Proliferation inhibitor

Abstract

The purpose of the present invention is to provide a method for removing non-endocrine extracorporeal cells of interest coexisting with insulin-secreting cells obtained by differentiation induction. The present invention relates to a method for producing an insulin-producing cell population, the method comprising a step for treating an insulin-producing cell population obtained by inducing the differentiation of pluripotent stem cells with a PLK inhibitor.

Description

This application is a divisional application of the Chinese patent application with application number 202080073014.X (application date: October 20, 2020, invention name: proliferation inhibitor).

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-induced pluripotent stem cells. [Background Technology]

[0002] Research is currently underway to induce pluripotent stem cells, such as iPS cells and ES cells, to differentiate into insulin-producing cells or pancreatic β cells and apply them to the treatment of diabetes.

[0003] To date, 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 populations obtained through differentiation induction also contain non-endocrine cells in addition to the target insulin-secreting cells. To promote their use in diabetes treatment, a method for more efficiently obtaining the target insulin-secreting cells is desired.

Prior art literature

Non-patent literature

[0004] [Non-patent document 1] Stem Cell Research (2015) 14, 185-197 [Summary of the invention] [Technical problem to be solved by the invention]

[0005] The present inventors have discovered that, in addition to these insulin-secreting cells (insulin-producing cells or pancreatic β cells), a population of cells induced to differentiate from pluripotent stem cells into insulin-producing cells or pancreatic β cells also contains non-endocrine cells characterized by chromogranin A (hereinafter referred to as "CHGA") negativity (hereinafter sometimes referred to as "CHGA-negative cells"). Furthermore, the present inventors have discovered that CHGA-negative cells include highly proliferative cells characterized by Ki67 positivity (CHGA-negative and Ki67-positive cells).

[0006] When using differentiated insulin-secreting cells for diabetes treatment, for example, it is extremely important to strictly control the presence of cells other than insulin-secreting cells from a safety perspective. Furthermore, the infiltration or persistence of highly proliferative cells may negatively impact the recipient or the long-term survival of the transplanted insulin-secreting cells, which is not desirable.

[0007] Therefore, an object of the present invention is to provide a method for removing CHGA-negative cells that coexist with insulin-secreting cells induced to differentiate. Technical means used to solve the problem

[0008] As a result of the inventors' intensive research to solve the above-mentioned problems, they discovered 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.

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

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

[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 inducing differentiation of pluripotent stem cells to 15% or more.

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

[13] The cell population according to

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

[14] The cell population according to

[12] or

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

[15] The cell population according to any one of

[12] to

[14] , which is used for transplantation.

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

[12] to

[15] .

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

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

[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.

[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.

[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.

[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.

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

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

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

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

[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.

[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.

[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.

[28] The cell population according to any one of

[12] to

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

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

[28] .

[30] The method according to

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

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

[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.

[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.

[34] The cell population according to any one of

[12] to

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

[35] The cell population according to any one of

[12] to

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

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

[12] to

[14] ,

[34] and

[35] .

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

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

[39] The method according to

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

[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.

[41] The method according to

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

[42] The method according to

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

[43] The method according to any one of

[17] ,

[30] and

[39] to

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

[44] The method according to any one of

[17] ,

[30] and

[39] to

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

[45] The method according to any one of

[17] ,

[30] and

[39] to

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

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

[47] The cell population according to

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

[48] ​​The cell population according to

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

[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.

[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.

[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.

[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.

[53] The cell population according to any one of

[46] to

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

[54] The cell population according to any one of

[46] to

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

[55] The cell population according to any one of

[46] to

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

[56] The cell population according to any one of

[46] to

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

[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.

[58] The use according to

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

[59] The use according to

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

[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.

[61] The use according to

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

[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.

[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.

[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.

[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.

[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.

[67] The use according to any one of

[57] to

[62] , comprising insulin-positive and NKX6.1-positive cells at a ratio of 15% or more. This specification incorporates the contents described in the specification and / or drawings of Japanese Patent Application No. 2019-191876, which is the basis of priority for this application. All publications, patents, and patent applications cited in this specification are incorporated herein by reference. Effects of the invention

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

[0011] 1. Terminology The following describes the terms used in this specification.

[0012] In this specification, "about" means a value that fluctuates within plus or minus 25%, 20%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference value, respectively. Preferably, the terms "about" or "approximately" mean a value that fluctuates within plus or minus 15%, 10%, 5%, or 1% relative to a reference value, respectively.

[0013] In this specification, "include / comprise(s) or comprising" means including / comprising the elements following the sentence, but is not limited thereto. Therefore, although the inclusion / comprising of the elements following the sentence is implied, it does not imply the exclusion of any other elements.

[0014] In this specification, "consist(s) of or consisting of" means including and limited to all the elements following the phrase. Therefore, the phrase "consist(s) of" means that the listed elements are essential or necessary, and there are essentially no other elements.

[0015] In this specification, "not using feeder cells" means that feeder cells are substantially not contained, and no medium pretreated by culturing feeder cells is used, etc. Therefore, the culture medium does not contain substances such as growth factors and cytokines secreted by feeder cells.

[0016] In addition, "feeder cells" or "feeders" refer to cells that are co-cultured with other types of cells to provide an environment that supports and enables the growth of these cells. Feeder cells can be of the same origin as the cells they support, or of a different origin. For example, as feeders for human cells, human skin fibroblasts or human embryonic stem cells can be used, as can primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts. Feeder cells can be inactivated by irradiation or treatment with mitomycin C.

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

[0018] As used herein, "culture" refers to maintaining cells in an in vitro environment and causing them to grow and / or differentiate. "Cultivating" means causing cells to persist, 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).

[0019] In this specification, "enrichment" and "enrichment" refer to increasing the amount of a specific constituent 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 specific 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. The cell group related to the target cell type can also be enriched by cell selection and screening methods well known in the art. The cell group can also be enriched 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 enriched by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99%.

[0020] As used herein, "deplete" and "depletion" refer to reducing the amount of a specific component in a composition, such as a composition of cells; "depleted" refers to a cell population in which the amount of a specific component is reduced compared to the proportion of the component in the cell population before depletion, when used to describe a composition of cells, such as a cell population. For example, a composition, such as a cell population associated with 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 population before depletion. A cell population associated with a target cell type can also be depleted by cell selection and screening methods known in the art. A cell population can also be depleted by a specific screening or selection process described herein. In a specific embodiment of the present invention, the method for depleting a target cell population reduces (depletes) the cell population associated with the target cell population by at least 50%, 80%, 85%, 90%, 95%, 97%, 98% or 99%.

[0021] In this specification, "purify" and "purification" refer to the removal of impurities from a composition such as a composition of cells 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 when describing a composition such as a cell group for cells. 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 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 described 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%, or can be reached to the extent that impurities (including mixed cells) cannot be detected.

[0022] In this specification, "does not reduce 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 decrease in the number of cells may occur for reasons not caused by the implementation of the method of the present invention (for example, natural cell death that usually occurs in the steps of cell culture and differentiation known in the past). Therefore, "does not reduce the number of cells" also includes situations 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.

[0023] As used herein, "inhibiting proliferation" means that the cell number does not significantly increase as a result of the method of the present invention, and means that there is no significant increase in the cell number before and after the method. Therefore, "inhibiting proliferation" also includes situations where the rate of cell increase after the method of the present invention is 30% or less, 20% or less, 10% or less, or 5% or less compared to before the method of the present invention.

[0024] As used herein, "marker" refers to a cellular antigen or gene specifically expressed by a given cell type, such as a "marker protein" or "marker gene." Preferably, the marker is a cell surface marker, in which case concentration, separation, and / or detection of viable cells can be performed. The marker can be a positive or negative selection marker.

[0025] The detection of the marker protein can be performed by immunological assay using a specific antibody 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.

[0026] In this specification, "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter in a cell.

[0027] In this specification, "a factor having CDK8 / 19 inhibitory activity" refers to any substance that has CDK8 / 19 inhibitory activity. Compared to other proteins in the same CDK family, CDK8 is non-essential for cell proliferation, and inhibition of CDK8 has no significant effect under normal conditions. Similar to CDK8, inhibition of CDK19 is often accompanied by inhibition of CDK8.

[0028] "Growth factors" are endogenous proteins that promote the differentiation and / or proliferation of specific cells. Examples of "growth 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), and other cytokines effective for stem cells, such as stem cell factor (SCF) and erythropoietin (Epo).

[0029] In this specification, the term "ROCK inhibitor" refers to 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. ROCK inhibitors are not particularly limited as long as they have the above-mentioned function, and examples thereof include N-(4-pyridyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (also referred to as Y-27632 in this 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). ROCK inhibitors are not limited to those mentioned above. Antisense oligonucleotides or siRNA against ROCK mRNA, antibodies binding to ROCK, dominant negative ROCK mutants, etc. can also be used as ROCK inhibitors. These are commercially available or can be synthesized according to known methods.

[0030] In this specification, a "GSK3β inhibitor" refers to a substance that has inhibitory activity against 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, apoptosis, and the maintenance of stem cells. GSK3 exists in 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 may also be a substance that has GSK3α inhibitory activity in combination with GSK3β inhibitory activity.

[0031] Examples of GSK3β inhibitors include CHIR98014 (2-[[2-[(5-nitro-6-aminopyridin-2-yl)amino]ethyl]amino)-4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidine), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl)-amino]ethyl]amino 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, Kenpaul 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), 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. Antisense oligonucleotides or siRNAs targeting GSK3β mRNA, antibodies that bind to GSK3β, dominant-negative GSK3β mutants, etc. can also be used as GSK3β inhibitors. These are commercially available or can be synthesized according to known methods.

[0032] Examples of the "serum replacement" 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 mixtures thereof (e.g., ITS-G). B-27 supplement, KSR, Serum Replacement, ITS-G, is used as a serum substitute. When serum replacer is added to culture medium, the concentration in the culture medium is 0.01% to 10% by weight, preferably 0.1% to 2% by weight. In the present invention, serum replacer is preferably used to replace serum.

[0033] 2. Insulin-producing cell populations with inhibited proliferation of CHGA-negative cells 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. The present invention also relates to methods of inhibiting the proliferation of CHGA-negative cells by treating them with a PLK inhibitor.

[0034] "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.

[0035] "CHGA-negative cells" may include cells characterized by confirmed expression of Ki67 as a marker (ie, CHGA-positive and Ki67-positive cells). Ki67 is a cell cycle-associated nuclear protein. It is widely known as a marker for cell proliferation and the cell cycle, as it is expressed in the G1, S, G2, and M phases of proliferating cells, but not in the G0 phase, when proliferation is stopped. Therefore, cells that are both CHGA-positive and Ki67-positive are highly proliferative.

[0036] The "insulin-producing cell population" of the present invention refers to a cell population comprising insulin-producing cells derived by differentiation induction of pluripotent stem cells. "Insulin-producing cells" refer to cells characterized by expression of a marker for insulin (i.e., insulin-positive cells). "Insulin-producing cells" may be cells expressing a marker for NK6 homeobox 1 (NKX6.1), preferably cells expressing markers for both insulin and NKX6.1 (i.e., insulin-positive and NKX6.1-positive cells).

[0037] The "insulin-producing cell population" of the present invention has a low content of CHGA-negative cells compared to the insulin-producing cell population obtained by differentiation and induction of pluripotent stem cells according to a conventionally known method, and its content (sometimes described as "ratio" in this specification) is 40% and less or 30% and less, preferably 20% and less, more preferably 15% and less, and further preferably 10% and less, for example, 9% and less, 8% and less, 7% and less, 6% and less, 5% and less, 4% and less, 3% and less, 2% and less, or 1% and less. The lower limit of the content is not particularly limited and is 0% and more, 0.1% and more, or 0.5% and more. The content can be expressed using two numerical values ​​selected from the aforementioned upper and lower limits, for example, a content of 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 in insulin-positive cells, particularly insulin-positive and NKX6.1-positive cells, compared to insulin-producing cell populations derived by differentiation-induced pluripotent stem cells according to conventional methods. The content of insulin-positive and NKX6.1-positive cells is 14% or more, preferably 15% or more, more preferably 20% or more, and even more preferably 25% or more, for example, 30% or more, 35% or more, 40% or more, or 45% or more. The upper limit of this content is not particularly limited and is 70% or less, 60% or less, or 50% or less. This content can be expressed using two numerical values ​​selected from the upper and lower limits described above, for example, 14%-50%, preferably 15%-50%, more preferably 20%-50%, and even more preferably 25%-50%.

[0038] 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, derived from pluripotent stem cells, or a cell population at a subsequent differentiation stage thereof, with a PLK inhibitor. Treatment of a cell population at a predetermined differentiation stage with a PLK inhibitor can inhibit the proliferation of CHGA-negative cells, reduce their cell content, and enrich for insulin-positive cells, preferably insulin-positive and NKX6.1-positive cells.

[0039] It is known that during the differentiation process of pluripotent stem cells into pancreatic beta cells, cells with different characteristics appear depending on the differentiation stage (WO 2009 / 012428, WO 2016 / 021734). For example, these differentiation stages can be roughly divided into pluripotent stem cells, definitive endoderm cells, gastrointestinal cells, posterior foregut cells, pancreatic progenitor cells, endocrine progenitor cells, insulin-producing cells, and pancreatic beta cells, in order of relative undifferentiation.

[0040] "Pluripotency" as used herein refers to the ability to differentiate into various tissues or cells with distinct morphologies or functions, and the ability to differentiate into cells of any of the three germ layers. "Pluripotency" is incapable of differentiation into the blastoderm, and therefore, lacks the ability to form an individual, distinguishing it from "totipotency," which is the ability to differentiate into all tissues of an organism, including the blastoderm.

[0041] "Multipotency" as used herein refers to the ability to differentiate into a limited number of cell layers. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent, not pluripotent.

[0042] As used herein, "pluripotent stem cells" refer to embryonic stem cells (ES cells) and cells that potentially possess the same differentiation pluripotency, i.e., the ability to differentiate into various tissues of an organism (including all of the endoderm, mesoderm, and ectoderm). Examples of cells that possess the same differentiation pluripotency as ES cells include "induced pluripotent stem cells" (also referred to as "iPS cells" in this specification). Preferably, in the present invention, pluripotent stem cells are human pluripotent stem cells.

[0043] If mouse ES cells are used as "ES cells," various mouse ES cell lines established by inGenious, RIKEN (RIKEN), and the like 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, NIH strains CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28; WiCell Research Institute strains H1 and H9; and RIKEN strains KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 can be used as ES cell lines.

[0044] “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 the four factors Oct3 / 4, Sox2, Klf4 and c-Myc into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), there are also 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 using 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 produced 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), and induced pluripotent stem cells prepared by Sakurada et al. (Japanese Patent Application Laid-Open No. 2008-307007) can also be used.

[0045] 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, such as those described in WO 2008 / 151058, WO 2009 / 006930, WO 2009 / 006997, and WO 2009 / 007852.

[0046] As the induced pluripotent cell line, various iPS cell lines established by NIH, the RIKEN Institute (RIKEN), Kyoto University, and the like can be used. For example, human iPS cell lines include: RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and 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, and 648A1; and CDI's MyCell iPS cells(21525.102.10A) strain, MyCell iPS cells(21526.101.10A) strain, etc.

[0047] The term "pancreatic precursor cell population" as used herein refers to a cell population comprising pancreatic precursor cells. In this 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.

[0048] The pancreatic progenitor cell population is a cell population comprising pancreatic progenitor 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 progenitor cells, the pancreatic progenitor cell population may also contain other cells (e.g., endocrine progenitor cells, insulin-producing cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0049] The "endocrine precursor cell population" in this specification refers to a cell population containing endocrine precursor cells. In this specification, endocrine precursor cells refer to cells characterized by the expression of at least one marker selected from the group consisting of CHGA, NeuroD, and NGN3, and the absence of markers for pancreatic hormones (e.g., insulin). Endocrine precursor cells may also express markers such as PAX-4, NKX2.2, Islet-1, PDX-1, and PTF-1α.

[0050] The endocrine precursor cell population is a cell population comprising 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 even more preferably 70% or more. In addition to endocrine precursor cells, the endocrine precursor cell population may also include other cells (e.g., pancreatic precursor cells, insulin-producing cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0051] 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.

[0052] As described above, "insulin-producing cells" and "insulin-producing cell populations" typically comprise insulin-producing cells at a ratio of 5% or more, preferably 10% or more, more preferably 15% or more, even more preferably 20% or more, even more preferably 25% or more, and particularly preferably 30% or more. In addition to insulin-producing cells, the cell population may also include 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.).

[0053] The term "pancreatic β cells" as used herein refers to cells that are more mature than "insulin-producing cells," and specifically refers to cells that express at least one of MAFA, UCN3, and IAPP, markers of pancreatic β cell maturation, or that exhibit an increased insulin secretion response to glucose stimulation.

[0054] A "pancreatic β cell population" is a cell population comprising pancreatic β cells that can be obtained by differentiating and maturing an insulin-producing cell population, preferably by differentiating and maturing the cells 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.).

[0055] Cell populations at various stages of differentiation can be obtained using known methods for inducing pluripotent stem cells to differentiate into pancreatic β cells. Specifically, the following differentiation induction steps can be used to obtain the desired cell populations: Step 1) inducing differentiation of pluripotent stem cells into definitive endoderm cells; Step 2) inducing the definitive endoderm cells to differentiate into gastrointestinal cells; Step 3) inducing the differentiation of gastrointestinal cells into posterior foregut cells; Step 4) inducing the differentiation of posterior foregut cells into pancreatic progenitor cells; Step 5) inducing differentiation of pancreatic precursor cells into endocrine precursor cells; and Step 6) Inducing differentiation of endocrine precursor cells into insulin-producing cells. Each step will be described below, but the differentiation induction of each cell is not limited to these methods.

[0056] Step 1) Differentiation into definitive endoderm cells 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, but is 22,000-150,000 cells / cm 2 , preferably 22,000-100,000 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.

[0057] 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, approximately 5%. The culture can be performed in two-dimensional or three-dimensional culture.

[0058] 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 / 20mM Glucose / NaHCO3 / FAF-BSA / ITS-X / GlutaMAX medium can be used. TM / Ascorbic acid / Penisilin Streptomycin medium etc. are used as the basic medium for culturing mammalian cells.

[0059] The concentration of activin A in the culture medium is generally 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. 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. 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.

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

[0061] The concentration of the ROCK inhibitor in the culture 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 generally 5 μM to 20 μM, preferably 5 μM to 15 μM, and particularly preferably about 10 μM.

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

[0063] In a specific embodiment, after culturing for one day in a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, the culture is continued for two days while the medium is exchanged daily with a medium containing only activin A. Alternatively, pluripotent stem cells can be produced by first culturing them in a medium containing 0.01 μM to 20 μM insulin in the presence of a low dose of activin A, followed by a second culturing in a medium without insulin.

[0064] Step 2) Differentiation into gastrointestinal cells 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.

[0065] 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, approximately 5%. The culture can be performed in two-dimensional or three-dimensional culture.

[0066] The culture medium can be a basal medium for culturing mammalian cells, similar to step 1). In addition to growth factors, serum replacements, vitamins, antibiotics, etc. can also be appropriately added to the culture medium.

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

[0068] The concentration of the growth factor in the culture medium is appropriately adjusted depending on the type of growth factor used, but is generally about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. When EGF is used, its concentration is about 5 ng / mL to 2000 ng / mL (i.e., about 0.8 nM to 320 nM), preferably about 5 ng / mL to 1000 ng / mL (i.e., about 0.8 nM to 160 nM), and more preferably about 10 ng / mL to 1000 ng / mL (i.e., about 1.6 nM to 160 nM). When FGF10 is used, its concentration is about 5 ng / mL to 2000 ng / mL (i.e., about 0.3 nM to 116 nM), preferably about 10 ng / mL to 1000 ng / mL (i.e., about 0.6 nM to 58 nM). 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.

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

[0070] The culture temperature is not particularly limited, but is 30° C. to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture container is, for example, approximately 5%.

[0071] The culture medium can be a basal medium for culturing mammalian cells, similar to step 1). In addition to growth factors, serum replacements, vitamins, antibiotics, etc. can also be appropriately added to the culture medium.

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

[0073] The concentration of the growth factor in the culture medium is appropriately adjusted depending on the type of growth factor used, but is generally about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. When EGF is used, its concentration is about 5 ng / mL to 2000 ng / mL (i.e., about 0.8 nM to 320 nM), preferably about 5 ng / mL to 1000 ng / mL (i.e., about 0.8 nM to 160 nM), and more preferably about 10 ng / mL to 1000 ng / mL (i.e., about 1.6 nM to 160 nM). When FGF10 is used, its concentration is about 5 ng / mL to 2000 ng / mL (i.e., about 0.3 nM to 116 nM), preferably about 10 ng / mL to 1000 ng / mL (i.e., about 0.6 nM to 58 nM). 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.

[0074] The concentration of cyclopamine in the culture 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.

[0075] 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.

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

[0077] 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, and 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 in step 4).

[0078] The culture medium can be a basal medium for culturing mammalian cells, similar to step 1). In addition to growth factors, serum replacements, vitamins, antibiotics, etc. can also be appropriately added to the culture medium.

[0079] The aforementioned compounds or salts thereof can be used as factors having CDK8 / 19 inhibitory activity. The amount added to the culture medium is appropriately determined depending on the compound or salt used, typically ranging from about 0.00001 μM to 5 μM, preferably 0.00001 μM to 1 μM. The concentration of the factor having CDK8 / 19 inhibitory activity in the culture medium is preferably such that the inhibitory activity against CDK8 / 19 is 50% or greater.

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

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

[0082] 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.

[0083] In addition, the culture medium may also contain a protein kinase C (PKC) activator. Examples of PKC activators include, but are not limited to, PDBu (PKC activator II) and TPB (PKC activator V). The PKC activator is added at a concentration of approximately 0.1 ng / mL to 100 ng / mL, preferably approximately 1 ng / mL to 50 ng / mL, and more preferably approximately 3 ng / mL to 10 ng / mL.

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

[0085] 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-antifungal (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 can be performed by two-dimensional culture or three-dimensional culture.

[0086] In addition, in the case that cell culture is two-dimensional culture, feeder cells are not used, and culture is carried out by adhesion. During cultivation, culture vessels such as the cell culture sheet using dish, flask, microplate, OptiCell (product name) (Nunc company) etc. are used. Preferably, culture vessel is surface treated, to improve its adhesiveness (hydrophilicity) to cell, collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, matrigel (for example, BD matrigel (Becton Dickinson, Japan), vitronectin etc. are coated thereon for the matrix of 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.

[0087] The culture temperature is not particularly limited, but is 30° C. to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture container is, for example, approximately 5%.

[0088] The pancreatic progenitor cells obtained in step 4) can be further purified using glycoprotein 2 (GP2), a well-known surface marker, etc. The purification can be performed by a known method, for example, using beads immobilized with an anti-GP2 antibody.

[0089] Step 5) Differentiation into endocrine precursor cells 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 in 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 resulting dispersion is centrifuged, the recovered cells are resuspended in a small amount of fresh culture medium, and this 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.

[0090] The culture medium can be a basal medium for culturing mammalian cells, similar to that used in step 1). SANT1, retinoic acid, ALK5 inhibitor II, T3, and LDN can be added to the culture medium as previously reported (Nature Biotechnology 2014; 32: 1121-1133). Furthermore, appropriate amounts of Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum replacements, vitamins, and antibiotics can also be added to the culture medium.

[0091] The culture is performed by non-adhesive culture without using feeder cells. For culture, dishes, flasks, microplates, multi-well plates (Nunc) or bioreactors are used. The culture container is preferably surface treated to reduce its adhesion to the cells.

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

[0093] Step 6) Differentiation into insulin-producing cells 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.

[0094] 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, ZnSO4, heparin, N-acetylcysteine, Trolox, and R428 can be added to the culture medium.

[0095] 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, dishes, flasks, microplates, multi-well plates (Nunc Corporation) or bioreactors are used. Preferably, the culture container is surface treated to reduce its adhesion to the cells.

[0096] The culture temperature is not particularly limited, but is 30° C. to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture container is, for example, approximately 5%.

[0097] Differentiation into pancreatic β cells The cells obtained in the above steps 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 body.

[0098] The "animal" is preferably a mammal, and examples thereof include humans, non-human primates, pigs, cows, horses, sheep, goats, llamas, dogs, cats, rabbits, mice, and guinea pigs, among which humans are preferred.

[0099] Transplantation is preferably performed in a region of the body where the cell population can be fixed. For example, it can be performed subcutaneously, intraperitoneally, or beneath the membranes of various organs such as the peritoneum, omentum, adipose tissue, muscle tissue, pancreas, and kidney. 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 recipient, the size of the transplant site, and the severity of the disease. For example, 10×10 4 cells-10×10 11The transplanted cell population is induced to differentiate into the target cell population, preferably pancreatic β-cell population, in the in vivo environment, and can then be recovered or directly left in the body.

[0100] During transplantation, the cell population can be embedded in a gel containing a biocompatible material for transplantation. For example, the cell population embedded in the gel containing a biocompatible material can be enclosed in a device such as a capsule, a bag or a chamber and then transplanted into the body.

[0101] The term "embedding" in the present invention means that an endocrine precursor cell population or a cell population at a later differentiation stage is dispersed and contained in a gel containing a biocompatible material.

[0102] The "biocompatible material" in this specification means any material that does not induce a significant immune response or harmful biological response (for example, toxic reaction, coagulation, etc.) when transplanted into a living body 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, polypropylene fumarate, etc. esters, 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 ester, collagen, cellulose, fibroin, keratin, gelatin, fibrin, pullulan, laminin, gellan gum, polysilicone, urethane, elastin, etc. and their deformations and combinations thereof. If necessary, the surface of the "biocompatible material" may 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 modification with functional groups known to control cell proliferation, differentiation, and function (e.g., amino, carboxyl, hydroxyl, methacrylic, acrylic, etc.) to enable cell adhesion. In certain embodiments, alginate or alginate ester can be appropriately used as the "biocompatible material."

[0103] 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. can be suitably used.

[0104] Alginate (also known as propylene glycol alginate) is a derivative formed by ester-bonding propylene glycol to the carboxyl groups of alginic acid.

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

[0106] The gel using alginate or alginate ester can be prepared according to known methods (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.

[0107] 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 capable of dissolving the alginate or alginate ester, and water, physiological saline, or the like may be used.

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

[0109] Gels containing alginate or alginate esters may include nanofibers. Nanofibers are natural or synthetic fibers with diameters in the nanometer range. Examples of natural nanofibers include materials containing one or more polysaccharides such as collagen, cellulose, fibroin, keratin, gelatin, and chitosan. Examples of synthetic nanofibers include polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), polylactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), and poly(ethylene-vinyl acetate) (PEVA). Nanofibers may be included in gels containing alginate 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 included in a gel containing alginate or alginate esters is not particularly limited and may be 0.05% or more by weight, preferably 0.1% or more by weight.

[0110] Embedding of the cell population in the gel containing alginate or alginate ester can be performed by any method without particular limitation. For example, the method can be performed by mixing the cell population in a solution of alginate or alginate ester and causing the mixture to form a gel.

[0111] 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.

[0112] The gelation of an alginate or alginate ester solution containing a cell population can be achieved by adding a crosslinker to the solution. The amount of crosslinker added can be selected from 0.1% to 5% by weight of the solution, for example, 0.1% to 1% by weight. Gelation can be performed in a container having a predetermined structure and / or shape for cell culture or cell transplantation, or in a mold designed to produce a gel that conforms to the container.

[0113] 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 population can be added dropwise to a solution of a crosslinking agent. The size of the droplets can be adjusted according to the shape of the nozzle during the dropwise addition and the dropwise addition method, thereby determining the size of the gel capsule containing alginic acid. 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, or 500 μm and below. The crosslinking agent solution can contain a crosslinking agent selected from an amount of 0.1% to 10% by weight, for example, an amount of 0.1% to 5% by weight.

[0114] 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 are known: PLK1, PLK2, PLK3, and PLK4. The PLK inhibitor used in the present invention can be any one of PLK1, PLK2, PLK3, and PLK4, as long as it can inhibit the proliferation of CHGA-negative cells. The inhibitor of each PLK can also be an inhibitor of other PLKs. The PLK inhibitor used in the present invention can be any one of PLK1, PLK2, PLK3, and PLK4, as long as it can inhibit the proliferation of CHGA-negative cells. It can also be a substance that combines with PLK inhibitory activity and has other activities (such as inhibitory activity). Preferably, the "PLK inhibitor" in the present invention is an inhibitor that has inhibitory activity against PLK1 or PLK4, and preferably an inhibitor with higher selectivity for PLK1 or PLK4. For example, substances with a 50% inhibitory concentration (IC50) against PLK1 or PLK4 of 1 μM or less, preferably 100 nM or less, more preferably 50 nM or less, even more preferably 10 nM or less, particularly preferably 5 nM or less, and particularly preferably 3 nM or less can be suitably used. Methods for determining PLK inhibitory activity can be selected from known methods, such as those using Serine / Threonine Kinase Assay Kits (Medical & Biological Laboratories Co., Ltd.). "PLK inhibitors" in the present invention can be determined using conventionally known methods and can be found in patent and non-patent literature.

[0115] PLK inhibitors that can be used in the present invention include, but are 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, this compound is sometimes described as "CAS 929095-18-1"), (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, this compound is sometimes described as "CAS 1338800-06-8"), 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-benzofuro[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-propan-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. Furthermore, as long as these compounds have PLK inhibitory activity and preferably have a 50% inhibitory concentration (IC50) against PLK1 or PLK4 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 some of their partial structures (substituents, rings, etc.) may be modified.

[0116] Preferably, the PLK inhibitors of the present invention are (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).

[0117] 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. These are commercially available or can be synthesized according to known methods.

[0118] 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.

[0119] Treatment of a pancreatic precursor cell population, preferably an endocrine precursor cell population, more preferably an insulin-producing cell population, derived from induced differentiation of pluripotent stem cells, or a cell population at a subsequent differentiation stage, with a PLK inhibitor can be performed by contacting the cell population with the PLK inhibitor. For example, the treatment can be performed by culturing the cell population in a culture medium supplemented with the PLK inhibitor. The PLK inhibitor can be included in the culture medium in any amount sufficient to inhibit PLK activity, for example, at 10 μM or less, or 5 μM or less, preferably 4 μM or less, more preferably 3 μM or less, for example, at 2 μM or less, or 1 μM or less. The lower limit of the amount of the PLK inhibitor added is not particularly limited and can be 0.1 μM or greater, preferably 0.5 μM or greater. The amount of the PLK inhibitor added is between 10 μM and 0.1 μM or greater, preferably between 5 μM and 0.5 μM or greater, and particularly preferably between 3 μM and 0.5 μM or greater. 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 can also be exchanged during treatment with a PLK inhibitor, and the culture medium can be exchanged with a culture medium having the same composition as before the addition of the PLK inhibitor, or with a culture medium having a different composition, depending on the culture schedule.

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

[0121] 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. 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.

[0122] According to this method, treatment with a PLK inhibitor can reduce 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. Thus, the resulting cell population can be depleted of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells.

[0123] Specifically, the proportion of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, in the resulting cell population can be reduced compared to cells cultured and / or differentiated without treatment with a PLK inhibitor. The proportion of CHGA-negative cells in the resulting 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 this proportion is not particularly limited, and for example, it is 0% or more, 0.1% or more, or 0.5% or more. The ratio can be expressed using two numerical values ​​selected from the aforementioned upper and lower limits, for example, 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 resulting 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 this proportion is not particularly limited, and for example, it is 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, 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%.

[0124] Furthermore, according to this method, a pancreatic precursor cell population, preferably an endocrine precursor cell population, more preferably an insulin-producing cell population, or a cell population at a later stage of differentiation, derived from pluripotent stem cells treated with a PLK inhibitor is differentiated into insulin-producing cells or pancreatic β cells. This inhibits the proliferation of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, thereby yielding a cell population enriched in insulin-producing cells or pancreatic β cells. Specifically, the proportion of insulin-producing cells or pancreatic β cells in the cell population obtained after differentiation induction can be increased compared to cells obtained without treatment with a PLK inhibitor. The proportion of insulin-producing cells or pancreatic β cells in the resulting cell population is 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or 90% or greater. The upper limit of this proportion is not particularly limited, but is 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, or 95% or less. This ratio can be expressed using two numerical values ​​selected from the upper and lower limits described above, for example, 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% or greater, preferably 15% or greater, more preferably 20% or greater, and even more preferably 25% or greater, for example, 30% or greater, 35% or greater, 40% or greater, or 45% or greater. The upper limit of this ratio is not particularly limited and is 50% or less. This ratio can be expressed using two numerical values ​​selected from the upper and lower limits described above, for example, 14%-50%, preferably 15%-50%, more preferably 20%-50%, and even more preferably 25%-50%.

[0125] The insulin-producing cells or pancreatic beta cells obtained by this method can be transplanted into an animal and directly retained as insulin-secreting cells during differentiation. The insulin-producing cells or pancreatic beta cells obtained by this method can avoid the proliferation of CHGA-negative cells, particularly CHGA-negative and Ki67-positive cells, thereby ensuring safe and long-term survival of the transplanted cells.

[0126] 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 cell medicine for treating diabetes, particularly type 1 diabetes, by being transplanted directly or encapsulated into the affected area.

[0127] Furthermore, the cell population of the present invention may be a prodrug. In this specification, a prodrug refers to a cell population that differentiates after being transplanted into a living body and changes into cells that have a function of treating a disease.

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

[0129] Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples. [Example]

[0130] Differentiation induction from pluripotent stem cells into endocrine precursor cell populations was carried out according to the above steps 1)-5) and previous reports (Stem Cell Research (2015) 14, 185-197), etc. Differentiation induction into insulin-producing cells was carried out according to the above step 6), etc.

[0131] 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) 1. Methods 1) Endocrine progenitor cell populations induced by iPS cell differentiation were 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 insulin-producing cell populations. Subsequently, a PLK1 inhibitor (CAS929095-18-1, 3 μM) or a PLK4 inhibitor (CAS1338800-06-8, 3 μM) was added to the differentiation induction medium (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. 2) The endocrine progenitor 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 without a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8) to induce differentiation into an insulin-producing cell population.

[0132] 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.

[0133] 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 rate of insulin-positive and NKX6.1-positive cells, which were the target cells in each method.

[0134] 2. Results Two experiments were conducted using each method. Table 1 shows the rates of CHGA-negative (and Ki67-positive or Ki67-negative) cells and insulin-positive and NKX6.1-positive cells obtained when treated with a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8) during the insulin-producing cell production step.

[0135] The researchers confirmed that treatment with a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8) during the final four days of the differentiation induction step toward insulin-producing cells significantly and reproducibly reduced the rate of CHGA-negative cells compared to controls in both Ki67-positive and Ki67-negative cells. This result suggests that treatment with a PLK1 inhibitor (CAS929095-18-1) or a PLK4 inhibitor (CAS1338800-06-8) during the insulin-producing cell production process can reduce the number of off-target cells (CHGA-negative cells) in the cell population or inhibit their proliferation. 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.

[0136]

Table 1

[0137] The above results indicate that by treating a cell population at the final differentiation stage of the insulin-producing cell production step with a PLK1 inhibitor or a PLK4 inhibitor, the number of 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 indicate that a cell population enriched in target cells (insulin-positive and NKX6.1-positive cells) can be obtained.

[0138] 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) 1. Methods Endocrine progenitor cell populations derived from iPS cell differentiation induction were cultured in the same manner as in Example 1, except that a 30 mL culture reactor was used and the insulin-producing cell population was treated with a PLK inhibitor for 7 days. Specifically, the endocrine progenitor cell population derived from iPS cell differentiation induction was cultured in a 30 mL culture reactor for 4 days in a differentiation induction medium to induce differentiation into an insulin-producing cell population. Subsequently, the cells were cultured for 7 days in a differentiation induction medium supplemented with a PLK1 inhibitor (CAS929095-18-1, 3 μM) or a PLK4 inhibitor (CAS1338800-06-8, 3 μM). As a control, a population of endocrine progenitor cells obtained by differentiation induction of iPS cells was cultured in a differentiation induction medium in a 30 mL culture reactor for 11 days to induce differentiation into an insulin-producing cell population.

[0139] 2. Results Even in a system using a 30 mL culture reactor, treatment with a PLK1 inhibitor or PLK4 inhibitor during the differentiation induction step toward insulin-producing cells reproducibly and significantly reduced the CHGA-negative cell rate compared to both Ki67-positive and Ki67-negative controls. Furthermore, compared to the control, the insulin-positive (and NKX6.1-positive) cell rate reproducibly and significantly increased, accompanied by a decrease in off-target cells. Furthermore, compared to Example 1, the treatment time with the PLK1 inhibitor or PLK4 inhibitor was prolonged, and its effect could not be confirmed. However, it was confirmed that treatment with the PLK1 inhibitor or PLK4 inhibitor should preferably be performed on at least the cell population at the final differentiation stage in the insulin-producing cell production step.

[0140] Example 3: Single-cell RNA-seq expression analysis of insulin-producing cell populations According to a previous report (Stem Cell Research (2015) 14, 185-197), single-cell RNA-seq expression analysis of insulin-producing cell populations derived from pluripotent stem cell differentiation and induction, revealed that the PLK gene is highly expressed in CHGA-negative cells, which are considered off-target cells. Therefore, when a PLK-targeted inhibitor is used to reduce or inhibit the proliferation of off-target cells, the significant effects described above are observed.

Claims

1. A method for producing an insulin-producing cell population, wherein the cell population comprises 3% or less of CHGA-negative and Ki67-positive cells, 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. The PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor.

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 the 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 present in a population of insulin-producing cells induced by differentiation of pluripotent stem cells, The method comprises: Inducing differentiation of pluripotent stem cells into insulin-producing cell populations, and The insulin-producing cell population derived from pluripotent stem cells was treated with PLK inhibitors. The PLK inhibitor is a PLK1 inhibitor or a PLK4 inhibitor. 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 proportion of CHGA-negative and Ki67-positive cells in an insulin-producing cell population to 3% or less. 10 . The method according to claim 6 , which increases the proportion of insulin-positive and NKX6.1-positive cells in an insulin-producing cell population to 15% or more.

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