Proliferation inhibitors

Abstract

The present invention aims to provide a method for removing non-endocrine target extracellular cells coexisting with differentiation-induced insulin-secreting cells. The present invention relates to a method for producing an insulin-producing cell population, the method comprising treating the insulin-producing cell population obtained by differentiation-induced pluripotent stem cells with a PLK inhibitor.

Description

[Technical field]

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

[0002] Currently, research is being conducted on inducing the differentiation of pluripotent stem cells such as iPS cells and ES cells into insulin-secreting cells called insulin-producing cells or pancreatic β cells and applying them to the treatment of diabetes.

[0003] So far, various methods for inducing differentiation of pluripotent stem cells into insulin-secreting cell populations have been developed and reported (Non-Patent Document 1). However, the insulin-secreting cell population obtained by differentiation induction contains non-endocrine cells in addition to the target insulin-secreting cells; in order to promote the use of diabetes treatment, a method for obtaining the target insulin-secreting cells more efficiently is desired.

[0004] [Prior art literature]

[0005]

Non-patent literature

[0006] [Non-patent document 1] Stem Cell Research (2015) 14, 185-197 [Summary of the invention]

[0007] [Technical problem to be solved by the invention]

[0008] The present inventors have found that in a cell population induced to differentiate from pluripotent stem cells into insulin-producing cells or pancreatic β cells, in addition to these insulin-secreting cells (insulin-producing cells or pancreatic β cells), there are non-endocrine cells characterized by chromogranin A (hereinafter, described as "CHGA") negative (hereinafter, sometimes described as "CHGA-negative cells"). It has also been found that CHGA-negative cells contain highly proliferative cells characterized by Ki67 marker positivity (CHGA-negative and Ki67-positive cells).

[0009] When insulin-secreting cells obtained by differentiation induction are intended to be used for diabetes treatment, etc., it is extremely important to strictly control cells other than insulin-secreting cells from the perspective of safety. In addition, the mixing / residual existence of cells with high proliferative potential may have a negative impact on the recipient or affect the long-term survival of the transplanted insulin-secreting cells, which is not preferred.

[0010] Here, an object of the present invention is to provide a method for removing CHGA-negative cells that coexist with insulin-secreting cells obtained by differentiation induction.

[0011] [Technical means used to solve the problem]

[0012] As a result of the inventors' intensive research to solve the above-mentioned problems, they found that by treating a pancreatic precursor cell group, preferably an endocrine precursor cell group, more preferably an insulin-producing cell group, or a cell group in a subsequent differentiation stage obtained by differentiation induction of pluripotent stem cells with a PLK inhibitor, the proliferation of CHGA-negative cells can be inhibited, and a pancreatic precursor cell group, an endocrine precursor cell group, an insulin-producing cell group, or a cell group in a subsequent differentiation stage in which the content of these cells has been reduced can be obtained.

[0013] The present invention is based on these new findings and includes the following inventions.

[0014] [1] A method for producing an insulin-producing cell population, the method comprising treating an insulin-producing cell population obtained by differentiation induction of pluripotent stem cells with a PLK inhibitor.

[0015] [1-1] A method for producing an insulin-producing cell population, the method comprising treating an endocrine precursor cell population obtained by differentiation induction of pluripotent stem cells with a PLK inhibitor.

[0016] [1-2] A method for producing an insulin-producing cell population, the method comprising treating a pancreatic progenitor cell population obtained by differentiation induction of pluripotent stem cells with a PLK inhibitor.

[0017] [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.

[0018] [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.

[0019] [4] The production method according to any one of [1] to [3], wherein the produced cell population contains 15% or more of insulin-positive and NKX6.1-positive cells.

[0020] [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.

[0021] [6] The production method according to any one of [1] to [5], wherein the production method comprises treating with a PLK inhibitor at a concentration of 3 μM or less.

[0022] [7] A method for inhibiting the proliferation of CHGA-negative cells present in a population of insulin-producing cells obtained by differentiation induction of pluripotent stem cells,

[0023] The method comprises treating the cell population with a PLK inhibitor.

[0024] [7-1] A method for inhibiting the proliferation of CHGA-negative cells present in a population of endocrine precursor cells induced by differentiation of pluripotent stem cells,

[0025] The method comprises treating the cell population with a PLK inhibitor.

[0026] [7-2] A method for inhibiting the proliferation of CHGA-negative cells present in a pancreatic precursor cell population obtained by differentiation induction of pluripotent stem cells,

[0027] The method comprises treating the cell population with a PLK inhibitor.

[0028] [8] The method according to [7], wherein the insulin-producing cell population is treated with a PLK inhibitor at 3 μM or less.

[0029] [9] The method according to [7] or [8], wherein the method reduces the CHGA-negative cells present in the insulin-producing cell population to a ratio of 20% or less.

[0030]

[10] The method according to any one of [7] to [9], wherein the method reduces the proportion of CHGA-negative and Ki67-positive cells present in a cell population obtained by differentiation induction of pluripotent stem cells to 3% or less.

[0031]

[11] The 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 obtained by differentiation induction of pluripotent stem cells to 15% or more.

[0032]

[12] A cell population comprising CHGA-negative cells at a ratio of 20% or less, and is an insulin-producing cell population obtained by differentiation induction of pluripotent stem cells treated with a PLK inhibitor, or a cell population in a subsequent differentiation stage.

[0033]

[13] The cell population according to

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

[0034]

[14] The cell population according to

[12] or

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

[0035]

[15] The cell population according to any one of

[12] to

[14] , which is used for transplantation.

[0036]

[16] A drug for treating diabetes, comprising the cell population described in any one of

[12] to

[15] .

[0037]

[17] A method for treating or preventing diabetes, comprising the step of transplanting a population of insulin-producing cells induced by differentiation of pluripotent stem cells treated with a PLK inhibitor, or a population of cells in a subsequent differentiation stage.

[0038]

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

[0039]

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

[0040]

[20] The 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]vinyl]-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.

[0041]

[21] The method according to any one of [1] to [6], wherein the PLK1 inhibitor has a 50% inhibitory concentration (IC 50 ) less than 5 nM of the substance.

[0042]

[22] The production method according to 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.

[0043]

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

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

[0044]

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

[11] , wherein the PLK4 inhibitor is a 50% inhibitory concentration (IC 50 ) less than 5 nM of the substance.

[0045]

[25] The 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]vinyl]-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.

[0046]

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

[11] , wherein the PLK1 inhibitor has a 50% inhibitory concentration (IC 50 ) less than 5 nM of the substance.

[0047]

[27] The 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.

[0048]

[28] The cell population according to any one of

[12] to

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

[0049]

[29] A drug for treating diabetes, comprising the cell population described in

[28] .

[0050]

[30] The method according to

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

[0051]

[31] The method according to [5], wherein the step of differentiating the insulin-producing cell population treated with a PLK inhibitor is performed by transplanting into an animal.

[0052]

[32] The method according to any one of [7] to

[11] , which reduces the absolute number of CHGA-negative cells present in the cell population.

[0053]

[33] The method according to any one of [7] to

[11] , which does not reduce the number of cells other than the aforementioned CHGA-negative cells present in the cell population.

[0054]

[34] The cell population according to any one of

[12] to

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

[0055]

[35] The cell population according to any one of

[12] to

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

[0056]

[36] A prodrug comprising the cell population described in any one of

[12] to

[14] ,

[34] and

[35] .

[0057]

[37] A method for producing an insulin-producing cell population or a pancreatic β cell population, the method comprising:

[0058] (1) treating an insulin-producing cell population or a pancreatic β-cell population with a PLK inhibitor; and (2) embedding the insulin-producing cell population in a gel containing a biocompatible material.

[0059]

[38] A method for producing an insulin-producing cell population or a pancreatic β cell population, the method comprising:

[0060] (0) a step of purifying the target cell population so that the purity of the target cell population becomes at least 70% or more;

[0061] (1) treating an insulin-producing cell population or a pancreatic β-cell population with a PLK inhibitor; and

[0062] (2) A step of differentiating the insulin-producing cell population treated with a PLK inhibitor.

[0063]

[39] The method according to

[30] , wherein the PLK4 inhibitor is a 50% inhibitory concentration (IC 50 ) less than 5 nM of the substance.

[0064]

[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]vinyl]-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.

[0065]

[41] The method according to

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

[0066]

[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.

[0067]

[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.

[0068]

[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.

[0069]

[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.

[0070]

[46] A cell population for use in a method for treating or preventing diabetes, the cell population being an insulin-producing cell population obtained by differentiation induction of pluripotent stem cells treated with a PLK inhibitor, or a cell population in a subsequent differentiation stage.

[0071]

[47] The cell population according to

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

[0072]

[48] ​​The cell population according to

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

[0073]

[49] The cell population according to

[47] , wherein the PLK4 inhibitor is (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]vinyl]-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.

[0074]

[50] The cell population according to

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

[0075]

[51] The cell population according to

[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.

[0076]

[52] The cell population according to any one of

[46] to

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

[0077]

[53] The cell population according to any one of

[46] to

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

[0078]

[54] The cell population according to any one of

[46] to

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

[0079]

[55] The cell population according to any one of

[46] to

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

[0080]

[56] The cell population according to any one of

[46] to

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

[0081]

[57] A use of a cell population for the manufacture of a drug for treating or preventing diabetes, wherein the cell population is an insulin-producing cell population obtained by differentiation induction of pluripotent stem cells treated with a PLK inhibitor, or a cell population in a subsequent differentiation stage.

[0082]

[58] The use according to

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

[0083]

[59] The use according to

[58] , wherein the PLK4 inhibitor is a 50% inhibitory concentration (IC 50 ) less than 5 nM of the substance.

[0084]

[60] The use according to

[58] , wherein the PLK4 inhibitor is (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]vinyl]-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.

[0085]

[61] The use according to

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

[0086]

[62] The use according to

[58] , 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.

[0087]

[63] The use according to any one of

[57] to

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

[0088]

[64] The 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.

[0089]

[65] The 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.

[0090]

[66] The 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.

[0091]

[67] The use according to any one of

[57] to

[62] , comprising 15% or more of insulin-positive and NKX6.1-positive cells.

[0092] This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2019-191876 which is the basis of priority of this application.

[0093] All publications, patents and patent applications cited in this specification are directly incorporated into this specification as reference.

[0094] [Effects of the invention]

[0095] According to the present invention, a method for removing CHGA-negative cells coexisting with differentiation-induced insulin-producing cells can be provided. [Specific implementation method]

[0096] 1. Terminology

[0097] The following is a description of the terms used in this specification.

[0098] In this specification, "about" means a value fluctuating within plus or minus 25%, 20%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference value. Preferably, the term "about" or "approximately" means a range of plus or minus 15%, 10%, 5% or 1% relative to a reference value.

[0099] In this specification, "comprise(s) or comprising~" means including / containing the elements following the sentence, but is not limited thereto. Therefore, although it implies including / containing the elements following the sentence, it does not imply excluding any other elements.

[0100] In this specification, "consist(s) of or consisting of" means including and limited to all the elements following the sentence. Therefore, the sentence "consist(s) of" means that the listed elements are essential or necessary, and there are substantially no other elements.

[0101] In this specification, "not using feeder cells" means that feeder cells are substantially not contained, and a medium pretreated by culturing feeder cells is not used, etc. Therefore, the medium does not contain substances such as growth factors and cytokines secreted by feeder cells.

[0102] In addition, "feeder cells" or "feeders" refer to cells that are co-cultured with other types of cells and provide an environment that can support the cells and make them grow. Feeder cells can be of the same source as the cells they support, or of different sources. For example, as feeders for human cells, human skin fibroblasts or human embryonic stem cells can be used, and primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts can also be used. Feeder cells can be inactivated by radiation or mitomycin C treatment.

[0103] In this specification, "adhesion" refers to the attachment of cells to a container, for example, cells adhere to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of an appropriate culture medium. There are also cells that cannot be maintained or grown in culture without adhering to the cell culture container. In contrast, non-adherent cells can still be maintained and proliferated in culture without adhering to the container.

[0104] In this specification, "culture" means maintaining cells in an in vitro environment and causing them to grow and / or differentiate. "Cultivating" means causing cells to continue, proliferate and / or differentiate in a tissue or in vitro, such as in a cell culture dish or flask. Cultivation includes two-dimensional culture (planar culture) and three-dimensional culture (suspension culture).

[0105] In this specification, "enrichment" and "enrichment" refer to increasing the amount of specific constituents in a composition such as a composition of a cell, and "enriched" refers to a cell group in which the amount of a specific constituent is increased compared to the ratio of the constituent in the cell group before enrichment when describing a composition such as a cell group for a cell. For example, a composition such as a cell group related to a target cell type can be enriched, so that the ratio of the target cell type is increased compared to the ratio of the target cells present in the cell group before enrichment. Cell groups related to target cell types can also be enriched by cell selection and screening methods known in the art. Cell groups can also be enriched by specific screening or selection processes recorded in this specification. In a specific embodiment of the present invention, a cell group related to a target cell group is enriched by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99%.

[0106] In this specification, "deplete" and "depletion" refer to reducing the amount of a specific constituent in a composition such as a composition of cells; "depleted" refers to a cell group in which the amount of a specific constituent is reduced compared to the proportion of the constituent in the cell group before depletion when used to describe a composition of cells such as a cell group. For example, a composition such as a cell group related to a target cell type can be depleted, so that the proportion of the target cell type is reduced compared to the proportion of target cells present in the cell group before depletion. The cell group related to the target cell type can also be depleted by cell selection and screening methods known in the art. The cell group can also be depleted by a specific screening or selection process described in this specification. In a specific embodiment of the present invention, the cell group related to the target cell group is reduced (depleted) by at least 50%, 80%, 85%, 90%, 95%, 97%, 98% or 99% by a method for depleting the target cell group.

[0107] In this specification, "purify" and "purification" refer to the removal of impurities in a composition such as a composition of a cell, so that a specific constituent becomes pure, and "purified" refers to a cell group in which the amount of impurities is reduced compared to the proportion of the constituent in the cell group before purification, and the purity of the specific constituent is improved. For example, a composition such as a cell group related to a target cell type can be purified, so that the proportion of the target cell type is increased compared to the proportion of the target cells present in the cell group before purification. The cell group related to the target cell type can also be purified by cell selection and screening methods known in the art. The cell group can also be purified by a specific screening or selection process recorded in this specification. In a specific embodiment of the present invention, the purity of the target cell group is at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% by a method for purifying the target cell group, or it can be reached to the extent that impurities (including mixed cells) cannot be detected.

[0108] In this specification, "not reducing the number of cells" means that the number of cells will not be significantly reduced due to the implementation of the method of the present invention, which means that there is no significant difference between the number of cells before and after the implementation of the method. However, a reduction in the number of cells may occur for reasons not caused by the implementation of the method of the present invention (for example, the natural death of cells that usually occurs in the steps of cell culture and differentiation known in the past). Therefore, "not reducing the number of cells" also includes the situation where the reduction rate of cells after implementation is 30% or less, 20% or less, 10% or less, or 5% or less compared to before the implementation of the method of the present invention.

[0109] In this specification, "inhibiting proliferation" means that the number of cells will not increase significantly due to the implementation of the method of the present invention, which means that there is no significant increase between the number of cells before and after the implementation of the method. Therefore, "inhibiting proliferation" also includes the case where the increase rate of cells 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 of the present invention.

[0110] In this specification, "marker" means a cell antigen or its gene that is specifically expressed by a specified cell type, such as a "marker protein" or a "marker gene". Preferably, the marker is a cell surface marker, in which case, concentration, separation and / or detection of surviving cells can be performed. The marker can be a positive selection marker or a negative selection marker.

[0111] The detection of the marker protein can be performed by immunological assay using specific antibodies to the marker protein, for example, by ELISA, immunostaining, or flow cytometry. The detection of the marker gene can be performed by nucleic acid amplification methods and / or nucleic acid detection methods known in the art, for example, by RT-PCR, microarrays, biochips, etc. In this specification, the marker protein is "positive" means that it is detected as positive by flow cytometry, and "negative" means that it is below the detection limit by flow cytometry. In addition, in this specification, the marker gene is "positive" means that it is detected by RT-PCR, and "negative" means that it is below the detection limit by RT-PCR.

[0112] In the present specification, "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter in a cell.

[0113] In this specification, "factors having CDK8 / 19 inhibitory activity" means all substances having CDK8 / 19 inhibitory activity. Compared with other proteins in the same CDK family, CDK8 is not essential for cell proliferation, and inhibition of CDK8 has no significant effect under normal conditions. CDK19 is similar to CDK8, and CDK8 inhibition is usually accompanied by CDK19 inhibition.

[0114] "Proliferation factors" are endogenous proteins that promote differentiation and / or proliferation of specific cells. Examples of "proliferation factors" include 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 effective for stem cells, such as stem cell factor (SCF) and erythropoietin (Epo).

[0115] In the present specification, "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. There is no particular limitation on the ROCK inhibitor as long as it has the above-mentioned function, and examples thereof include N-(4-pyridyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (sometimes referred to as Y-27632 in the present specification), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolyl)sulfonyl]hexahydro-1H-1,4-diazepine (H-1152), 4β-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzene-1α-carboxamide (Wf-536), N-(1H-pyrrolo[2,3-b] [4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A). The ROCK inhibitor is not limited to the above, and antisense oligonucleotides or siRNA against ROCK mRNA, antibodies that bind to ROCK, dominant negative ROCK mutants, etc. can also be used as ROCK inhibitors, which are commercially available or can be synthesized according to known methods.

[0116] In this specification, "GSK3β inhibitor" refers to a substance that has inhibitory activity on GSK3β (glycogen synthase kinase 3β). GSK3 (glycogen synthase kinase 3) is a type of serine / threonine protein kinase that is involved in many signal transduction pathways related to glycogen production or apoptosis, maintenance of stem cells, etc. GSK3 has two isoforms, α and β. The "GSK3β inhibitor" used in the present invention is not particularly limited as long as it has GSK3β inhibitory activity, and can also be a substance that is combined with GSK3β inhibitory activity and has GSK3α inhibitory activity.

[0117] 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 ) Nicotine nitrile, 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, Kenpaulone lone), 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, CT9 9021, CT20026, BIO, BIO-acetone oxime, pyridocarbazole-cyclopentadienylruthenium complex, OTDZT, α-4-dibromoacetophenone, lithium, etc. GSK3β is not limited to the above, and antisense oligonucleotides or siRNAs against GSK3β mRNA, antibodies bound to GSK3β, dominant negative GSK3β mutants, etc. can also be used as GSK3β inhibitors, which can be commercially available or synthesized according to known methods.

[0118] Examples of "serum substitutes" in this specification include: Knockout TM Serum Replacement(KSR:Thermo Fisher Scientific), 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'-mercaptoglycerol or a mixture thereof (e.g., ITS-G). Preferably, B-27 supplement, KSR, Serum Replacement, ITS-G as a serum substitute. When the serum substitute is added to the culture medium, the concentration in the culture medium is 0.01%-10% by weight, preferably 0.1%-2% by weight. In the present invention, it is preferred to use "serum substitute" to replace serum.

[0119] 2. Insulin-producing cell populations with inhibited proliferation of CHGA-negative cells

[0120] The present invention relates to insulin-producing cell populations in which the proliferation of CHGA-negative cells is suppressed, and these cell populations can be obtained by treating them with a PLK inhibitor.

[0121] The present invention also relates to methods of inhibiting the proliferation of CHGA-negative cells by treating them with a PLK inhibitor.

[0122] "CHGA-negative cells" refer to non-endocrine cells that are present in a pancreatic precursor cell population, preferably an endocrine precursor cell population, more preferably an insulin-producing cell population, or a cell population at a subsequent differentiation stage induced by differentiation of pluripotent stem cells into pancreatic β cells, and are characterized by negative expression of the CHGA marker.

[0123] "CHGA-negative cells" may include cells characterized by confirmed expression of Ki67 as a marker (ie, CHGA-negative and Ki67-positive cells).

[0124] Ki67 is known to be a cell cycle-related nuclear protein, and is widely known as a marker of cell proliferation and cell cycle because it is expressed in the G1 phase, S phase, G2 phase, and M phase of proliferating cells, but not in the G0 phase when proliferation stops. Therefore, CHGA-negative and Ki67-positive cells are cells that show high proliferation.

[0125] The "insulin-producing cell population" of the present invention refers to a cell population comprising insulin-producing cells induced by differentiation of pluripotent stem cells. "Insulin-producing cells" refer to cells characterized by the expression of insulin markers (i.e., insulin-positive cells). "Insulin-producing cells" may be cells expressing a marker of NK6 homeobox 1 (NKX6.1), preferably cells expressing markers of both insulin and NKX6.1 (i.e., insulin-positive and NKX6.1-positive cells).

[0126] The "insulin-producing cell group" of the present invention has a low content of CHGA-negative cells compared to the insulin-producing cell group obtained by differentiation and induction of pluripotent stem cells according to the previously known method, and its content (sometimes described as "ratio" in this specification) is 40% and below or 30% and below, preferably 20% and below, more preferably 15% and below, and further preferably 10% and below, for example, 9% and below, 8% and below, 7% and below, 6% and below, 5% and below, 4% and below, 3% and below, 2% and below, or 1% and below. The lower limit of the content is not particularly limited, and is 0% and above, 0.1% and above, or 0.5% and above. The content can be expressed using two numerical values ​​selected from the aforementioned upper limit and lower limit, for example, the content is 0.5%-40% or 0.5%-30%, preferably 0.5%-20%, more preferably 0.5%-15%, and further preferably 0.5%-10%. On the other hand, the "insulin-producing cell population" of the present invention is a cell population enriched with insulin-positive cells, particularly insulin-positive and NKX6.1-positive cells, compared to the insulin-producing cell population obtained by differentiation induction of pluripotent stem cells according to a conventionally known method, and the content of insulin-positive and NKX6.1-positive cells is 14% or more, preferably 15% or more, more preferably 20% or more, and further 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 is not particularly limited, and is 70% or less, 60% or less, or 50% or less. The content can be expressed using two numerical values ​​selected from the aforementioned upper limit and lower limit, for example, the content is 14%-50%, preferably 15%-50%, more preferably 20%-50%, and further preferably 25%-50%.

[0127] The "insulin-producing cell population" of the present invention can be obtained by treating a pancreatic precursor cell population, preferably an endocrine precursor cell population, more preferably an insulin-producing cell population, or a cell population at a subsequent differentiation stage obtained by differentiation induction of 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 inhibited, the cell content thereof can be reduced, and insulin-positive cells, preferably insulin-positive and NKX6.1-positive cells, can be enriched.

[0128] It is known that in the process of differentiation from pluripotent stem cells to pancreatic β cells, cells with different characteristics appear according to the differentiation stage (WO 2009 / 012428, WO 2016 / 021734). For example, this differentiation stage can be roughly divided into pluripotent stem cells, definitive endoderm cells, gastrointestinal cells, posterior foregut cells, pancreatic precursor cells, endocrine precursor cells, insulin-producing cells, and pancreatic β cells in a relatively undifferentiated order.

[0129] "Pluripotency" in this specification means the ability to differentiate into various tissues or cells with different morphologies or functions, and the ability to differentiate into cells of any of the three germ layers. "Pluripotency" cannot differentiate into a blastoderm, and therefore, from the perspective of not having the ability to form an individual, it is distinguished from "totipotency" which includes a blastoderm and can differentiate into all tissues of an organism.

[0130] "Multipotency" in this specification means the ability to differentiate into multiple cells of a limited number of layers. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent, not pluripotent.

[0131] "Pluripotent stem cells" in this specification refer to embryonic stem cells (ES cells) and cells that potentially have the same differentiation pluripotency, that is, the ability to differentiate into various tissues of an organism (entoderm, mesoderm, ectoderm). "Induced pluripotent stem cells" (also referred to as "iPS cells" in this specification) can be cited as cells that have the same differentiation pluripotency as ES cells. Preferably, in the present invention, pluripotent stem cells are human pluripotent stem cells.

[0132] If mouse ES cells are used as "ES cells", various mouse ES cell lines established by inGenious, RIKEN (RIKEN), etc. can be used; if human ES cells are used as "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, CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains, etc. of NIH; H1 strains and H9 strains of WiCell Research Institute; KhES-1 strains, KhES-2 strains, KhES-3 strains, KhES-4 strains, KhES-5 strains, SSES1 strains, SSES2 strains, SSES3 strains, etc. of RIKEN can be used as ES cell lines.

[0133] “Induced pluripotent stem cells” refer to cells obtained by introducing specific factors (nuclear reprogramming factors) into mammalian somatic cells or undifferentiated stem cells and reprogramming them. Currently, there are many types of "induced pluripotent stem cells". In addition to the iPS cells established by Yamanaka et al. by introducing Oct3 / 4, Sox2, Klf4 and c-Myc factors 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 screening with Nanog expression as an indicator after the introduction of the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells made by a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al., Nature Biotechnology, (2008) 26, 101-106), iPS cells established by introducing six factors by a virus-free method (Okita K et al., Nat. Methods 2011 May; 8 (5): 409-12, Okita K et al., Stem Cells. 31 (3): 458-66.). In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG and LIN28, prepared by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells prepared by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells prepared by Sakurada et al. (Japanese Patent Laid-Open No. 2008-307007), etc. can also be used.

[0134] In addition, all published papers (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol 3, Issue 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No 7, 795-797) or patents (e.g., Japanese Patent Application Laid-Open No. 2008-307007, Japanese Patent Application Laid-Open No. 2008-283972, US 2008 / 2336610, US 2009 / 047263, WO 2007 / 069666, WO 2008 / 118220, WO 2008 / 124133, WO Any of the induced pluripotent stem cells known in the art described in WO 2008 / 151058, WO 2009 / 006930, WO 2009 / 006997, WO 2009 / 007852).

[0135] As the induced pluripotent cell line, various iPS cell lines established by NIH, RIKEN (RIKEN), Kyoto University, etc. can be used. For example, human iPS cell lines include: RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, Nips-B2, Kyoto University's Ff-WJ-18, Ff-I01s01, Ff-I01s02, Ff-I01s04, Ff-I01s06, Ff-I14s03, Ff-I14s04, QHJI01s01, QHJI01s04, QHJI14s03, QHJI14s04, 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, and CDI's MyCell iPS cells(21525.102.10A) strain, MyCell iPS cells(21526.101.10A) strain, etc.

[0136] The term "pancreatic precursor cell population" in the present specification refers to a cell population comprising pancreatic precursor cells. In the present specification, pancreatic precursor cells refer to cells characterized by expression of at least one marker selected from the group consisting of PDX-1, NKX6.1, PTF-1α, GATA4, and SOX9.

[0137] The pancreatic precursor cell population is a cell population containing pancreatic precursor cells at a ratio of 30% or more, preferably 40% or more, more preferably 50% or more, further preferably 60% or more, and even more preferably 70% or more. In addition to pancreatic precursor cells, the pancreatic precursor cell population may also contain other cells (e.g., endocrine precursor cells, insulin-producing cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0138] The "endocrine precursor cell population" in this specification means a cell population including endocrine precursor cells.

[0139] In this specification, endocrine precursor cells refer to cells characterized by the expression of at least one marker of CHGA, NeuroD and NGN3, and the lack of expression of markers of pancreatic-associated hormone systems (e.g., insulin, etc.). Endocrine precursor cells may also express markers such as PAX-4, NKX2.2, Islet-1, PDX-1, and PTF-1α.

[0140] The endocrine precursor cell population is a cell population that contains endocrine precursor cells at a ratio of 30% or more, preferably 40% or more, more preferably 50% or more, further preferably 60% or more, and further preferably 70% or more. In addition to endocrine precursor cells, the endocrine precursor cell population may also contain other cells (e.g., pancreatic precursor cells, insulin-producing cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0141] The proportion of specific cells in a cell population can be determined based on a known method capable of counting the number of cells, such as flow cytometry.

[0142] As described above, "insulin-producing cells" and "insulin-producing cell populations" generally contain insulin-producing cells at a ratio of 5% or more, preferably 10% or more, more preferably 15% or more, further preferably 20% or more, further preferably 25% or more, and particularly preferably 30% or more. In addition to insulin-producing cells, the cell population may also contain other cells (e.g., endocrine precursor cells; other pancreatic hormone-producing cells expressing at least one marker of glucagon, somatostatin, and pancreatic polypeptide; Ki67-positive cells, CHGA-negative cells, etc.).

[0143] The "pancreatic β cells" in this specification refer to cells that are more mature than "insulin-producing cells", and specifically, refer to cells that express at least one of MAFA, UCN3 and IAPP, which are markers of pancreatic β cell maturation, or are characterized by an increased insulin secretion response induced by glucose stimulation.

[0144] "Pancreatic β cell population" refers to a cell population comprising pancreatic β cells that can be obtained by differentiating / maturating an insulin-producing cell population, preferably by differentiating / maturating it in vivo. In addition to pancreatic β cells, the cell population may also include other cells (e.g., insulin-producing cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0145] Cell populations at various differentiation stages can be obtained using a known method for inducing differentiation of pluripotent stem cells into pancreatic β cells. That is, each target cell population can be obtained using the following differentiation induction steps:

[0146] Step 1) inducing differentiation of pluripotent stem cells into definitive endoderm cells;

[0147] Step 2) inducing the definitive endoderm cells to differentiate into gastrointestinal cells;

[0148] Step 3) inducing differentiation of gastrointestinal cells into posterior foregut cells;

[0149] Step 4) inducing differentiation of posterior foregut cells into pancreatic progenitor cells;

[0150] Step 5) inducing differentiation of pancreatic precursor cells into endocrine precursor cells; and

[0151] Step 6) Inducing differentiation of endocrine precursor cells into insulin-producing cells.

[0152] Each step will be described below, but the differentiation induction of each cell is not limited to these methods.

[0153] Step 1) Differentiation into definitive endoderm cells

[0154] First, pluripotent stem cells are differentiated into definitive endoderm cells. Methods for inducing definitive endoderm from pluripotent stem cells are well known, and any of these methods may be used. Preferably, pluripotent stem cells are cultured in a medium containing activin A, more preferably in a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, so as 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 22000-100000 cells / cm 2 , more preferably 22000-80000 cells / cm 2 The culture time is 1 to 4 days, preferably 1 to 3 days, and particularly preferably 3 days.

[0155] The culture temperature is not particularly limited, and is performed at 30° C. to 40° C. (eg, 37° C.). The carbon dioxide concentration in the culture container is, for example, about 5%. The culture can be performed in two-dimensional culture or three-dimensional culture.

[0156] As the culture medium used in this step, RPMI 1640 medium, MEM medium, iMEM medium, DMEM / F12 medium, modified MEM Zinc Option medium, modified MEM / 1% B-27 / PenisilinStreptomycin medium, MCDB131 / 20 mM Glucose / NaHCO medium can be used. 3 / FAF-BSA / ITS-X / GlutaMAX TM / Ascorbic acid / Penisilin Streptomycin medium etc. are used as the basic medium for culturing mammalian cells.

[0157] The concentration of activin A in the culture medium is usually 30 ng / mL to 200 ng / mL, preferably 50 ng / mL to 150 ng / mL, more preferably 70 ng / mL to 120 ng / mL, particularly preferably about 100 ng / mL.

[0158] In another embodiment, activin A may be included in the culture medium at a low amount, for example, 5 ng / mL-100 ng / mL, preferably 5 ng / mL-50 ng / mL, more preferably 5 ng / mL-10 ng / mL.

[0159] In yet another embodiment, the concentration of activin A in the culture medium is about 0.1 ng / mL-100 ng / mL, preferably about 1 ng / mL-50 ng / mL, more preferably about 3 ng / mL-10 ng / mL.

[0160] The concentration of the GSK3β inhibitor in the culture medium is appropriately set depending on the type of the GSK3β inhibitor used. For example, when CHIR99021 is used as the GSK3β inhibitor, the concentration is usually 2 μM to 5 μM, preferably 2 μM to 4 μM, and particularly preferably about 3 μM.

[0161] The concentration of the ROCK inhibitor in the medium is appropriately set depending on the type of ROCK inhibitor used. For example, when Y27632 is used as the ROCK inhibitor, the concentration is usually 5 μM to 20 μM, preferably 5 μM to 15 μM, and particularly preferably about 10 μM.

[0162] Insulin may be further added to the culture medium. Insulin may be contained in the culture medium in an amount of 0.01 μM-20 μM, preferably 0.1 μM-10 μM, more preferably 0.5 μM-5 μM. The insulin concentration in the culture medium may be the insulin concentration contained in the added B-27 supplement, but is not limited thereto.

[0163] In a specific embodiment, after culturing for 1 day in a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, the culture is continued for 2 days while the medium is exchanged every day with a medium containing only activin A. Alternatively, the pluripotent stem cells can be produced by first culturing in a medium containing 0.01 μM to 20 μM insulin in the presence of a low amount of activin A, and then second culturing in a medium without insulin.

[0164] Step 2) Differentiation into gastrointestinal cells

[0165] The definitive endoderm cells obtained in step 1) are further cultured in a culture medium containing proliferation factors to induce differentiation into gastrointestinal cells. The culture time is 2 to 8 days, preferably about 4 days.

[0166] The culture temperature is not particularly limited, and is performed at 30° C. to 40° C. (eg, 37° C.). The carbon dioxide concentration in the culture container is, for example, about 5%. The culture can be performed in two-dimensional culture or three-dimensional culture.

[0167] The medium may be a basal medium for culturing mammalian cells in the same manner as in step 1). In addition to growth factors, serum replacements, vitamins, antibiotics, etc. may be appropriately added to the medium.

[0168] As the growth factor, EGF, KGF, and FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is further preferred.

[0169] The concentration of the growth factor in the culture medium is appropriately set according to the type of growth factor used, but is generally about 0.1nM-1000μM, preferably about 0.1nM-100μM. When EGF is used, its concentration is about 5ng / mL-2000ng / mL (i.e., about 0.8nM-320nM), preferably about 5ng / mL-1000ng / mL (i.e., about 0.8nM-160nM), and more preferably about 10ng / mL-1000ng / mL (i.e., about 1.6nM-160nM). When FGF10 is used, its concentration is about 5ng / mL-2000ng / mL (i.e., about 0.3nM-116nM), preferably about 10ng / mL-1000ng / mL (i.e., about 0.6nM-58nM). For example, when KGF is used as a growth factor, the concentration is generally 5 ng / mL to 150 ng / mL, preferably 30 ng / mL to 100 ng / mL, and particularly preferably about 50 ng / mL.

[0170] Step 3) Differentiation into posterior foregut cells

[0171] The gastrointestinal cells obtained in step 2) are further cultured in a culture medium containing growth factors, cyclopamine, noggin, etc., and differentiated into posterior foregut cells. The culture time is 1 to 5 days, preferably about 2 days. The culture can be carried out by two-dimensional culture or three-dimensional culture.

[0172] The culture temperature is not particularly limited, but is 30° C. to 40° C. (eg, 37° C.) The carbon dioxide concentration in the culture container is, for example, about 5%.

[0173] The medium may be a basal medium for culturing mammalian cells in the same manner as in step 1). In addition to growth factors, serum replacements, vitamins, antibiotics, etc. may be appropriately added to the medium.

[0174] As the growth factor, EGF, KGF, and FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is further preferred.

[0175] The concentration of the growth factor in the culture medium is appropriately set according to the type of growth factor used, but is generally about 0.1nM-1000μM, preferably about 0.1nM-100μM. When EGF is used, its concentration is about 5ng / mL-2000ng / mL (i.e., about 0.8nM-320nM), preferably about 5ng / mL-1000ng / mL (i.e., about 0.8nM-160nM), and more preferably about 10ng / mL-1000ng / mL (i.e., about 1.6nM-160nM). When FGF10 is used, its concentration is about 5ng / mL-2000ng / mL (i.e., about 0.3nM-116nM), preferably about 10ng / mL-1000ng / mL (i.e., about 0.6nM-58nM). For example, when KGF is used as a growth factor, the concentration is generally 5 ng / mL to 150 ng / mL, preferably 30 ng / mL to 100 ng / mL, and particularly preferably about 50 ng / mL.

[0176] The concentration of cyclopamine in the medium is not particularly limited, but is usually 0.5 μM to 1.5 μM, preferably 0.3 μM to 1.0 μM, and particularly preferably about 0.5 μM.

[0177] The concentration of Noggin in the culture medium is not particularly limited, but is usually 10 ng / mL to 200 ng / mL, preferably 50 ng / mL to 150 ng / mL, and particularly preferably about 100 ng / mL.

[0178] Step 4) Differentiation into pancreatic progenitor cells

[0179] The posterior foregut cells obtained in step 3) are further cultured in a culture medium containing a factor having CDK8 / 19 inhibitory activity, preferably in a culture medium containing a factor having CDK8 / 19 inhibitory activity and a proliferation factor, and differentiated into pancreatic progenitor cells. The culture time is 2 to 10 days, preferably about 5 days. The culture can be carried out by two-dimensional culture or three-dimensional culture.

[0180] In the case of two-dimensional culture, according to previous reports (Toyoda et al., Stem cell Research (2015) 14, 185-197), the posterior foregut cells obtained in step 3) were treated with 0.25% trypsin-EDTA and dispersed by pipetting, the obtained dispersion was centrifuged, the recovered cells were resuspended in a small amount of fresh culture medium, and this cell suspension was re-inoculated into the new culture medium of step 4).

[0181] The medium may be a basal medium for culturing mammalian cells in the same manner as in step 1). In addition to growth factors, serum replacements, vitamins, antibiotics, etc. may be appropriately added to the medium.

[0182] The aforementioned various compounds or salts thereof can be used as factors having CDK8 / 19 inhibitory activity, and the amount added to the culture medium is appropriately determined according to the compound or salt used, usually about 0.00001 μM-5 μM, preferably 0.00001 μM-1 μM. The concentration of the factor having CDK8 / 19 inhibitory activity in the culture medium preferably reaches a concentration of 50% or more of the inhibitory activity on CDK8 / 19.

[0183] As the growth factor, EGF, KGF, and FGF10 are preferred, KGF and / or EGF are more preferred, and KGF and EGF are further preferred.

[0184] The concentration of the growth factor in the culture medium is appropriately set according to the type of growth factor used, but is generally about 0.1nM-1000μM, preferably about 0.1nM-100μM. When EGF is used, its concentration is about 5ng / mL-2000ng / mL (i.e., about 0.8nM-320nM), preferably about 5ng / mL-1000ng / mL (i.e., about 0.8nM-160nM), and more preferably about 10ng / mL-1000ng / mL (i.e., about 1.6nM-160nM). When FGF10 is used, its concentration is about 5ng / mL-2000ng / mL (i.e., about 0.3nM-116nM), preferably about 10ng / mL-1000ng / mL (i.e., about 0.6nM-58nM). For example, when KGF and EGF are used as growth factors, the concentration of EGF is usually 5 ng / mL-150 ng / mL, preferably 30 ng / mL-100 ng / mL, and particularly preferably about 50 ng / mL; the concentration of KGF is usually 10 ng / mL-200 ng / mL, preferably 50 ng / mL-150 ng / mL, and particularly preferably about 100 ng / mL.

[0185] The first day of culturing in step 4) may be performed in the presence of a ROCK inhibitor, and thereafter culturing may be performed in a medium not containing a ROCK inhibitor.

[0186] In addition, the culture medium may also contain a protein kinase C (PKC) activator. PDBu (PKC activator II), TPB (PKC activator V), etc. can be used as the PKC activator, but are not limited thereto. The concentration of the PKC activator is about 0.1 ng / mL-100 ng / mL, preferably about 1 ng / mL-50 ng / mL, and more preferably about 3 ng / mL-10 ng / mL.

[0187] In addition, dimethyl sulfoxide and / or activin (1 ng / mL-50 ng / mL) may be added to the culture medium.

[0188] In any step, in addition to the above-mentioned components, serum replacements (e.g., B-27 supplement, ITS-G) may be added to the culture medium. In addition, as needed, amino acids, L-glutamine, GlutaMAX (product name), non-essential amino acids, vitamins, nicotine amide, 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 also be added. When antibiotics are added to the culture medium, their concentration in the culture medium is generally 0.01%-20% by weight, preferably 0.1%-10% by weight. Cultivation may be performed by two-dimensional culture or three-dimensional culture.

[0189] In addition, in the case where cell culture is two-dimensional culture, feeder cells are not used, and culture is carried out by adhesion. During cultivation, culture vessels such as cell culture sheets of dish, flask, microplate, OptiCell (product name) (Nunc company) etc. are used. Preferably, culture vessel is surface treated, to improve its adhesion (hydrophilicity) to cells, collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, matrigel (for example, BD matrigel (Beckton Dickinson, Japan), vitronectin etc. are coated thereon with the matrix for cell adhesion. Preferably, the culture vessel coated with type I collagen, matrigel, fibronectin, vitronectin or poly-D-lysine etc. is used as culture vessel, more preferably, the culture vessel coated with matrigel or poly-D-lysine is used as culture vessel.

[0190] The culture temperature is not particularly limited, but is 30° C. to 40° C. (eg, 37° C.) The carbon dioxide concentration in the culture container is, for example, about 5%.

[0191] The pancreatic progenitor cells obtained in step 4) may be further purified using glycoprotein 2 (GP2), a well-known surface marker, etc. The purification may be performed by a method known per se, for example, using beads to which an anti-GP2 antibody is immobilized.

[0192] Step 5) Differentiation into endocrine precursor cells

[0193] The pancreatic precursor cells obtained in step 4) are further cultured in a culture medium containing proliferation factors to induce differentiation into endocrine precursor cells. The culture can be carried out by two-dimensional culture or three-dimensional culture. In the case of two-dimensional culture, the pancreatic precursor cells obtained in step 4) are treated with 0.25% trypsin-EDTA, dispersed by pipetting, centrifuged, treated with 0.25% trypsin-EDTA and dispersed by pipetting, the obtained dispersion is centrifuged, the recovered cells are resuspended in a small amount of fresh culture medium, and the cell suspension is re-inoculated into the new culture medium of step 5). The culture time is 2 to 3 days, preferably about 2 days.

[0194] The culture medium can be a basal medium for culturing mammalian cells in the same manner as in step 1). According to previous reports (Nature Biotechnology 2014; 32: 1121-1133), SANT1, retinoic acid, ALK5 inhibitor II, T3, and LDN are added to the culture medium. Furthermore, Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum replacements, vitamins, antibiotics, etc. can be added to the culture medium in appropriate amounts.

[0195] The culture is performed by non-adhesive culture without using feeder cells. During the culture, a dish, flask, microplate, multi-well plate (Nunc) or a bioreactor is used. The culture container is preferably surface treated to reduce the adhesion of the cells.

[0196] The culture temperature is not particularly limited, but is 30° C. to 40° C. (eg, 37° C.) The carbon dioxide concentration in the culture container is, for example, about 5%.

[0197] Step 6) Differentiation into insulin-producing cells

[0198] The endocrine precursor cells obtained in step 5) are further cultured in a culture medium containing growth factors to induce differentiation into insulin-producing cells. The culture time is 10 to 30 days, preferably about 10 to 20 days.

[0199] The culture medium can be a basal medium for culturing mammalian cells in the same manner as in step 1). According to a previous report (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 are added to the culture medium. Furthermore, Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum replacements, vitamins, antibiotics, etc. can be added to the culture medium in appropriate amounts. For example, ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, and ascorbic acid can be added to the culture medium, or T3, ALK5 inhibitor II, ZnSO 4 , heparin, N-acetylcysteine, Trolox and R428 were added to the culture medium.

[0200] Cultivation can be performed by two-dimensional culture or three-dimensional culture. Cultivation does not use feeder cells. In the case of three-dimensional culture, non-adhesive culture is used. During cultivation, a dish, flask, microplate, multi-well plate (Nunc company) or a bioreactor is used. Preferably, the culture container is surface treated to reduce its adhesion to the cells.

[0201] The culture temperature is not particularly limited, but is 30° C. to 40° C. (eg, 37° C.) The carbon dioxide concentration in the culture container is, for example, about 5%.

[0202] Differentiation into pancreatic β cells

[0203] The cells obtained in the above step can be induced to differentiate into pancreatic β cells. The step of differentiating into pancreatic β cells can be performed by transplanting an endocrine precursor cell group or a cell group at a later differentiation stage, preferably an insulin-producing cell group, into an animal organism.

[0204] The "animal" is preferably a mammal, and examples thereof include humans, non-human primates, pigs, cows, horses, sheep, goats, llamas, dogs, cats, rabbits, mice, guinea pigs, etc., and preferably humans.

[0205] Transplantation is preferably performed in a region of the body where the cell population can be fixed at a certain position, for example, it can be performed subcutaneously, intraperitoneally, in the peritoneal epithelium, omentum, adipose tissue, muscle tissue, pancreas, kidney, etc. The number of cells to be transplanted can vary depending on important factors such as the differentiation stage of the transplanted cells, the age and weight of the transplanted subject, the size of the transplanted site, the severity of the disease, etc., and is not particularly limited. For example, it can be 10×10 4 cells-10×10 11The transplanted cell population is induced to differentiate in the in vivo environment and can be differentiated into a target cell population, preferably a pancreatic β cell population, and can then be recovered or directly left in the body.

[0206] During transplantation, the cell population may be embedded in a gel containing a biocompatible material for transplantation. For example, the cell population embedded in a gel containing a biocompatible material may be enclosed in a device such as a capsule, a bag or a chamber and then transplanted into the body.

[0207] The term "embedding" in the present invention means that an endocrine precursor cell population or a cell population at a subsequent differentiation stage is dispersed and contained in a gel containing a biocompatible material.

[0208] The "biocompatible material" in this specification means any material that does not induce significant immune response or harmful biological response (for example, toxic response, coagulation, etc.) when transplanted into a living body and left for a short or long term. In addition, the "biocompatible material" is preferably a biodegradable material. Such materials can be listed as: polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), polyethylene glycol (PEG), polyhydroxyethyl methacrylate, polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), poly(ethylene-vinyl acetate copolymer) (PEVA), polyacrylamide, polyethylene oxide, polyvinylamine, polyhydroxybutyric acid, poly(N-vinyl pyrrolidone), polyvinyl alcohol, poly(propylene fumarate), polyvinyl alcohol, polyvinyl acrylate, ... Ester, polyacrylic acid, poly e-caprolactone, polymethacrylic acid, polyvinylidene fluoride (PVDF), pectic acid, hyaluronic acid, heparan sulfate, chondroitin sulfate, heparan sulfate proteoglycan, heparin, chitin, chitosan, xanthan gum, carboxymethyl cellulose, carboxymethyl chitosan, alginate, alginate, collagen, cellulose, fibroin, keratin, gelatin, fibrin, pullulan, laminin, gellan gum, polysilicone, urethane, elastin, etc. and their deformations and their combinations. If necessary, the surface of the "biocompatible material" may also be modified by surface modification (e.g., by coating with a matrix for cell adhesion (collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, matrigel, vitronectin, etc.)) or by using functional groups known to control cell proliferation, differentiation and function (e.g., amino, carboxyl, hydroxyl, methacrylic, acrylic, etc.) to enable cell adhesion. In a specific embodiment, alginate or alginate ester may be appropriately used as the "biocompatible material".

[0209] The alginate may be any water-soluble salt, and may be a metal salt, an ammonium salt, etc. For example, sodium alginate, calcium alginate, ammonium alginate, etc. may be used appropriately.

[0210] Alginate (also known as propylene glycol alginate) is a derivative in which propylene glycol is esterified with the carboxyl group of alginic acid.

[0211] The ratio of mannuronic acid to guluronic acid (M / G ratio) contained in alginate is arbitrary. Generally speaking, when M>G, a highly flexible gel can be formed, and when M<G, a firm gel can be formed. In the present invention, alginate containing guluronic acid in a ratio of 10%-90%, 20%-80%, 30%-70% or 40%-60% can be used.

[0212] The gel using alginate or alginate ester can be prepared according to a known method (WO 2010 / 032242, WO 2011 / 154941), and can be obtained by adding a cross-linking agent to an alginate or alginate ester solution to cause gelation.

[0213] The solvent may contain 0.05% to 10% by weight, preferably 0.1% to 5% by weight, and more preferably 0.5% to 3% by weight of alginate or alginate ester. The solvent may be any solvent that can dissolve alginate or alginate ester, and water, physiological saline, etc. may be used.

[0214] The cross-linking agent is not particularly limited, as long as it can gel the solution of alginate or alginate ester, and a polyvalent metal cation can be used. The polyvalent metal cation is preferably a divalent metal cation, more preferably a calcium ion, a strontium ion, or a barium ion. The cross-linking 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 cross-linking agent.

[0215] The gel containing alginate or alginate may include nanofibers. Nanofibers are natural or synthetic fibers with a diameter in the nanometer range. Natural nanofibers can be listed as: materials containing one or more polysaccharides such as collagen, cellulose, fibroin, keratin, gelatin, chitosan, etc. Synthetic nanofibers can be listed as: polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), polylactic acid-co-glycolic acid (PLGA), poly (3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), poly (ethylene-vinyl acetate copolymer) (PEVA), etc. The nanofibers can be included in the gel containing alginic acid in an amount of less than 1% by weight, for example, 0.9%, 0.8%, 0.7%, 0.6%, 0.5% or less by weight. The lower limit of the amount of nanofibers contained in the gel containing alginate or alginate is not particularly limited, and can be 0.05% and above by weight, preferably 0.1% and above by weight.

[0216] The embedding of the cell population in the gel containing alginate or alginate ester can be performed by any method without particular limitation, and can be performed, for example, by mixing the cell population in a solution of alginate or alginate ester and causing the mixture to form a gel.

[0217] The cell population can be selected from 1×10 4 -1×10 9 cells / mL, preferably 1×10 7 -1×10 8 The amount of cells / mL is contained in the alginate or alginate ester solution.

[0218] The gelation of the alginate or alginate solution containing the cell population can be carried out by adding a cross-linking agent to the solution. The amount of the cross-linking agent added can be selected from 0.1%-5% by weight, for example, 0.1%-1% by weight, relative to the solution. The gelation can be carried out in a container having a prescribed structure and / or shape for cell culture or cell transplantation, or in a mold designed to obtain a gel that conforms to the container.

[0219] Alternatively, it can be carried out by forming a gel capsule containing alginic acid according to a known method (WO 2010 / 010902). That is, a solution of alginate or alginate ester containing a cell group can be added dropwise to a solution of a crosslinking agent. The size of the droplet can be adjusted according to the shape of the nozzle during the dropwise addition and the dropwise addition method, and the size of the gel capsule containing alginic acid can be determined. The dropwise addition method is not particularly limited, but can be carried out by air spraying, airless spraying, electrostatic spraying, etc. The size of the gel capsule containing alginic acid is not particularly limited, and its diameter can be 5 mm and below, 1 mm and below, 500 μm and below. The crosslinking agent solution can contain a crosslinking agent selected from an amount of 0.1%-10% by weight, for example, an amount of 0.1%-5% by weight.

[0220] The "PLK inhibitor" in the present invention is a substance that has inhibitory activity against Polo-like kinase (PLK). PLK is a family of serine / threonine kinases preserved in eukaryotes. It is a kinase responsible for the M phase of the cell cycle. Four types of PLK1, PLK2, PLK3 and PLK4 are known. The PLK inhibitor used in the present invention can be an inhibitor of any one of PLK1, PLK2, PLK3 and PLK4 as long as it can inhibit the proliferation of CHGA-negative cells, and the inhibitor of each PLK can also be an inhibitor of other PLKs. The PLK inhibitor used in the present invention can be a substance that is combined with PLK inhibitory activity and has other activities (inhibitory activity, etc.) as long as it can inhibit the proliferation of CHGA-negative cells. Preferably, the "PLK inhibitor" in the present invention is an inhibitor that has inhibitory activity against PLK1 or PLK4, preferably an inhibitor with higher selectivity for 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, further preferably 10 nM or less, particularly preferably 5 nM or less, and particularly preferably 3 nM or less for PLK1 or PLK4 can be appropriately used. A method for determining PLK inhibitory activity can be selected from known methods, such as the determination method using Serine / Threonine Kinase Assay Kits (MEDICAL&BIOLOGICAL LABORATORIES CO., LTD.). The "PLK inhibitor" in the present invention can be found in patent literature or non-patent literature using a conventionally known method.

[0221] PLK inhibitors that can be used in the present invention can be exemplified (but not limited to): (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, the compound is sometimes described as "CAS929095-18-1"), (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]vinyl]-1H-indazol-6-yl]-5'-methoxy-spiro[cyclopropane-1,3'-[ 3H]indol]-2'(1'H)-one (CAS No.1338800-06-8) (in the present specification, the compound is sometimes described 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-pyrimidinamine (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-pyrimidinamine (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]diazepine-2-yl)amino]-2-fluoro-5-methoxy-N-(1-methyl-4-piperidinyl)benzamide hydrochloride (CAS No.2108449-45-0), 3-(1,3-benzodicyclopenten-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-benzofurano[2,3-c]azepine-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-propane-1-one (CAS No.82-08-6), N-[[4-[(6-chloro-3-pyridyl)methoxy]-3-methoxyphenyl]methyl]-3,4-dimethoxy-phenylethaneamine 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-pyridyl)vinyl]phenyl]-acetamide (CAS No. 173529-46-9), sodium (E)-2-((2-methoxy-5-(((2,4,6-trimethoxyphenyl)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]diazepine-2-yl)amino)-2-fluoro-5-methoxy-N-(1-methylpiperidin-4-yl)benzamide (CAS No.1137868-52-0), 4-((6-fluoro-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]diazepine-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 their salts. In addition, as long as these compounds have PLK inhibitory activity and preferably have a 50% inhibition concentration (IC50) of 1 μM or less, preferably 100 nM or less, more preferably 50 nM or less, further preferably 10 nM or less, particularly preferably 5 nM or less, and particularly preferably 3 nM or less, they may have one or more substituents, and a part of the partial structure (substituent, ring, etc.) may be transformed. .

[0222] Preferably, the PLK inhibitor of the present invention 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, (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]vinyl]-1H-indazol-6-yl]-5'-methoxy-spiro[cyclopropane-1,3'-[ 3 H]indol]-2'(1'H)-one (CAS No. 1338800-06-8).

[0223] PLK inhibitors are not limited to the compounds shown above. Antisense oligonucleotides or siRNA against ROCK mRNA, antibodies that bind to PLK, dominant negative PLK mutants, etc. can also be used as PLK inhibitors. They are commercially available or can be synthesized according to known methods.

[0224] The aforementioned various compounds or salts thereof can be used as PLK inhibitors. The amount added to the culture medium is appropriately determined according to the compound or salt used, and is generally about 0.00001 μM-100 μM, preferably 0.01 μM-10 μM, more preferably 0.1 μM-μM, and particularly preferably 0.1 μM-3 μM.

[0225] The treatment of a pancreatic precursor cell group, preferably an endocrine precursor cell group, more preferably an insulin-producing cell group or a cell group in a subsequent differentiation stage obtained by differentiation induction of pluripotent stem cells with a PLK inhibitor can be carried out by contacting the PLK inhibitor with the cell group. For example, it can be carried out by culturing the cell group in a culture medium to which a PLK inhibitor is added. The PLK inhibitor can be included in the culture medium in any amount capable of inhibiting PLK activity, for example, in an amount of 10 μM and less or 5 μM and less, preferably 4 μM and less, more preferably 3 μM and less, for example, in an amount of 2 μM and less or 1 μM and less. The lower limit of the amount of the PLK inhibitor added is not particularly limited, and can be 0.1 μM and more, preferably 0.5 μM and more. The amount of the PLK inhibitor added is 10 μM and less and 0.1 μM and more, preferably 5 μM and less and 0.5 μM and more, and particularly preferably 3 μM and less and 0.5 μM and more. Cultivation in the presence of a PLK inhibitor can be performed 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. Cultivation in the presence of a PLK inhibitor is preferably performed for 4 days or more. The culture medium may also be exchanged during treatment with a PLK inhibitor, and may be exchanged with a culture medium having the same composition before the exchange to which the PLK inhibitor was added or with a culture medium having a different composition according to the culture schedule.

[0226] The pancreatic precursor cell group, preferably the endocrine precursor cell group, more preferably the insulin-producing cell group or the cell group in the differentiation stage after that obtained by differentiation induction of pluripotent stem cells can be subjected to a step of further differentiating them into a target cell group together with treatment with a PLK inhibitor. Here, "treatment with a PLK inhibitor" also includes the case where the step of treatment with a PLK inhibitor and the step of differentiation are carried out at the same time, the case where the step of differentiation is carried out after treatment with a PLK inhibitor, and the case where the step of treatment with a PLK inhibitor is carried out after the step of differentiation. Therefore, the culture medium used when treating with a PLK inhibitor can be different from the culture medium used when differentiating the cell group, and the PLK inhibitor can also be further added to the culture medium used in the differentiation step.

[0227] This method does not inhibit the proliferation of teratomas, but can reduce or inhibit the number of remaining proliferative cells included in the pancreatic lineage.

[0228] This method does not inhibit the proliferation of iPS cells (for example, it may not reduce the number of alkaline phosphatase-positive cells), but can reduce or inhibit the number of remaining proliferative cells included in the pancreatic lineage.

[0229] According to the present method, the absolute number of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, in an endocrine precursor cell population or a cell population at a subsequent differentiation stage can be reduced by treating with a PLK inhibitor. Therefore, the CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, in the obtained cell population can be depleted.

[0230] That is, the proportion of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, in the obtained cell population can be reduced compared to the case where the cells are cultured and / or differentiated without treatment with a PLK inhibitor. The proportion 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 proportion is not particularly limited, for example, 0% or more, 0.1% or more, or 0.5% or more. The ratio can be expressed by two values ​​selected from the aforementioned upper limit and lower limit, for example, the ratio is 0.1%-40%, 0.1%-30%, 0.1%-20%, 0.1%-15% or 0.1%-10%, preferably 0.1%-20%, 0.1%-15% or 0.1%-10%. Alternatively, the ratio is 0.5%-40%, 0.5%-30%, 0.5%-20%, 0.5%-15% or 0.5%-10%, preferably 0.5%-20%, 0.5%-15% or 0.5%-10%. In particular, the proportion of CHGA-negative and Ki67-positive cells in the obtained cell population 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, preferably 3% or less, 2% or less, 1% or less or 0.5% or less. The lower limit of the proportion is not particularly limited, for example, 0% or more or 0.1% or more. The ratio can be expressed using two numerical values ​​selected from the aforementioned upper limit value and lower limit value respectively, for example, the ratio is 0.1%-8%, 0.1%-7%, 0.1%-6%, 0.1%-5%, 0.1%-4%, 0.1%-3%, 0.1%-2%, 0.1%-1% or 0.1%-0.5%, preferably 0.1%-3%, 0.1%-2%, 0.1%-1% or 0.1%-0.5%.

[0231] In addition, according to the present method, a pancreatic precursor cell group, preferably an endocrine precursor cell group, more preferably an insulin-producing cell group or a cell group in a subsequent differentiation stage obtained by differentiation induction of pluripotent stem cells treated with a PLK inhibitor is differentiated into insulin-producing cells or pancreatic β cells, thereby inhibiting the proliferation of CHGA-negative cells, especially CHGA-negative and Ki67-positive cells, and a cell group enriched with insulin-producing cells or pancreatic β cells can be obtained. That is, compared with the case where the PLK inhibitor is not used for treatment, the proportion of insulin-producing cells or pancreatic β cells in the cell group obtained after differentiation induction can be increased. The proportion of insulin-producing cells or pancreatic β cells in the obtained cell group is 40% and above, 50% and above, 60% and above, 70% and above, 80% and above or 90% and above. The upper limit of this ratio is not particularly limited, and is 100% and below, 99% and below, 98% and below, 97% and below, 96% and below or 95% and below. The ratio can be expressed using two values ​​selected from the aforementioned upper limit and lower limit, for example, the ratio is 40%-95%, 50%-95%, 60%-95%, 70%-95%, 80%-95% or 90%-95%. In particular, the ratio of insulin-positive and NKX6.1-positive cells is 14% and above, preferably 15% and above, more preferably 20% and above, and further preferably 25% and above, for example, 30% and above, 35% and above, 40% and above or 45% and above. The upper limit of the ratio is not particularly limited and is 50% and below. The ratio can be expressed using two values ​​selected from the aforementioned upper limit and lower limit, for example, the ratio is 14%-50%, preferably 15%-50%, more preferably 20%-50%, and further preferably 25%-50%.

[0232] The insulin-producing cells or pancreatic β cells obtained by this method can be transplanted into an animal organism and directly retained as insulin-secreting cells during differentiation in the animal organism. The insulin-producing cells or pancreatic β cells obtained by this method can avoid the proliferation of CHGA-negative cells, especially CHGA-negative and Ki67-positive cells, and achieve the effect of safety and long-term survival of transplanted cells.

[0233] According to the present invention, an insulin-producing cell population or a pancreatic β-cell population (the cell population of the present invention) from which CHGA-negative cells, particularly highly proliferative CHGA-negative and Ki67-positive cells, have been removed can be used as a cellular medicine for treating diabetes, particularly type 1 diabetes, by being transplanted directly or after being encapsulated into the affected area.

[0234] Furthermore, the cell population of the present invention may be a prodrug. In the present specification, a prodrug refers to a cell population that differentiates after being transplanted into a living body and changes into cells having a function of treating a disease.

[0235] 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, cattle, sheep, monkeys, humans) by preparing a pharmaceutical composition directly or by mixing with a pharmaceutically acceptable carrier.

[0236] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

[0237] [Example]

[0238] Differentiation induction from pluripotent stem cells to endocrine precursor cell populations was carried out according to the above steps 1)-5), previous reports (Stem Cell Research (2015) 14, 185-197), etc. Differentiation induction to insulin-producing cells was carried out according to the above step 6), etc.

[0239] Example 1: Reduction of off-target cells (CHGA-negative cells) and increase of 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)

[0240] 1. Methods

[0241] 1) The endocrine precursor cell population obtained by differentiation induction of iPS cells was cultured for 7 days in a differentiation induction medium (modified MEM / 1% B-27 / Penisilin Streptomycin medium) containing differentiation factors (ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, ascorbic acid) to induce differentiation into an insulin-producing cell population. Next, a PLK1 inhibitor (CAS929095-18-1, 3 μM) or a PLK4 inhibitor (CAS1338800-06-8, 3 μM) was added to a differentiation induction medium (modified 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.

[0242] 2) The endocrine precursor cell population induced by differentiation of iPS cells was cultured for 11 days using a differentiation induction medium (modified MEM / 1% B-27 / Penisilin Streptomycin medium) containing differentiation factors (ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, ascorbic acid) but not containing a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8), and differentiated into an insulin-producing cell population.

[0243] The number of CHGA-negative (and Ki67-positive or Ki67-negative) cells in the cell populations obtained by the above methods 1) and 2) was counted by flow cytometry to determine the rate of off-target cells, i.e., CHGA-negative (and Ki67-positive or Ki67-negative) in each cell population.

[0244] Furthermore, the number of insulin-positive and NKX6.1-positive cells in the cell populations obtained by the methods 1) and 2) above was counted by flow cytometry to determine the target cells in each method, namely, the rate of insulin-positive and NKX6.1-positive cells.

[0245] 2. Results

[0246] The results of the CHGA-negative (and Ki67-positive or Ki67-negative) cell rate and the insulin-positive and NKX6.1-positive cell rate obtained by treating with PLK1 inhibitor (CAS929095-18-1) or PLK4 inhibitor (CAS1338800-06-8) in the production step of insulin-producing cells are shown in Table 1.

[0247] It was confirmed that when the PLK1 inhibitor (CAS929095-18-1) or PLK4 inhibitor (CAS1338800-06-8) was used for the last 4 days of the differentiation induction step to insulin-producing cells, the CHGA-negative cell rate was significantly reduced with excellent reproducibility compared with any control of Ki67-positive and Ki67-negative cells. This result indicates that in the process of producing insulin-producing cells, by treating with a PLK1 inhibitor (CAS929095-18-1) or PLK4 inhibitor (CAS1338800-06-8), the off-target cells (CHGA-negative cells) in the cell population can be reduced or the proliferation of these off-target cells can be suppressed.

[0248] On the other hand, it was confirmed that in the production process of insulin-producing cells, by treating with a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8), the rate of target cells (insulin-positive and NKX6.1-positive cells) in the cell population was significantly increased with excellent reproducibility, accompanied by a decrease in off-target cells, compared with the control.

[0249]

Table 1

[0250]

[0251] Regarding each cell rate, the upper part shows the first experimental result, and the lower part shows the second experimental result.

[0252] From the above results, it can be seen that by treating the cell population in the final differentiation stage of the insulin-producing cell production step with a PLK1 inhibitor or a PLK4 inhibitor, the off-target cells (CHGA-negative cells) present in the cell population can be reduced or the proliferation of these off-target cells can be inhibited. The results show that a cell population enriched with target cells (insulin-positive and NKX6.1-positive cells) can be obtained.

[0253] Example 2: Reduction of off-target cells (CHGA-negative cells) and increase of 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)

[0254] 1. Methods

[0255] The endocrine precursor cell group obtained by differentiation induction of 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 group by the PLK inhibitor was performed for 7 days. That is, in a 30 mL culture reactor, the endocrine precursor cell group obtained by differentiation induction of iPS cells was cultured in a differentiation induction medium for 4 days to induce differentiation into an insulin-producing cell group. Then, the cells were cultured in a differentiation induction medium supplemented with a PLK1 inhibitor (CAS929095-18-1, 3 μM) or a PLK4 inhibitor (CAS1338800-06-8, 3 μM) for 7 days.

[0256] As a control, an endocrine precursor cell group obtained by differentiation induction of iPS cells was cultured in a differentiation induction medium for 11 days in a 30 mL culture reactor to induce differentiation into an insulin-producing cell group.

[0257] 2. Results

[0258] It was confirmed that even in a system using a 30 mL culture reactor, by using a PLK1 inhibitor or a PLK4 inhibitor for treatment in the differentiation induction step to insulin-producing cells, the CHGA-negative cell rate was significantly reduced with excellent reproducibility compared to any control of Ki67-positive and Ki67-negative cells; in addition, compared to the control, the insulin-positive (and NKX6.1-positive) cell rate was significantly increased with excellent reproducibility along with the reduction of off-target cells. In addition, compared with the above-mentioned Example 1, the treatment time with the PLK1 inhibitor or the PLK4 inhibitor was extended, and its influence could not be confirmed, but it was confirmed that the treatment with the PLK1 inhibitor or the PLK4 inhibitor should be preferably performed on at least the cell population in the final differentiation stage of the insulin-producing cell production step.

[0259] Example 3: Single cell RNA-seq expression analysis of insulin-producing cell populations

[0260] According to previous reports (Stem Cell Research (2015) 14, 185-197), the above steps 1)-6) performed single cell RNA-seq expression analysis on the insulin-producing cell population obtained by differentiation and induction of pluripotent stem cells, and the results showed that the PLK gene was highly expressed in CHGA-negative cells, which are off-target cells. Therefore, when a PLK targeted inhibitor is used to reduce / inhibit the proliferation of off-target cells, a significant effect as described above can be observed.

Claims

1. A method for producing an insulin-producing cell population, wherein the cell population contains CHGA-negative and Ki67-positive cells at a ratio of 3% or less, wherein the method comprises the steps of inducing differentiation of pluripotent stem cells into an insulin-producing cell population, and treating the insulin-producing cell population obtained by differentiation of the pluripotent stem cells with a PLK inhibitor, wherein the PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor, 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, wherein the PLK4 inhibitor is (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]vinyl]-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.

2. The manufacturing method according to claim 1, wherein: The produced cell population contained CHGA-negative cells at a ratio of 20% or less.

3. The manufacturing method according to any one of claims 1 to 2, wherein: The produced cell population contained insulin-positive and NKX6.1-positive cells at a ratio of 15% or more. The production method according to claim 1 , further comprising a step of differentiating the insulin-producing cell population treated with the PLK inhibitor. The production method according to claim 1 , wherein the insulin-producing cell population is treated with a PLK inhibitor at a concentration of 3 μM or less.

6. A method for inhibiting the proliferation of CHGA-negative cells, wherein the CHGA-negative cells are present in a population of insulin-producing cells obtained by differentiation induction of pluripotent stem cells, The method comprises: Inducing differentiation of pluripotent stem cells into insulin-producing cell populations, and The insulin-producing cell population obtained by differentiation and induction of pluripotent stem cells is treated with a PLK inhibitor, wherein the PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor, 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, wherein the PLK4 inhibitor is (1S,2R)-2-[3-[(1E)-2-[4-[[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl]phenyl]vinyl]-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. 7 . The method according to claim 6 , wherein the insulin-producing cell population is treated with a PLK inhibitor at a concentration of 3 μM or less.

8. The method according to claim 6 or 7, which reduces the CHGA-negative cells present in the insulin-producing cell population to a ratio of 20% or less.

9. The method according to claim 6, which reduces the CHGA-negative and Ki67-positive cells present in the insulin-producing cell population to a ratio of 3% or less. 10 . The method according to claim 6 , which increases the proportion of insulin-positive and NKX6.1-positive cells present in an insulin-producing cell population to 15% or more.

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