Maturation Agent

Abstract

The purpose of the present invention is to provide a novel method for inducing differentiation of an insulin-positive cell population from pluripotent stem cells. Provided is a method for producing an insulin-positive cell population, the method including differentiating a pancreatic progenitor cell population, or a cell population thereof at a later stage of differentiation, in medium containing a CDK8 / 19 inhibitor.

Description

[Technical Field]

[0001] The present invention relates to a method for producing an insulin-positive cell population from pluripotent stem cells. More specifically, the present invention relates to a method for producing an insulin-positive cell population, which comprises treating a pancreatic progenitor cell population obtained by inducing differentiation from pluripotent stem cells, or a cell population at a later differentiation stage, with a factor that has inhibitory activity against cyclin-dependent kinase 8 and / or cyclin-dependent kinase 19 (hereinafter sometimes referred to as "CDK8 / 19"). [Background of the invention]

[0002] Research is underway to induce differentiation of insulin-positive cells and pancreatic beta cells from pluripotent stem cells such as iPS cells and ES cells, and to apply this to the treatment of diabetes.

[0003] To date, various methods have been developed and reported for inducing differentiation from pluripotent stem cells into insulin-positive cell populations (Non-Patent Document 1). However, the insulin-positive cell population obtained by differentiation induction contains various cells in addition to the desired insulin-positive cells (particularly insulin-positive and NKX6.1-positive cells, etc.). When attempting to apply insulin-positive cell populations to diabetes treatment, it is extremely important from a safety standpoint to strictly control the types of cells contained in the cell population. Therefore, there has been a strong demand for a new method for inducing differentiation of insulin-positive cell populations that allows for the inclusion of a larger number of desired cells. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Stem Cell Research(2015)14, 185-197 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel method for inducing differentiation of an insulin-positive cell population from pluripotent stem cells. [Means for solving the problem]

[0006] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they found that, in the process of inducing an insulin-positive cell population from pluripotent stem cells, by applying a factor having CDK8 / 19 inhibitory activity (hereinafter sometimes referred to as a "CDK8 / 19 inhibitor") to a pancreatic progenitor cell population or a cell population at a later differentiation stage, it is possible to efficiently induce the differentiation of insulin-positive cells, particularly insulin-positive and NKX6.1-positive cells, and to obtain an insulin-positive cell population containing a higher number of such cells than with conventional methods.

[0007] The present invention is based on these new findings and includes the following inventions. [1] A method for producing an insulin-positive cell population, comprising: A method comprising differentiating a population of pancreatic progenitor cells, or a population of cells at a later differentiation stage, in a medium containing a CDK8 / 19 inhibitor. [2] The method according to [1], wherein the medium is substantially free of ALK5 inhibitory activity. [3] IC of the CDK8 / 19 inhibitor against ALK5 50 The method of [1] or [2], wherein the amount of the α-glutamyltransferase is 1 μM or more. [4] CDK8 / 19 inhibitors include diethyl (E)-(4-(3-(5-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)acrylamido)benzyl)phosphonate, 2-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide, 4-((2-(6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl)ethyl)amino)quinazoline-6-carbonitrile, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide ...isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide, 4-(4-iso The method according to any one of [1] to [3], wherein the compound is one or more selected from the group consisting of (oxin-6-yl)-1H-pyrazol-3-yl)benzene-1,3-diol, 3-(2-(imidazo[1,2-b]pyridazin-6-ylthio)ethyl)-4-(naphthalen-1-ylsulfonyl)-3,4-dihydroquinoxalin-2(1H)-one, and (E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(4-(morpholinomethyl)phenyl)acrylamide. [5] Any of the methods [1] to [4], wherein the pancreatic progenitor cell population or a cell population at a later differentiation stage is produced by inducing differentiation of pluripotent stem cells. [6] A differentiation medium for a pancreatic progenitor cell population or a cell population undergoing further differentiation, comprising a CDK8 / 19 inhibitor. [7] The medium according to [6], which is substantially free of ALK5 inhibitory activity. [8] IC of CDK8 / 19 inhibitors against ALK5 50 The medium according to [6] or [7], wherein the amount of α-glucan is 1 μM or more. [9] CDK8 / 19 inhibitors include diethyl (E)-(4-(3-(5-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)acrylamido)benzyl)phosphonate, 2-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide, 4-((2-(6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl)ethyl)amino)quinazoline-6-carbonitrile, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide ...4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide, 4-(4-(2,3-dihydrobenzo[b][1,4]diphenyl)benzyl)phosphonate, 4-(4-(4-(isoquinolin-4-yl The medium of any of [6] to [8], wherein the compound is one or more selected from the group consisting of (E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(4-(morpholinomethyl)phenyl)acrylamide, 3-(2-(imidazo[1,2-b]pyridazin-6-ylthio)ethyl)-4-(naphthalen-1-ylsulfonyl)-3,4-dihydroquinoxalin-2(1H)-one, and (E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(4-(morpholinomethyl)phenyl)acrylamide. This specification includes the contents disclosed in the specification and / or drawings of Japanese Patent Application No. 2020-193454, filed on November 20, 2020, which is the priority basis of this application. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Effects of the Invention]

[0008] The present invention provides a novel method for inducing differentiation of an insulin-positive cell population from pluripotent stem cells. That is, the present invention makes it possible to efficiently induce differentiation of insulin-positive cells, particularly insulin-positive and NKX6.1-positive cells, in the process of inducing an insulin-positive cell population from pluripotent stem cells, and to obtain an insulin-positive cell population containing a larger number of such cells than with conventional methods. [Brief explanation of the drawings]

[0009] [Figure 1]Figure 1 is a graph showing the percentage of insulin-positive and NKX6.1-positive (indicated as "INS+ / NKX+") cells and the percentage of insulin-positive and NKX6.1-negative (indicated as "INS+ / NKX-") cells in an insulin-positive cell population obtained by treating a pancreatic progenitor cell population with a differentiation-inducing medium containing ALK5 inhibitor II or a CDK8 / 19 inhibitor (compound 1, compound 2, or compound 3). [Figure 2] Figure 2 shows the results of flow cytometric expression analysis of insulin and NKX6.1 in insulin-positive cell populations obtained by treating pancreatic progenitor cell populations with differentiation-inducing medium containing a predetermined concentration of ALK5 inhibitor II or a CDK8 / 19 inhibitor (compound 1, compound 2, or compound 3). DETAILED DESCRIPTION OF THE INVENTION

[0010] 1. Terminology The terms used in this specification will be explained below.

[0011] As used herein, "about" or "approximately" refers to a value that varies by plus or minus 25%, 20%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%, respectively, from the reference value. Preferably, the term "about" or "approximately" refers to a range of plus or minus 15%, 10%, 5%, or 1%, respectively, from the reference value.

[0012] As used herein, "comprise(s)" or "comprising" means the inclusion, but not limitation, of the elements that follow the phrase. Thus, it implies the inclusion of the elements that follow the phrase, but not the exclusion of any other elements.

[0013] As used herein, the phrase "consist(s) of" or "consisting of" means inclusive of and limited to all elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, with other elements being substantially absent.

[0014] As used herein, "feeder cell-free" means that feeder cells are not essentially contained, and that a medium preconditioned by culturing feeder cells is not used. Therefore, the medium does not contain substances such as growth factors and cytokines secreted by feeder cells.

[0015] The term "feeder cells" or "feeders" refers to cells that are co-cultured with other types of cells and provide an environment in which the cells can grow and support them. Feeder cells may be derived from the same or a different species as the cells they support. For example, human skin fibroblasts or human embryonic stem cells may be used as feeders for human cells, as well as primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts. Feeder cells can be inactivated by irradiation or mitomycin C treatment, for example.

[0016] As used herein, "adherent" refers to cells that are attached to a container, e.g., cells that are attached to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of an appropriate culture medium. Some cells cannot be maintained or grown in culture unless they are attached to a cell culture container. In contrast, non-adherent cells can be maintained and grown in culture without being attached to a container.

[0017] As used herein, "culture" refers to maintaining, growing, and / or differentiating cells in an in vitro environment. "Culturing" refers to maintaining, growing, and / or differentiating cells in a tissue or outside the body, for example, in a cell culture dish or flask. Culture includes two-dimensional culture (plate culture) and three-dimensional culture (suspension culture).

[0018] As used herein, "enrich" and "enrichment" refer to increasing the amount of a particular component in a composition, such as a composition of cells, and "enriched," when used to describe a composition of cells, e.g., a cell population, refers to a cell population in which the amount of a particular component is increased compared to the proportion of such component in the cell population prior to enrichment. For example, a composition, such as a cell population, can be enriched for a target cell type, thus increasing the proportion of the target cell type compared to the proportion of target cells present in the cell population prior to enrichment. Cell populations can also be enriched for a target cell type by cell selection and sorting methods known in the art. Cell populations can also be enriched by certain sorting or selection processes described herein. In certain embodiments of the present invention, a method of enriching a target cell population results in a cell population that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% enriched for the target cell population.

[0019] As used herein, "deplete" and "depletion" refer to reducing the amount of a particular component in a composition, such as a composition of cells, and "depleted," when used to describe a composition of cells, e.g., a cell population, refers to a cell population in which the amount of a particular component is reduced compared to the proportion of such component in the cell population prior to depletion. For example, a composition, such as a cell population, can be depleted of a target cell type, such that the proportion of the target cell type is reduced compared to the proportion of target cells present in the cell population prior to depletion. Cell populations can also be depleted of a target cell type by cell selection and sorting methods known in the art. Cell populations can also be depleted by certain sorting or selection processes described herein. In certain embodiments of the present invention, a method of depleting a target cell population results in a cell population that is at least 50%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% reduced (depleted) in the target cell population.

[0020] As used herein, "purify" and "purification" refer to the removal of impurities from a composition, such as a composition of cells, to render the composition pure for a particular component. "Purified," when used to describe a composition of cells, e.g., a cell population, refers to a cell population in which the amount of impurities is reduced compared to the proportion of such component in the cell population prior to purification, thereby increasing the purity of the particular component. For example, a composition, such as a cell population, can be purified with respect to a target cell type, thus increasing the proportion of the target cell type compared to the proportion of target cells present in the cell population prior to purification. Cell populations can also be purified for a target cell type by cell selection and sorting methods known in the art. Cell populations can also be purified by specific sorting or selection processes described herein. In certain embodiments of the invention, the method of purifying a target cell population can result in a target cell population that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% pure, or with no detectable impurities (including contaminating cells).

[0021] As used herein, the term "marker" refers to a cellular antigen or its gene that is specifically expressed by a given cell type, such as a "marker protein" or a "marker gene." Preferably, the marker is a cell surface marker, which allows for enrichment, isolation, and / or detection of viable cells. The marker may be a positive selection marker or a negative selection marker.

[0022] Marker proteins can be detected by immunological assays using antibodies specific to the marker proteins, such as ELISA, immunostaining, and flow cytometry. Marker genes can be detected by nucleic acid amplification methods and / or nucleic acid detection methods known in the art, such as RT-PCR, microarrays, and biochips. As used herein, a marker protein being "positive" means that it is detected as positive by flow cytometry, and "negative" means that it is below the detection limit by flow cytometry. As used herein, a marker gene being "positive" means that it is detected by RT-PCR, and "negative" means that it is below the detection limit by RT-PCR.

[0023] As used herein, "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter within a cell.

[0024] As used herein, "factors having CDK8 / 19 inhibitory activity" or "CDK8 / 19 inhibitors" refer to any substance that has CDK8 / 19 inhibitory activity. In contrast to other proteins in the same CDK family, CDK8 is not required for cell proliferation, and CDK8 inhibition does not have a significant effect under normal conditions. CDK19 and CDK8 are similar, and CDK8 inhibition is usually accompanied by CDK19 inhibition.

[0025] As used herein, a "growth factor" refers to an endogenous protein that promotes 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, etc.), and other cytokines that have effects on stem cells, such as stem cell factor (SCF) and erythropoietin (Epo).

[0026] As used herein, 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. The ROCK inhibitor is not particularly limited as long as it has the above-mentioned function, and examples thereof include N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (sometimes referred to as Y-27632 in the present specification), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl]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]pyridin-4-yl)-4PER(R)-1-aminoethyl]cyclohexane-1α-carboxamide (Y-30141), N-(3-{[2-(4-amino-1,2,5-oxadiazole-3 Examples of ROCK inhibitors include N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A), N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A). ROCK inhibitors are not limited to these, and antisense oligonucleotides and siRNAs against ROCK mRNA, antibodies that bind to ROCK, dominant-negative ROCK mutants, and the like can also be used as ROCK inhibitors. These are commercially available or can be synthesized according to known methods.

[0027] As used herein, the term "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, and is involved in many signal pathways related to glycogen production, apoptosis, stem cell maintenance, and the like. 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 be a substance that has both GSK3β inhibitory activity and GSK3α inhibitory activity.

[0028] 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)pyrimidine), (2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), TWS-119 (3-[6-(3-aminophenyl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), Examples include 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (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, CT99021, CT20026, BIO, BIO-acetoxime, pyridocarbazole-cyclopentadienyl ruthenium complex, OTDZT, alpha-4-dibromoacetophenone, and lithium. GSK3β is not limited to these, and antisense oligonucleotides and siRNAs against GSK3β mRNA, antibodies that bind to GSK3β, dominant-negative GSK3β mutants, etc. can also be used as GSK3β inhibitors, and are commercially available or can be synthesized according to known methods.

[0029] As used herein, the term "serum substitute" refers to, for example, KnockOut TMExamples of serum replacements include Serum Replacement (KSR: Thermo Fisher Scientific), StemSure® Serum Replacement (Wako), B-27 supplement, N2-supplement, albumin (e.g., lipid-rich albumin), insulin, transferrin, fatty acids, collagen precursors, trace elements (e.g., zinc, selenium (e.g., sodium selenite)), 2-mercaptoethanol, 3'-thiolglycerol, and mixtures thereof (e.g., ITS-G). Preferred serum replacements include B-27 supplement, KSR, StemSure® Serum Replacement, and ITS-G. When serum replacements are added to the medium, their concentration in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight. In the present invention, it is preferable to use a "serum replacement" instead of serum.

[0030] 2. Method for producing insulin-positive cell population The present invention provides a method for producing an insulin-positive cell population, which comprises differentiating a pancreatic progenitor cell population, or a cell population at a later differentiation stage, in the presence of a CDK8 / 19 inhibitor.

[0031] CDK8 / 19 inhibitors act on pancreatic progenitor cell populations or cell populations at a later differentiation stage to produce insulin-positive cell populations enriched in insulin-positive cells, particularly insulin-positive and NKX6.1-positive cells.

[0032] The "CDK8 / 19 inhibitor" used in the present invention is not particularly limited as long as it has inhibitory activity against CDK8 / 19, and any factor that directly or indirectly inhibits the function of CDK8 / 19 can be used. Preferably, in the present invention, the "CDK8 / 19 inhibitor" refers to a factor that inhibits CDK8 / 19 by 50% or more. The method for determining the presence or absence of CDK8 / 19 inhibitory activity can be selected from known methods, such as the method described in Example 1 of the present specification in detail below.

[0033] In the present invention, known "CDK8 / 19 inhibitors" can also be used, and such CDK8 / 19 inhibitors can be found in patent literature or non-patent literature. For example, among the compounds described in US2012 / 0071477, WO2015 / 159937, WO2015 / 159938, WO2013 / 116786, WO2014 / 0038958, WO2014 / 134169, JP2015 / 506376, US2015 / 0274726, US2016 / 0000787, WO2016 / 009076, WO2016 / 0016951, WO2016 / 018511, WO2016 / 100782, and WO2016 / 182904, compounds (or salts thereof) having CDK8 / 19 inhibitory activity can be used as the "CDK8 / 19 inhibitor" in the present invention. In particular, among the above compounds, compounds (or salts thereof) that have selective inhibitory activity against CDK8 / 19 can be suitably used.

[0034] When a "CDK8 / 19 inhibitor" has inhibitory activity against ALK5 (activin receptor-like kinase 5), the concentration of the CDK8 / 19 inhibitor required to show 50% inhibition of ALK5 (IC 50 Preferably, the CDK8 / 19 inhibitor has a concentration (value) of 1 μM or more. By using such a CDK8 / 19 inhibitor, it becomes possible to make a medium supplemented with the CDK8 / 19 inhibitor substantially free of ALK5 inhibitory activity. "Substantially free of ALK5 inhibitory activity" not only means that the medium has absolutely no ALK5 inhibitory activity, but also includes cases in which the ALK5 inhibition rate is less than 50%, preferably 40% or less, more preferably 30% or less, even more preferably 20% or less, still more preferably 10% or less, and particularly preferably 5% or less.

[0035] More specifically, CDK8 / 19 inhibitors that can be used in the present invention include, but are not limited to, the following compounds or salts thereof: These compounds may have one or more substituents, or a partial structure (such as a substituent or ring) may be modified as long as they have CDK8 / 19 inhibitory activity.

[0036] [Table 1]

[0037] Furthermore, CDK8 / 19 inhibitors that can be used in the present invention are not particularly limited, but for example, compounds 7 to 13 shown below or salts thereof can be used. Compounds 7 to 11 are preferably in the free form, and compounds 12 and 13 are preferably trifluoroacetate salts.

[0038] [Table 2]

[0039] The CDK8 / 19 inhibitors of the present invention are not limited to the compounds listed above, and antisense oligonucleotides and siRNAs against CDK8 / 19 mRNA, antibodies that bind to CDK8 / 19, dominant-negative CDK8 / 19 mutants, etc. can also be used as CDK8 / 19 inhibitors.

[0040] The above CDK8 / 19 inhibitors are commercially available or can be synthesized according to known methods.

[0041] In the present invention, an "insulin-positive cell population" refers to a cell population containing insulin-positive cells obtained by inducing differentiation from pluripotent stem cells. "Insulin-positive cells" refer to cells characterized by the expression of an insulin marker. "Insulin-positive cells" may express the NK6 homeobox 1 (NKX6.1) marker, and are preferably cells expressing both the insulin and NKX6.1 markers (i.e., insulin-positive and NKX6.1-positive cells).

[0042] The "insulin-positive cell population" of the present invention is a cell population that is enriched in insulin-positive cells, particularly insulin-positive and NKX6.1-positive cells, compared to an insulin-positive cell population obtained by inducing differentiation from pluripotent stem cells according to a conventionally known method (i.e., a method comprising a step of culturing a pancreatic progenitor cell population or a cell population at a later differentiation stage in the presence of an ALK5 inhibitor (e.g., ALK5 inhibitor II) (Nature Biotechnology 2014;32:1121-1133, etc.)). The content of insulin-positive and NKX6.1-positive cells in the insulin-positive cell population of the present invention is 33% or more, preferably 34% or more, more preferably 35% or more, even more preferably 36% or more, and particularly preferably 37% or more. The upper limit of this content is not particularly limited, but is preferably 70% or less, 60% or less, or 50% or less. The content can be expressed using two numerical values ​​selected from the upper and lower limit values, respectively, and for example, the content is 33% to 50%, preferably 34% to 50%, more preferably 35% to 50%, even more preferably 36% to 50%, and particularly preferably 37% to 50%.

[0043] The "insulin-positive cell population" of the present invention can be obtained by treating a pancreatic progenitor cell population obtained by inducing differentiation from pluripotent stem cells, or a cell population at a later differentiation stage, with a CDK8 / 19 inhibitor. Treating a pancreatic progenitor cell population, or a cell population at a specific differentiation stage, with a CDK8 / 19 inhibitor, can yield an insulin-positive cell population enriched in insulin-positive cells, preferably insulin-positive and NKX6.1-positive cells. In addition to insulin-positive cells, the insulin-positive cell population may also contain other cells (e.g., endocrine precursor cells; other pancreatic hormone-producing cells expressing at least one marker of glucagon, somatostatin, and pancreatic polypeptide; Ki67-positive cells; CHGA-negative cells, etc.).

[0044] In the present invention, "a pancreatic precursor cell population, or a cell population at a later differentiation stage" means a pancreatic precursor cell population, an endocrine precursor cell population, or both a pancreatic precursor cell population and an endocrine precursor cell population.

[0045] As used herein, "pluripotency" refers to the ability to differentiate into tissues and cells with a variety of different morphologies and functions, and to differentiate into cells of any lineage of the three germ layers. "Pluripotency" cannot differentiate into the germ disc and therefore does not have the ability to form an individual, and is therefore distinct from "totipotency," which can differentiate into any tissue of the body, including the germ disc.

[0046] As used herein, "multipotency" refers to the ability to differentiate into cells of multiple, limited number of lineages. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent but not pluripotent.

[0047] As used herein, the term "pluripotent stem cells" refers to embryonic stem cells (ES cells) and cells that have the same pluripotency, i.e., the potential to differentiate into various tissues in the body (all of the endoderm, mesoderm, and ectoderm). Cells that have the same pluripotency as ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in this specification). Preferably, in the present invention, the pluripotent stem cells are human pluripotent stem cells.

[0048] As for "ES cells," various mouse ES cell lines established by inGenious, Inc., RIKEN, and the like can be used, and various human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis, Inc. can be used. For example, ES cell lines that can be used include 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.

[0049] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells," including iPS cells established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPS cells established by selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPS cells created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106)), and virus-free iPS cells established by introducing six factors (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318:1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451:141-146), and induced pluripotent stem cells created by Sakurada et al. (JP Patent Publication No. 2008-307007) can also be used.

[0050] 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., Schooler HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No. Any of the induced pluripotent stem cells known in the art and described in the Japanese Patent Laid-Open Nos. 2008-307007, 2008-283972, US2008 / 2336610, US2009 / 047263, WO2007 / 069666, WO2008 / 118220, WO2008 / 124133, WO2008 / 151058, WO2009 / 006930, WO2009 / 006997, and WO2009 / 007852 can be used.

[0051] As induced pluripotent cell lines, various iPS cell lines established by the NIH, the RIKEN Institute, Kyoto University, and others are available. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain; Kyoto University's Ff-WJ-18 strain, Ff-I01s01 strain, Ff-I01s02 strain, Ff-I01s04 strain, Ff-I01s06 strain, Ff-I14s03 strain, Ff-I14s04 strain, QHJI01s01 strain, QHJI01s04 strain, QHJI14s03 strain, QHJI14s04 strain, 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, and 648A1 strain; and CDI's MyCell iPS Cells (21525.102.10A) strain, MyCell iPS Cells (21526.101.10A) strain, etc.

[0052] As used herein, the term "pancreatic progenitor cell population" refers to a cell population containing pancreatic progenitor cells. As used herein, pancreatic progenitor cells are characterized by the expression of the marker NKX6.1 (i.e., they are NKX6.1-positive). Pancreatic progenitor cells may further express at least one of the markers PDX-1, PTF-1α, GATA4, and SOX9. Preferably, pancreatic progenitor cells are characterized by the expression of NKX6.1 and PDX-1 (i.e., they are NKX6.1-positive and PDX-1-positive).

[0053] In one embodiment, the "pancreatic progenitor cell population" of the present invention is a cell population corresponding to the culture after step 4) or the culture in step 5) in the process of inducing the differentiation of pluripotent stem cells into pancreatic β cells, as described in detail below.

[0054] The "pancreatic progenitor cell population" of the present invention contains pancreatic progenitor cells at a ratio of 30% or more, preferably 40% or more, more preferably 50% or more, even more 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 precursor cells, insulin-positive cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0055] The proportion of specific cells in the cell population described herein can be determined based on known techniques that can calculate the number of cells, such as flow cytometry.

[0056] As used herein, the term "endocrine precursor cell population" refers to a cell population containing endocrine precursor cells. As used herein, endocrine precursor cells refer to cells characterized by the expression of at least one marker, CHGA, NeuroD, and NGN3, and the absence of a marker of the pancreatic hormone system (e.g., insulin). Endocrine precursor cells may express markers such as PAX-4, NKX2.2, Islet-1, and PDX-1.

[0057] In one embodiment, the "endocrine precursor cell population" of the present invention is a cell population corresponding to the culture after step 5) or the culture in step 6) in the process of inducing differentiation of pluripotent stem cells into pancreatic beta cells, as described in detail below.

[0058] The "endocrine precursor cell population" of the present invention contains endocrine precursor cells at a rate of 30% or more, preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, and even more preferably 70% or more. In addition to endocrine precursor cells, the endocrine precursor cell population may also contain other cells (e.g., pancreatic precursor cells, insulin-positive cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0059] It is known that during the differentiation process from pluripotent stem cells to insulin-positive cells, cells with different characteristics emerge depending on the differentiation stage (WO2009 / 012428, WO2016 / 021734). For example, this differentiation stage can be broadly classified into pluripotent stem cells, definitive endoderm cells, primitive gut cells, posterior foregut cells, pancreatic progenitor cells, endocrine progenitor cells, and insulin-positive cells, in order of relative immaturity.

[0060] Pancreatic progenitor cell populations or cell populations at later differentiation stages can be obtained using known techniques for inducing differentiation of pluripotent stem cells into insulin-positive cells. That is, cell populations at each desired differentiation stage can be obtained using the following differentiation induction steps: Step 1) inducing differentiation of pluripotent stem cells into definitive endoderm cells; Step 2) inducing differentiation of definitive endoderm cells into primitive gut cells; Step 3) Induce differentiation of primitive gut cells into posterior foregut cells; Step 4) Induce differentiation of posterior foregut cells into pancreatic progenitor cells; Step 5) inducing differentiation of pancreatic progenitor cells into endocrine progenitor cells; Step 6) Inducing differentiation of endocrine precursor cells into insulin-positive cells. Each step will be explained below, but the differentiation induction into each cell type is not limited to these methods.

[0061] Step 1) Differentiation into definitive endoderm cells Pluripotent stem cells are first differentiated into definitive endoderm cells. Methods for inducing definitive endoderm from pluripotent stem cells are already known, and any of these methods may be used. Preferably, pluripotent stem cells are cultured in a medium containing activin A, more preferably in a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, to differentiate into definitive endoderm cells. The number of cells at the start of culture is not particularly limited, and may be 22,000 to 150,000 cells / cm. 2 , preferably 22,000 to 100,000 cells / cm 2 , more preferably 22,000 to 80,000 cells / cm 2 The culture period is 1 to 4 days, preferably 1 to 3 days, and particularly preferably 3 days.

[0062] The culture temperature is not particularly limited, but is typically 30 to 40°C (for example, 37°C). The carbon dioxide concentration in the culture vessel is, for example, about 5%. Culture may be performed in either two-dimensional or three-dimensional culture.

[0063] The media used in this process include RPMI 1640 medium, MEM medium, iMEM medium, DMEM / F12 medium, Improved MEM Zinc Option medium, Improved MEM / 1% B-27 / Penisilin Streptomycin medium, and MCDB131 / 20mM Glucose / NaHCO3 / FAF-BSA / ITS-X / GlutaMAX. TM A basal medium used for culturing mammalian cells, such as a medium containing ascorbic acid and penisilin / streptomycin, can be used.

[0064] The concentration of activin A in the medium is usually 30 to 200 ng / mL, preferably 50 to 150 ng / mL, more preferably 70 to 120 ng / mL, and particularly preferably about 100 ng / mL. In another embodiment, activin A can be contained in the medium at a low dose, for example, at an amount of 5 to 100 ng / mL, preferably 5 to 50 ng / mL, and more preferably 5 to 10 ng / mL. In yet another embodiment, the concentration of activin A in the medium is about 0.1 to 100 ng / mL, preferably about 1 to 50 ng / mL, and more preferably about 3 to 10 ng / mL.

[0065] The concentration of the GSK3β inhibitor in the medium is set appropriately depending on the type of GSK3β inhibitor used. For example, when CHIR99021 is used as the GSK3β inhibitor, the concentration is usually 2 to 5 μM, preferably 2 to 4 μM, and particularly preferably about 3 μM.

[0066] The concentration of the ROCK inhibitor in the medium is appropriately set depending on the type of ROCK inhibitor used. For example, when Y27632 is used as the ROCK inhibitor, the concentration is usually 5 to 20 μM, preferably 5 to 15 μM, and particularly preferably about 10 μM.

[0067] Insulin can also be added to the medium. Insulin can be contained in the medium at a concentration of 0.01 to 20 μM, preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM. The concentration of insulin in the medium may be, but is not limited to, the concentration of insulin contained in the added B-27 supplement.

[0068] In a specific embodiment, after culturing for one day in a medium containing activin A, a ROCK inhibitor, and a GSK3β inhibitor, the cells are further cultured for two days in a medium containing only activin A, with the medium being changed every day. Alternatively, pluripotent stem cells can be produced by first culturing in a medium containing 0.01 to 20 μM insulin in the presence of a low dose of activin A, followed by second culturing in a medium without insulin.

[0069] Step 2) Differentiation into primitive gut cells The definitive endoderm cells obtained in step 1) are further cultured in a medium containing growth factors to induce differentiation into primitive gut cells. The culture period is 2 to 8 days, preferably about 4 days.

[0070] The culture temperature is not particularly limited, but is typically 30 to 40°C (for example, 37°C). The carbon dioxide concentration in the culture vessel is, for example, about 5%. Culture may be performed in either two-dimensional or three-dimensional culture.

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

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

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

[0074] Step 3) Differentiation into posterior foregut cells The primitive gut cells obtained in step 2) are further cultured in a medium containing growth factors, cyclopamine, noggin, etc., to induce differentiation into posterior foregut cells. The culture period is 1 to 5 days, preferably about 2 days. Culture may be performed in either two-dimensional or three-dimensional culture.

[0075] The culture temperature is not particularly limited, but is typically 30 to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0076] The medium may be a basal medium used for culturing mammalian cells, as in step 1. In addition to growth factors, serum substitutes, vitamins, antibiotics, etc. may be added to the medium as appropriate.

[0077] As the growth factor, EGF, KGF, and FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is even more preferred.

[0078] The concentration of growth factors in the medium is determined appropriately depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / mL (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / mL (i.e., about 0.8 to 160 nM), and more preferably about 10 to 1000 ng / mL (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / mL (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / mL (i.e., about 0.6 to 58 nM). For example, when KGF is used as a growth factor, the concentration is usually 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL.

[0079] The concentration of cyclopamine in the medium is not particularly limited, but is generally 0.5 to 1.5 μM, preferably 0.3 to 1.0 μM, and particularly preferably about 0.5 μM.

[0080] The concentration of Noggin in the medium is not particularly limited, but is generally 10 to 200 ng / mL, preferably 50 to 150 ng / mL, and particularly preferably about 100 ng / mL.

[0081] Step 4) Differentiation into pancreatic progenitor cells The posterior foregut cells obtained in step 3) are further cultured in a medium containing a factor having CDK8 / 19 inhibitory activity, preferably a medium containing a factor having CDK8 / 19 inhibitory activity and a growth factor, to induce differentiation into pancreatic progenitor cells. The culture period is 2 to 10 days, preferably about 5 days. Culture may be performed in either two-dimensional or three-dimensional culture.

[0082] In the case of two-dimensional culture, as previously reported (Toyoda et al., Stem cell Research (2015) 14, 185-197), the posterior foregut cells obtained in step 3) are treated with 0.25% trypsin-EDTA solution and dispersed in the solution by pipetting to obtain a cell dispersion, which is then centrifuged, and the recovered cells are resuspended in a small amount of new medium, and the cell suspension is replated in new medium in step 4).

[0083] The medium may be a basal medium used for culturing mammalian cells, as in step 1. In addition to growth factors, serum substitutes, vitamins, antibiotics, etc. may be added to the medium as appropriate.

[0084] The factor having CDK8 / 19 inhibitory activity can be any of the various compounds or salts thereof described above, and the amount added to the medium is determined appropriately depending on the compound or salt thereof used, but is usually 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 medium is preferably a concentration that achieves 50% or more inhibitory activity against CDK8 / 19.

[0085] As the growth factor, EGF, KGF, and FGF10 are preferred, KGF and / or EGF are more preferred, and KGF and EGF are even more preferred.

[0086] The concentration of growth factor in the medium is set appropriately depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / mL (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / mL (i.e., about 0.8 to 160 nM), more preferably about 10 to 1000 ng / mL (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / mL (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / mL (i.e., about 0.6 to 58 nM). For example, when KGF and EGF are used as growth factors, the concentrations of EGF are typically 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL, and KGF are typically 10 to 200 ng / mL, preferably 50 to 150 ng / mL, and particularly preferably about 100 ng / mL.

[0087] In step 4), the first day of culture may be performed in the presence of a ROCK inhibitor, and thereafter culture may be performed in a medium that does not contain a ROCK inhibitor.

[0088] The 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 about 0.1 to 100 ng / mL, preferably about 1 to 50 ng / mL, and more preferably about 3 to 10 ng / mL.

[0089] Furthermore, dimethyl sulfoxide and / or activin (1 to 50 ng / mL) may be added to the medium.

[0090] In any of the steps, in addition to the above-mentioned components, serum substitutes (e.g., B-27 supplement, ITS-G) may be added to the medium. Furthermore, if necessary, amino acids, L-glutamine, GlutaMAX (product name), non-essential amino acids, vitamins, nicotinamide, antibiotics (e.g., antibiotic-antimycotic, penicillin, streptomycin, or a mixture thereof), antibacterial agents (e.g., amphotericin B), antioxidants, pyruvic acid, buffers, inorganic salts, and the like may also be added. When antibiotics are added to the medium, the concentration in the medium is usually 0.01 to 20% by weight, preferably 0.1 to 10% by weight. Culture may be performed in either two-dimensional or three-dimensional culture.

[0091] In the case of two-dimensional cell culture, adherent culture is performed without the use of feeder cells. Culture vessels such as dishes, flasks, microplates, and cell culture sheets such as OptiCell (product name) (Nunc) are used during culture. The culture vessels are preferably surface-treated to improve cell adhesion (hydrophilicity) or coated with a cell adhesion substrate such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel (e.g., BD Matrigel (Becton Dickinson Japan)), or vitronectin. Culture vessels coated with type I collagen, Matrigel, fibronectin, vitronectin, or poly-D-lysine are preferred, with Matrigel or poly-D-lysine being more preferred.

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

[0093] The pancreatic progenitor cells obtained in step 4) can be further purified using a known surface marker such as glycoprotein 2 (GP2). The purification can be carried out by a known method, for example, using beads onto which anti-GP2 antibodies are immobilized.

[0094] Step 5) Differentiation into endocrine precursor cells The pancreatic progenitor cells obtained in step 4) are further cultured in a medium containing growth factors to induce differentiation into endocrine precursor cells. Culture may be performed in either two-dimensional or three-dimensional culture. In the case of two-dimensional culture, the pancreatic progenitor cells obtained in step 4) are treated with a 0.25% trypsin-EDTA solution and dispersed in the solution by pipetting to obtain a cell dispersion. The resulting dispersion is centrifuged, and the recovered cells are resuspended in a small amount of fresh medium. The cell suspension is then replated in fresh medium in step 5). The culture period is 2 to 3 days, preferably about 2 days.

[0095] As in step 1), the medium may be a basal medium used for culturing mammalian cells. According to a previous report (Nature Biotechnology 2014;32:1121-1133), SANT1, retinoic acid, ALK5 inhibitor II, T3, and LDN may be added to the medium, and further, Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum substitutes, vitamins, antibiotics, and the like may be added as appropriate. In the present invention, when a CDK8 / 19 inhibitor is used in step 5), an ALK5 inhibitor (such as ALK5 inhibitor II) need not be used, and preferably, an ALK5 inhibitor (such as ALK5 inhibitor II) is not used.

[0096] The culture is performed in a non-adherent culture without using feeder cells. During the culture, dishes, flasks, microplates, multi-hole plates (Nunc), or bioreactors are used. It is preferable that the culture vessels are surface-treated to reduce cell adhesion.

[0097] The culture temperature is not particularly limited, but is typically 30 to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0098] The endocrine precursor cells obtained in step 5) can be further purified using a known surface marker such as glycoprotein 2 (GP2). The purification can be carried out by a known method, for example, using beads on which anti-GP2 antibodies are immobilized.

[0099] Step 6) Differentiation into insulin-positive cells The endocrine precursor cells obtained in step 5) are further cultured in a medium containing growth factors to induce differentiation into insulin-positive cells. The culture period is 10 to 30 days, preferably about 10 to 20 days.

[0100] As in step 1), the medium may be a basal medium used for culturing mammalian cells. According to a previous report (Nature Biotechnology 2014;32:1121-1133), the medium may be supplemented with ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor XX, γ-secretase inhibitor RO, N-cysteine, AXL inhibitor, and ascorbic acid. Furthermore, Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum substitutes, vitamins, antibiotics, and the like may be added as appropriate. For example, the medium may be supplemented with ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, and ascorbic acid, or with T3, ALK5 inhibitor II, ZnSO, heparin, N-acetylcysteine, Trolox, and R428. In the present invention, when a CDK8 / 19 inhibitor is used in step 6), an ALK5 inhibitor (such as ALK5 inhibitor II) does not have to be used, and preferably an ALK5 inhibitor (such as ALK5 inhibitor II) is not used.

[0101] Culture may be performed in either two-dimensional or three-dimensional culture. No feeder cells are used for culture. Three-dimensional culture is performed in a non-adherent manner. Dishes, flasks, microplates, multi-hole plates (Nunc), or bioreactors are used for culture. It is preferable that the culture vessels are surface-treated to reduce cell adhesion.

[0102] The culture temperature is not particularly limited, but is typically 30 to 40° C. (for example, 37° C.) The carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0103] Treatment of a pancreatic progenitor cell population obtained by inducing differentiation from pluripotent stem cells, or a cell population at a later differentiation stage, with a CDK8 / 19 inhibitor can be carried out by contacting the cell population with the CDK8 / 19 inhibitor. For example, treatment can be carried out by culturing the cell population in a medium containing the CDK8 / 19 inhibitor. The CDK8 / 19 inhibitor can be added to the medium in any amount that is capable of inhibiting CDK8 / 19 activity, for example, 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, or 1 μM or less. In particular, the CDK8 / 19 inhibitor has inhibitory activity against ALK5, and is present at a concentration required to exhibit a 50% inhibition rate against ALK5 (IC 50 When the concentration (value) is 1 μM or more, it can be contained in an amount less than 1 μM. The lower limit of the amount of CDK8 / 19 inhibitor to be added is not particularly limited, but can be 0.1 μM or more, 0.2 μM or more, 0.3 μM or more, 0.4 μM or more, or 0.5 μM or more. For example, the amount of CDK8 / 19 inhibitor to be added is 10 μM or less and 0.1 μM or more, preferably 5 μM or less and 0.1 μM or more, more preferably 1 μM or less and 0.1 μM or more, for example, less than 1 μM and 0.1 μM or more.

[0104] Furthermore, a pancreatic progenitor cell population obtained by inducing differentiation from pluripotent stem cells, or a cell population at a later differentiation stage, can be cultured in the presence of a CDK8 / 19 inhibitor 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. Culture in the presence of a CDK8 / 19 inhibitor is preferably carried out for 4 days or more. The medium can be replaced during treatment with a CDK8 / 19 inhibitor, and can be replaced with a medium having the same composition as that before the addition of the CDK8 / 19 inhibitor, or with a medium having a different composition, according to the culture schedule.

[0105] A pancreatic progenitor cell population obtained by inducing differentiation from pluripotent stem cells, or a cell population at a later differentiation stage, can be treated with a CDK8 / 19 inhibitor and then further differentiated into a target cell population. Here, "while treating with a CDK8 / 19 inhibitor" includes cases where the treatment with a CDK8 / 19 inhibitor and the differentiation step are performed simultaneously, cases where treatment with a CDK8 / 19 inhibitor is followed by a differentiation step, and cases where the differentiation step is followed by a treatment with a CDK8 / 19 inhibitor. Therefore, the medium used for treatment with a CDK8 / 19 inhibitor and the medium used for differentiating the cell population may be separate, or a CDK8 / 19 inhibitor may be added to the medium used for the differentiation step.

[0106] In one embodiment of the present invention, in the process of inducing differentiation of pluripotent stem cells into insulin-positive cells, a CDK8 / 19 inhibitor is included in the culture medium for step 5 and thereafter, i.e., the culture medium for step 5, or the culture medium for step 6, or the culture medium for step 5 and the culture medium for step 6, and allowed to act on the cells.

[0107] The insulin-positive cell population obtained by the present invention can be induced to differentiate into a cell population containing pancreatic β cells (hereinafter referred to as a "pancreatic β cell population"). As used herein, "pancreatic β cells" refers to cells that are more mature than "insulin-positive cells," and specifically refers to cells that express at least one of the markers MAFA, UCN3, and IAPP, which are maturation markers of pancreatic β cells, or are characterized by an increased insulin secretion response to glucose stimulation. In addition to pancreatic β cells, the pancreatic β cell population may also contain other cells (e.g., insulin-positive cells, Ki67-positive cells, CHGA-negative cells, etc.).

[0108] The pancreatic β cell population can be obtained by differentiating and maturing an insulin-positive cell population, preferably in vivo in an animal.

[0109] 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, with humans being preferred.

[0110] The transplantation is preferably carried out in a region of the body where the cell population can be fixed at a certain position, and can be carried out, for example, subcutaneously, intraperitoneally, into the peritoneal epithelium, omentum, adipose tissue, muscle tissue, or under the capsule of each organ such as the pancreas or kidney of an animal. The number of cells to be transplanted can vary depending on factors such as the differentiation stage of the cells to be transplanted, the age, weight, size of the transplant site, and severity of the disease of the recipient, and is not particularly limited. For example, 10 × 10 4 Cells ~10×10 11 The transplanted cell population can be induced to differentiate in an in vivo environment into a target cell population, preferably a pancreatic β cell population, and then may be recovered or may be left in the body as is.

[0111] When transplanting, the cell population may be embedded in a gel containing a biocompatible material and then transplanted. For example, the cell population embedded in a gel containing a biocompatible material can be enclosed in a device such as a capsule, bag, or chamber and then transplanted into a living body.

[0112] As used herein, the term "embedding" refers to containing a population of endocrine precursor cells or a population of cells at a later differentiation stage dispersed in a gel containing a biocompatible material.

[0113] As used herein, the term "biocompatible material" refers to any material that does not induce significant immune responses or adverse biological reactions (e.g., toxic reactions, blood coagulation, etc.) when implanted in a living body and left in place for a short or long period of time. Furthermore, the "biocompatible material" is preferably a biodegradable material. Examples of such materials include polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), polyethylene glycol (PEG), polyhydroxyethyl methacrylate, polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), poly(ethylene-co-vinyl acetate) (PEVA), polyacrylamide, polyethylene oxide, polyethyleneamine, polyhydroxybutyric acid, poly(N-vinylpyrrolidone), polyvinyl alcohol, and polypropylene fumarate. Examples of the polymeric material include cellulose, polyacrylic acid, poly e-caprolactone, polymethacrylic acid, polyvinylidene difluoride (PVDF), pectinic acid, hyaluronic acid, heparin sulfate, chondroitin sulfate, heparan sulfate proteoglycan, heparin, chitin, chitosan, xanthan, carboxymethylcellulose, carboxymethylchitosan, alginate, alginate ester, collagen, cellulose, silk fibroin, keratin, gelatin, fibrin, pullulan, laminin, gellan, silicone, urethane, elastin, and modified forms thereof, as well as combinations thereof. The surface of the "biocompatible material" may be optionally modified to allow cell adhesion (for example, coated with a cell adhesion substrate (collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel, vitronectin, etc.)), or modified with a functional group known to regulate cell proliferation, differentiation, or function (for example, amino group, carboxyl group, hydroxyl group, methacrylic acid group, acrylic acid group, etc.). In a specific embodiment, alginate or alginate ester can be suitably used as the "biocompatible material".

[0114] The alginate may be any water-soluble salt, and metal salts, ammonium salts, etc. can be used. For example, sodium alginate, calcium alginate, ammonium alginate, etc. can be preferably used.

[0115] The alginate ester (also referred to as propylene glycol alginate) is a derivative in which propylene glycol is ester-bonded to the carboxyl group of alginic acid.

[0116] The ratio of mannuronic acid to guluronic acid (M / G ratio) contained in the alginate is arbitrary. Generally, when M > G, a gel rich in flexibility can be formed, and when M < G, a strong gel can be formed. In the present invention, those containing guluronic acid at a ratio of 10 - 90%, 20 - 80%, 30 - 70%, or 40 - 60% can be used.

[0117] The production of the gel using the alginate or alginate ester can be carried out according to known methods (WO2010 / 032242, WO2011 / 154941), and can be obtained by adding a cross-linking agent to a solution of the alginate or alginate ester to cause gelation.

[0118] The alginate or alginate ester can be included in the solvent in an amount of 0.05 - 10% by weight, preferably 0.1 - 5% by weight, more preferably 0.5 - 3% by weight. The solvent may be any one capable of dissolving the alginate or alginate ester, and water, physiological saline, etc. can be used.

[0119] The crosslinking agent may be any agent capable of gelling an alginate or alginate ester solution, and is not particularly limited thereto. A polyvalent metal cation may be used. Divalent metal cations are preferred as polyvalent metal cations, and calcium ions, strontium ions, and barium ions are more preferred. The crosslinking agent may be used in the form of a salt, and in the present invention, at least one selected from calcium chloride, strontium chloride, and barium chloride may be used as the crosslinking agent.

[0120] Gels containing alginate or alginate esters can contain nanofibers. Nanofibers are natural or synthetic fibers with diameters in the nanometer range. Natural nanofibers include those containing one or more polysaccharides, such as collagen, cellulose, silk fibroin, keratin, gelatin, and chitosan. Synthetic nanofibers include polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), poly(lactide-co-glycolide) (PLGA), poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), and poly(ethylene-co-vinyl acetate) (PEVA). The nanofibers can be present in the alginate-containing gel in an amount of less than 1% by weight, e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, or less. There is no particular lower limit to the amount of nanofibers contained in a gel containing alginate or alginate ester, but it can be 0.05% by weight or more, preferably 0.1% by weight or more.

[0121] Embedding of a cell population into a gel containing alginate or an alginate ester can be carried out by any means, including, but not limited to, for example, mixing the cell population into a solution of alginate or an alginate ester and allowing it to gel.

[0122] The cell population was grown at 1 × 10 in a solution of alginate or alginate ester. 4 cells ~1×10 9cells / mL, preferably 1 x 10 7 cells ~1×10 8 It can be included in an amount selected from cells / mL.

[0123] 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, for example, 0.1 to 1% by weight, based on the weight of the solution. Gelation can be achieved in a container having a specific structure and / or shape used for cell culture or cell transplantation, or in a mold designed to obtain a gel that fits the container.

[0124] Alternatively, gel capsules containing alginic acid may be formed according to a known method (WO2010 / 010902). Specifically, a solution of alginate or alginate ester containing a cell population may be added dropwise to a crosslinker solution. The size of the droplets can be adjusted depending on the nozzle shape and method used for dropping, thereby defining the size of the alginic acid-containing gel capsules. The dropping method is not particularly limited, but can be performed by air spraying, airless spraying, electrostatic spraying, or other methods. The size of the alginic acid-containing gel capsules is not particularly limited, but can have a diameter of 5 mm or less, 1 mm or less, or 500 μm or less. The crosslinker solution can contain a crosslinker in an amount selected from 0.1 to 10 wt %, for example, 0.1 to 5 wt %.

[0125] The insulin-positive cell population obtained by the present invention can be transplanted into an animal body, and when differentiated in the animal body, can be left in place and used as cells that produce and secrete insulin.

[0126] The insulin-positive cell population obtained by the present invention is useful as a cell medicine for treating diabetes, particularly type I diabetes, when transplanted directly or encapsulated into the affected area.

[0127] The insulin-positive cell population obtained by the present invention may also be a prodrug. As used herein, the term "prodrug" refers to a cell population that differentiates into cells with the function of treating a disease after transplantation into a living body.

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

[0129] 3. Differentiation Medium The present invention provides a differentiation medium for a pancreatic progenitor cell population, or a cell population undergoing further differentiation, comprising a CDK8 / 19 inhibitor.

[0130] The differentiation medium of the present invention can be used for inducing differentiation of a pancreatic progenitor cell population or a cell population at a later differentiation stage. The differentiation medium of the present invention can be used in step 5) or step 6) of the above-mentioned method for inducing differentiation of pluripotent stem cells into insulin-positive cells.

[0131] The differentiation media of the present invention include RPMI 1640 medium, MEM medium, iMEM medium, DMEM / F12 medium, Improved MEM Zinc Option medium, Improved MEM / 1% B-27 / Penisilin Streptomycin medium, and MCDB131 / 20 mM Glucose / NaHCO3 / FAF-BSA / ITS-X / GlutaMAX. TM The CDK8 / 19 inhibitor is contained in a basal medium used for culturing mammalian cells, such as a medium containing ascorbic acid / penisilin / streptomycin, in any amount capable of inhibiting CDK8 / 19 activity. The amount of the CDK8 / 19 inhibitor contained in the medium is as defined above.

[0132] In addition to the CDK8 / 19 inhibitor, the differentiation medium of the present invention further contains other factors required for steps 5) and 6), such as growth factors, various inhibitors, serum replacements, antibiotics, and vitamins. According to a previous report (Nature Biotechnology 2014;32:1121-1133), for example, the medium used in step 5) can be supplemented with predetermined amounts of SANT1, retinoic acid, T3, LDN, Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum replacements, vitamins, antibiotics, and the like. Furthermore, the medium used in step 6) can be supplemented with predetermined amounts of T3, LDN, γ-secretase inhibitor XX, γ-secretase inhibitor RO, N-cysteine, AXL inhibitor, ascorbic acid, Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum replacements, vitamins, antibiotics, ZnSO, heparin, N-acetylcysteine, Trolox, R428, and the like.

[0133] The differentiation medium of the present invention is substantially free of ALK5 inhibitory activity. "Substantially free of ALK5 inhibitory activity" is as defined above and does not only mean that the medium has no ALK5 inhibitory activity at all, but also includes cases where the ALK5 inhibition rate is less than 50%, preferably 40% or less, more preferably 30% or less, even more preferably 20% or less, still more preferably 10% or less, and particularly preferably 5% or less, and may contain a compound having ALK5 inhibitory activity (e.g., ALK5 inhibitor II, etc.) as long as it satisfies the above definition.

[0134] The differentiation medium of the present invention may be provided in a single form in which the basal medium, CDK8 / 19 inhibitor, and the above-mentioned other factors are all mixed together, or may be provided in any combination or separately in multiple forms and prepared immediately upon use.

[0135] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Example]

[0136] Example 1: Evaluation of ALK4 inhibitory activity, ALK5 inhibitory activity, CDK8 inhibitory activity, and CDK19 inhibitory activity The ALK4 inhibitory activity, ALK5 inhibitory activity, CDK8 inhibitory activity, and CDK19 inhibitory activity of the test compounds, diethyl (E)-(4-(3-(5-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)acrylamide)benzyl)phosphonate (Compound 1), 2-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide (Compound 2 (BI-1347)), and 4-((2-(6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl)ethyl)amino)quinazoline-6-carbonitrile (Compound 3 (Senexin B)), were evaluated by the following methods. In a kinase panel assay, test compounds were administered at 0.1 μM, 1 μM (i.e., -Log ) for each of the kinases ALK4, ALK5, CDK8, and CDK19. 10 The binding inhibitory activity was measured from 0 to 100%. The estimated pIC 50 The pIC was calculated and displayed as a value of 6-8. 50 = 6 means that no binding inhibitory activity was observed at either the added concentration of 0.1 μM or 1 μM in this measurement test, and the compound in question either has no inhibitory activity or is below pIC 50 This means that the compound was considered to have only a weak binding inhibitory activity of ≦6. 50 = 8 is the estimated pIC calculated from the binding inhibitory activity of 0.1 μM and 1 μM 50 Values ​​≥ 8, pIC 50 This means that the antibody showed a strong binding activity of ≧8.

[0137] The concentration of each test compound required to show 50% inhibition against ALK4, ALK5, CDK8, and CDK19 (estimated pIC 50 values) are shown in Table 3.

[0138] [Table 3]

[0139] From Table 3, it was confirmed that Compound 1, Compound 2, and Compound 3 strongly inhibit CDK8 and CDK19.

[0140] Example 2: Increase in target cells (insulin-positive and NKX6.1-positive cells) in a cell population obtained by treating a pancreatic progenitor cell population with a CDK8 / 19 inhibitor 1. Method The differentiation of iPS cells into a pancreatic progenitor cell population was performed according to steps 1)-4) above, as previously reported (Stem Cell Research (2015) 14, 185-197), etc. The differentiation of iPS cells into insulin-positive cells was performed according to steps 5) and 6) above, etc. Pancreatic progenitor cell populations obtained by inducing differentiation from iPS cells were cultured for 2 days in a differentiation induction medium (Improved MEM / 1% B-27 / Penisilin Streptomycin medium) containing, together with differentiation factors (SANT1, retinoic acid, T3, LDN, Wnt inhibitors, ROCK inhibitors, FGF2), ALK5 inhibitor II (10 μM) or CDK8 / 19 inhibitors (Compound 1, Compound 2, or Compound 3) at specified concentrations, or neither ALK5 inhibitor II nor CDK8 / 19 inhibitors, to induce differentiation into endocrine progenitor cell populations.

[0141] Next, the cells were cultured for 7 days in a differentiation-inducing medium (Improved MEM / 1% B-27 / Penisilin Streptomycin medium) containing a predetermined concentration of ALK5 inhibitor II (10 μM) or CDK8 / 19 inhibitor (Compound 1, Compound 2, or Compound 3) together with differentiation factors (T3, LDN, γ-secretase inhibitor RO, FGF receptor 1 inhibitor PD-166866), or without ALK5 inhibitor II or CDK8 / 19 inhibitor, as previously reported (Nature Biotechnology 2014;32:1121-1133), the cells were cultured for 4 days in a differentiation-inducing medium (MCDB131 / 20 mM Glucose / NaHCO3 / FAF-BSA / ITS-X / Glutamax / Penisilin Streptomycin medium) containing T3, LDN, γ-secretase inhibitor RO, N-acetylcysteine, AXL inhibitor R428, ascorbic acid, ROCK inhibitor, ZnSO4, heparin, and Trolox together with ALK5 inhibitor II (10 μM) or CDK8 / 19 inhibitor at the indicated concentrations, or without either ALK5 inhibitor II or CDK8 / 19 inhibitor.

[0142] The number of insulin-positive and NKX6.1-positive cells and the number of insulin-positive and NKX6.1-negative cells in the insulin-positive cell population obtained by the above method were counted by flow cytometry, and the percentage of target cells, i.e., insulin-positive and NKX6.1-positive cells, and the percentage of non-target cells, i.e., insulin-positive and NKX6.1-negative cells, in each cell population were calculated.

[0143] 2.Results The percentages of insulin-positive and NKX6.1-positive cells and insulin-positive and NKX6.1-negative cells obtained when cells were treated with a differentiation-inducing medium containing ALK5 inhibitor II (10 μM) or a CDK8 / 19 inhibitor (Compound 1, Compound 2, or Compound 3) at a specified concentration, or without either an ALK5 inhibitor II or a CDK8 / 19 inhibitor, are shown in Figure 1. Figure 2 also shows the flow cytometry results of the cell populations obtained when ALK5 inhibitor II (10 μM), Compound 1 at 0.3 μM, Compound 2 at 3 nM, and Compound 3 at 0.1 μM were used.

[0144] It was confirmed that when a CDK8 / 19 inhibitor was added to the differentiation-inducing medium, a cell population could be produced in which the proportion of the desired insulin-positive and NKX6.1-positive cells was equal to or greater than the proportion of the undesired insulin-positive and NKX6.1-negative cells.

[0145] In particular, when compound 1 at 0.3 μM, compound 2 at 3 nM, and compound 3 at 0.1 μM were used as CDK8 / 19 inhibitors, a cell population with a higher proportion of the desired insulin-positive and NKX6.1-positive cells was produced compared to when ALK5 inhibitor II (10 μM), which has traditionally been used to induce differentiation of insulin-positive cell populations from pancreatic progenitor cell populations, was used.

[0146] When ALK5 inhibitor II was used, the percentage of the desired insulin-positive and NKX6.1-positive cells in the resulting cell population was approximately 33%, which remained the same or lower even when the concentration of ALK5 inhibitor II added to the medium was increased to 30 μM (data not shown).

[0147] On the other hand, it was confirmed that by treating with a CDK8 / 19 inhibitor during the manufacturing process of inducing differentiation of an insulin-positive cell population from a pancreatic progenitor cell population, the proportion of target cells (insulin-positive and NKX6.1-positive cells) in the cell population was increased to more than 33%.

[0148] From the above results, it was revealed that by treating a cell population in the process of inducing differentiation of an insulin-positive cell population from a pancreatic progenitor cell population with a CDK8 / 19 inhibitor instead of ALK5 inhibitor II, it is possible to produce an insulin-positive cell population containing target cells (insulin-positive and NKX6.1-positive cells) at a higher rate than with conventional methods.

Claims

1. 1. A method for producing an insulin-positive cell population, comprising: A method comprising differentiating a population of pancreatic progenitor cells, or a population of cells at a later stage of differentiation, in a medium comprising a CDK8 / 19 inhibitor.

2. The method of claim 1 , wherein the medium does not contain ALK5 inhibitor II.

3. IC of the CDK8 / 19 inhibitor against ALK5 ５０ The method of claim 1 or 2, wherein the α-glutamyltransferase is 1 μM or more.

4. CDK8 / 19 inhibitors include diethyl (E)-(4-(3-(5-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)acrylamido)benzyl)phosphonate, 2-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide, 4-((2-(6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl)ethyl)amino)quinazoline-6-carbonitrile, 4-(4-(2,3-dihydrobenzo[b][1,4]dioxa[ ...

4. The method according to claim 1, wherein the compound is one or more selected from the group consisting of (E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(4-(morpholinomethyl)phenyl)acrylamide, 3-(2-(imidazo[1,2-b]pyridazin-6-ylthio)ethyl)-4-(naphthalen-1-ylsulfonyl)-3,4-dihydroquinoxalin-2(1H)-one, and (E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(4-(morpholinomethyl)phenyl)acrylamide.

5. The method according to any one of claims 1 to 4, wherein the pancreatic progenitor cell population or the cell population at a later differentiation stage is produced by inducing differentiation of pluripotent stem cells.

6. A differentiation medium for a pancreatic progenitor cell population, or a cell population undergoing further differentiation, comprising a CDK8 / 19 inhibitor.

7. The culture medium described in claim 6, which does not contain ALK5 inhibitor II.

8. IC of CDK8 / 19 inhibitors against ALK5 ５０ The medium according to claim 6 or 7, wherein the amount of ATP is 1 μM or more.

9. CDK8 / 19 inhibitors include diethyl (E)-(4-(3-(5-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)acrylamido)benzyl)phosphonate, 2-(4-(4-(isoquinolin-4-yl)phenyl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide, 4-((2-(6-(4-methylpiperazine-1-carbonyl)naphthalen-2-yl)ethyl)amino)quinazoline-6-carbonitrile, 4-(4-(2,3-dihydrobenzo[b][1,4]dioxa[ ...

9. The medium according to claim 6, wherein the compound is one or more selected from the group consisting of (E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(4-(morpholinomethyl)phenyl)acrylamide, 3-(2-(imidazo[1,2-b]pyridazin-6-ylthio)ethyl)-4-(naphthalen-1-ylsulfonyl)-3,4-dihydroquinoxalin-2(1H)-one, and (E)-3-(4-(1-cyclopropyl-1H-pyrazol-4-yl)pyridin-3-yl)-N-(4-(morpholinomethyl)phenyl)acrylamide.

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