the 50% inhibitory concentration (IC50) against PLK1 or PLK4 is 1 μM or less, preferably 100 nM or less, more preferably 50 nM or less, still more preferably 10 nM or less, particularly preferably 5 nM or less, and most preferably 3 nM or less, they may have one or more substituents or some partial structures (substituents, rings, etc.) may be converted.

[0116] Preferably, in the present invention, the PLK inhibitor is (R)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide (CAS No.: 929095-18-1) which can be used as a PLK1 inhibitor, and (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]ethenyl]-1H-indazol-6-yl]-5'-methoxy-spiro[cyclopropane-1,3'- 3 H]indol]-2'(1'H)-one (CAS No. 1338800-06-8) which can be used as a PLK4 inhibitor.

[0117] The PLK inhibitor is not limited to the compounds shown above, and antisense oligonucleotides or siRNAs against PLK mRNA, antibodies that bind to PLK, dominant negative PLK mutants, etc. can also be used as PLK inhibitors and can be commercially available or synthesized according to known methods.

[0118] As the PLK inhibitor, the various compounds or their salts described above can be used. Depending on the compound or its salt used, the addition amount to the medium is appropriately determined, but it is usually about 0.00001 μM to 100 μM, preferably 0.01 μM to 10 μM, more preferably 0.1 μM to 5 μM, and particularly preferably 0.1 μM to 3 μM.

[0119] Treatment of a pancreatic progenitor cell population, preferably an endocrine progenitor cell population, more preferably an insulin-producing cell population, or a cell population at a subsequent differentiation stage, obtained by inducing differentiation from pluripotent stem cells with a PLK inhibitor can be performed by contacting the cell population with the PLK inhibitor. For example, it can be performed by culturing the cell population in a medium supplemented with a PLK inhibitor. The PLK inhibitor can be included in the medium in any amount capable of inhibiting PLK activity, for example, in an amount of 10 μM or less, or 5 μM or less, preferably 4 μM or less, more preferably 3 μM or less, for example, in an amount of 2 μM or less or 1 μM or less. The lower limit of the addition amount of the PLK inhibitor is not particularly limited, but can be 0.1 μM or more, preferably 0.5 μM or more. The addition amount of the PLK inhibitor is 10 μM or less and 0.1 μM or more, preferably 5 μM or less and 0.5 μM or more, and particularly preferably 3 μM or less and 0.5 μM or more. Culturing in the presence of the PLK inhibitor can be carried out for at least 12 hours, preferably 24 hours or more, 2 days or more, 4 days or more, 8 days or more, 10 days or more, or 15 days or more. Culturing in the presence of the PLK inhibitor is preferably carried out for 4 days or more. During the treatment period with the PLK inhibitor, the medium can be exchanged, and according to the culture schedule, it can be exchanged with a medium having the same composition as before the exchange supplemented with the PLK inhibitor or a medium having a different composition.

[0120] A population of pancreatic progenitor cells, preferably a population of endocrine progenitor cells, more preferably a population of insulin-producing cells, or a cell population at a subsequent differentiation stage, obtained by inducing differentiation from pluripotent stem cells, can be treated with a PLK inhibitor and subjected to a step of further differentiating into a target cell population. Here, "treating with a PLK inhibitor" includes cases where the step of treating with a PLK inhibitor and the step of differentiating are performed simultaneously, cases where the step of differentiating is performed after treatment with a PLK inhibitor, and cases where the step of treating with a PLK inhibitor is performed after the step of differentiating. Therefore, the medium used for treating with a PLK inhibitor and the medium used for differentiating the cell population may be different, or a PLK inhibitor may be further added to the medium used for the differentiating step.

[0121] This method can reduce or suppress the remaining number of proliferating cells included in the pancreatic lineage, rather than suppressing the growth of teratomas. This method can reduce or suppress the remaining number of proliferating cells included in the pancreatic lineage, rather than suppressing the growth of iPS cells (for example, it is not necessary to reduce the number of alkaline phosphatase-positive cells).

[0122] According to this method, it is possible to reduce the absolute number of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, in a population of endocrine progenitor cells or a cell population at a subsequent differentiation stage by treatment with a PLK inhibitor. As a result, CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, in the obtained cell population can be depleted.

[0123] That is, it is possible to reduce the ratio of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, in the obtained cell population as compared to the case where the cells are cultured and / or differentiated without treatment with a PLK inhibitor. The ratio of CHGA-negative cells in the obtained cell population is 40% or less, 30% or less, 20% or less, 15% or less, or 10% or less, preferably 20% or less, 15% or less, or 10% or less, for example, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. The lower limit of the ratio is not particularly limited, but is, for example, 0% or more, 0.1% or more, or 0.5% or more. The ratio can be represented using two numerical values respectively selected from the numerical values of the above upper and lower limits. For example, the ratio is 0.1% to 40%, 0.1% to 30%, 0.1% to 20%, 0.1% to 15%, or 0.1% to 10%, preferably 0.1% to 20%, 0.1% to 15%, or 0.1% to 10%. Alternatively, the ratio is 0.5% to 40%, 0.5% to 30%, 0.5% to 20%, 0.5% to 15%, or 0.5% to 10%, preferably 0.5% to 20%, 0.5% to 15%, or 0.5% to 10%. Particularly, the ratio of CHGA-negative and Ki67-positive cells is 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less in the obtained cell population, preferably 3% or less, 2% or less, 1% or less, or 0.5% or less. The lower limit of the ratio is not particularly limited, but is, for example, 0% or more, or 0.1% or more. The ratio can be represented using two numerical values respectively selected from the numerical values of the above upper and lower limits. For example, the ratio is 0.1% to 8%, 0.1% to 7%, 0.1% to 6%, 0.1% to 5%, 0.1% to 4%, 0.1% to 3%, 0.1% to 2%, 0.1% to 1%, or 0.1% to 0.5%, preferably 0.1% to 3%, 0.1% to 2%, 0.1% to 1%, or 0.1% to 0.5%.

[0124] Moreover, according to this method, a pancreatic progenitor cell population, preferably an endocrine progenitor cell population, more preferably an insulin-producing cell population, or a cell population at a subsequent differentiation stage, obtained by inducing differentiation from pluripotent stem cells treated with a PLK inhibitor, is differentiated into insulin-producing cells or pancreatic β cells, whereby the proliferation of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, is suppressed, and a cell population enriched in insulin-producing cells or pancreatic β cells can be obtained. That is, it is possible to increase the ratio of insulin-producing cells or pancreatic β cells in the cell population obtained after induction of differentiation as compared with the case where it is obtained without treatment with a PLK inhibitor. The ratio of insulin-producing cells or pancreatic β cells in the obtained cell population is 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. The upper limit of the ratio is not particularly limited, but is 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, or 95% or less. The ratio can be represented using two numerical values respectively selected from the numerical values of the upper limit and the lower limit, for example, the ratio is 40% to 95%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, or 90% to 95%. In particular, the ratio of insulin-positive and NKX6.1-positive cells is 14% or more, preferably 15% or more, more preferably 20% or more, still more preferably 25% or more, for example, 30% or more, 35% or more, 40% or more, or 45% or more. The upper limit of the ratio is not particularly limited, but is 50% or less. The ratio can be represented using two numerical values respectively selected from the numerical values of the upper limit and the lower limit, for example, the ratio is 14% to 50%, preferably 15% to 50%, more preferably 20% to 50%, still more preferably 25% to 50%.

[0125] The insulin-producing cells or pancreatic β cells obtained by this method can be transplanted into an animal body and, when differentiated in the animal body, can be left as they are and used as insulin-secreting cells. According to the insulin-producing cells or pancreatic β cells obtained by this method, the proliferation of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, can be avoided, and safe and long-term engraftment of transplanted cells can be achieved.

[0126] According to the present invention, an insulin-producing cell population or pancreatic β-cell population (the cell population of the present invention) from which CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells having high proliferative ability, have been removed is useful as a cell medicine for treating diabetes, particularly type I diabetes, when transplanted into an affected part as it is or after encapsulation.

[0127] Further, the cell population of the present invention may be a prodrug. As used herein, a prodrug refers to a cell population that differentiates after transplantation into a living body and changes into a cell having a function of treating a disease.

[0128] The cell population of the present invention has low toxicity (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity), and can be safely administered to mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans) as it is or by mixing it with a pharmacologically acceptable carrier or the like to form a pharmaceutical composition.

[0129] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

Examples

[0130] Induction of differentiation from pluripotent stem cells into an endocrine progenitor cell population was carried out according to the above steps 1)-5), as reported previously (Stem Cell Research (2015) 14, 185-197), etc. Induction of differentiation into insulin-producing cells was carried out according to the above step 6), etc.

[0131] Example 1: Decrease in unwanted cells (CHGA-negative cells) and increase in target cells (insulin-positive and NKX6.1-positive cells) in a cell population obtained by treating an insulin-producing cell population with a PLK inhibitor (1) 1. Method 1) An endocrine progenitor cell population obtained by inducing differentiation from iPS cells was cultured in a differentiation induction medium (Improved MEM / 1%B-27 / Penisilin Streptomycin medium) containing differentiation factors (ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, ascorbic acid) for 7 days to induce differentiation into an insulin-producing cell population. Subsequently, a PLK1 inhibitor (CAS929095-18-1, 3 μM) or a PLK4 inhibitor (CAS1338800-06-8, 3 μM) was added to the differentiation induction medium (Improved MEM / 1%B-27 / Penisilin Streptomycin medium) containing differentiation factors (ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, ascorbic acid) and cultured for 4 days. 2) An endocrine progenitor cell population obtained by inducing differentiation from iPS cells was cultured in a differentiation induction medium (Improved MEM / 1%B-27 / Penisilin Streptomycin medium) containing differentiation factors (ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, ascorbic acid) and not containing a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8) for 11 days to induce differentiation into an insulin-producing cell population.

[0132] The number of CHGA-negative (and either Ki67-positive or Ki67-negative) cells in the cell populations obtained by the above methods 1) and 2) was counted by flow cytometry, and the rate of unwanted cells, i.e., CHGA-negative (and either Ki67-positive or Ki67-negative), in each cell population was determined.

[0133] Also, the number of insulin-positive and NKX6.1-positive cells in the cell populations obtained by the above methods 1) and 2) was counted by flow cytometry, and the rate of target cells, i.e., insulin-positive and NKX6.1-positive cells, in each method was determined.

[0134] 2. Results The experiment was conducted twice using each method. Table 1 shows the results of the CHGA-negative (and either Ki67-positive or Ki67-negative) cell rate and the insulin-positive and NKX6.1-positive cell rate obtained when treated with a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8) in the process of manufacturing insulin-producing cells.

[0135] When the last 4 days of the differentiation induction process into insulin-producing cells were treated with a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8), it was confirmed that the CHGA-negative cell rate decreased significantly and reproducibly compared to the control for both Ki67-positive and Ki67-negative cells. This result indicates that by treating with a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8) during the production process of insulin-producing cells, unwanted cells (CHGA-negative cells) in the cell population can be reduced or the growth of these unwanted cells can be suppressed. On the other hand, by treating with a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8) during the production process of insulin-producing cells, it was confirmed that the rate of target cells (insulin-positive and NKX6.1-positive cells) in the cell population increased significantly and reproducibly compared to the control along with the decrease in unwanted cells.

[0136]

Table 1

[0137] From the above results, it became clear that by treating the cell population at the final differentiation stage of the insulin-producing cell manufacturing process with a PLK1 inhibitor or a PLK4 inhibitor, it is possible to reduce unwanted cells (CHGA-negative cells) present in the cell population or suppress the growth of these unwanted cells, and as a result, a cell population enriched with target cells (insulin-positive and NKX6.1-positive cells) can be obtained.

[0138] Example 2: Decrease in unwanted cells (CHGA-negative cells) and increase in target cells (insulin-positive and NKX6.1-positive cells) in a cell population obtained by treating an insulin-producing cell population with a PLK inhibitor (2) 1. Method An endocrine progenitor cell population obtained by inducing differentiation from iPS cells was cultured in the same manner as in Example 1 above, except that a 30 mL culture reactor was used and the treatment of the insulin-producing cell population with the PLK inhibitor was carried out for 7 days. That is, an endocrine progenitor cell population obtained by inducing differentiation from iPS cells was cultured in a 30 mL culture reactor with a differentiation induction medium for 4 days to induce differentiation into an insulin-producing cell population. Subsequently, it was cultured for 7 days in a differentiation induction medium supplemented with a PLK1 inhibitor (CAS929095-18-1, 3 μM) or a PLK4 inhibitor (CAS1338800-06-8, 3 μM). As a control, an endocrine progenitor cell population obtained by inducing differentiation from iPS cells was cultured in a 30 mL culture reactor with a differentiation induction medium for 11 days to induce differentiation into an insulin-producing cell population.

[0139] 2. Results Even in the system using a 30 mL culture reactor, by treating with a PLK1 inhibitor or a PLK4 inhibitor in the differentiation induction step into insulin-producing cells, it was confirmed that the CHGA-negative cell rate was significantly decreased with good reproducibility compared to the control for both Ki67-positive and Ki67-negative cells. Also, it was confirmed that the insulin-positive (and NKX6.1-positive) cell rate was significantly increased with good reproducibility compared to the control along with the decrease in unwanted cells. Further, compared with Example 1 above, although the treatment period using the PLK1 inhibitor or the PLK4 inhibitor was lengthened, no such effect was observed, and it was confirmed that the treatment with the PLK1 inhibitor or the PLK4 inhibitor may be performed at least on the cell population at the final differentiation stage of the insulin-producing cell manufacturing process.

[0140] Example 3: Single-cell RNA-seq expression analysis of an insulin-producing cell population As previously reported (Stem Cell Research (2015) 14, 185-197), as a result of performing single-cell RNA-seq expression analysis on an insulin-producing cell population obtained by inducing differentiation from pluripotent stem cells according to the above steps 1)-6), etc., it was found that the PLK gene was highly expressed in CHGA-negative cells, which are unwanted cells. From this, in order to reduce / suppress the growth of unwanted cells, when an inhibitor targeting PLK was used, a remarkable effect was observed as described above.

Claims

**Claim 1** A method for producing a population of insulin-producing cells, comprising the step of treating a population of insulin-producing cells induced to differentiate from pluripotent stem cells with a PLK inhibitor having a 50% inhibitory concentration (IC50) of 10 nM or less against PLK1 or PLK4, wherein the produced population of cells contains insulin-positive and NKX6.1-positive cells at a ratio of 16.8% or more. **Claim 2** The production method according to claim 1, wherein the produced population of cells contains CHGA-negative cells at a ratio of 20% or less. **Claim 3** The production method according to claim 1 or 2, wherein the produced population of cells contains CHGA-negative and Ki67-positive cells at a ratio of 3% or less. **Claim 4** The production method according to any one of claims 1 to 3, further comprising the step of differentiating the population of insulin-producing cells treated with the PLK inhibitor. **Claim 5** The production method according to any one of claims 1 to 4, wherein the population of insulin-producing cells is treated with a PLK inhibitor having a 50% inhibitory concentration (IC50) of 3 nM or less against PLK1 or PLK4 at 3 μM or less. **Claim 6** A method for suppressing the proliferation of CHGA-negative cells present in a population of insulin-producing cells induced to differentiate from pluripotent stem cells, comprising treating the population of cells with a PLK inhibitor having a 50% inhibitory concentration (IC50) of 10 nM or less against PLK1 or PLK4, wherein the insulin-positive and NKX6.1-positive cells present in the population of insulin-producing cells are increased to a ratio of 16.8% or more. **Claim 7** The method according to claim 6, wherein the population of insulin-producing cells is treated with a PLK inhibitor having a 50% inhibitory concentration (IC50) of 3 nM or less against PLK1 or PLK4 at 3 μM or less. **Claim 8** The method according to claim 6 or 7, wherein the CHGA-negative cells present in the population of insulin-producing cells are reduced to a ratio of 20% or less. **Claim 9** The method according to any one of claims 6 to 8, wherein the CHGA-negative and Ki67-positive cells present in the population of insulin-producing cells are reduced to a ratio of 3% or less.

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