Method for producing biological tissue-like structure

Abstract

The purpose of the present invention is to provide a novel method for producing a biotissue-like structure including differentiated cells derived from pluripotent stem cells. The present invention provides a method for forming a biotissue-like structure with host-derived blood vessels or connective tissues by implanting, in biotissues, a composition including the state in which pluripotent stem cell-derived cells are disposed after being dispersed in a biocompatible material to induce the differentiation of cells.

Description

[Technical field]

[0001] The present invention relates to a biological tissue-like structure composed of differentiated cells derived from pluripotent stem cells, as well as a production method and use thereof. [Background technology]

[0002] Functional differentiated cells induced from pluripotent stem cells such as induced pluripotent cells and embryonic stem cells (ES cells) are expected to be a cell source for transplantation regenerative medicine. At present, attempts are being made to construct structures (hereinafter referred to as "tissue-like structures") that have functions equivalent to or similar to those of biological organs and tissues using such functional differentiated cells for transplantation therapy using tissue engineering. However, when attempting to fabricate a thick, three-dimensional structure, it may not be possible to sufficiently supply oxygen, nutrients, etc. to all cells, and it is not easy to maintain and manage the cells in the structure both in vitro and in vivo. For this reason, conventional structures have limitations in thickness and size, and may not be sufficient to exhibit functions equivalent to or similar to those of biological organs and tissues. In this field, a new method for easily fabricating a thick, three-dimensional tissue-like structure is still desired.

[0003] Research is being conducted on inducing differentiation of pluripotent stem cells such as artificial pluripotent cells and embryonic stem cells into endocrine cells that secrete hormones, such as pancreatic β cells and pancreatic α cells, and applying the cells to the treatment of diabetes. To date, a method has been reported in which endocrine cells are produced by inducing differentiation of pluripotent stem cells into pancreatic progenitor cells, transplanting the cells into a living body, and maturing the cells. For example, Non-Patent Document 1 describes a method for producing endocrine cells including insulin-positive cells and glucagon-positive cells by transplanting pancreatic progenitor cells, which have been induced to differentiate from pluripotent stem cells, into mice and maturing the cells. However, no three-dimensional biological tissue-like structure has been produced to date that contains endocrine cells such as insulin-positive cells and glucagon-positive cells, is thick, and does not contain pancreatic ducts or cysts. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Robert T et al,Stem Cell Reports.2018 Mar 13;10(3):739-750. 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 producing a biological tissue-like structure containing differentiated cells induced from pluripotent stem cells. [Means for solving the problem]

[0006] As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they discovered that by transplanting a composition containing cells derived from pluripotent stem cells dispersed in a biocompatible material into biological tissue, the cells can be engrafted, induced to differentiate and mature, and, together with host-derived blood vessels and connective tissue, a biological tissue-like structure can be constructed.

[0007] The present invention is based on these findings and includes the following inventions. [1] A composition for use in a method for creating a biological tissue-like structure in the biological tissue of a host animal, comprising a biocompatible material and cells derived from human pluripotent stem cells, wherein the cells derived from human pluripotent stem cells are dispersed and disposed in the biocompatible material. [2] The composition of [1], wherein the biocompatible material is fibrin gel. [3] The composition according to [2], wherein the fibrin gel is formed by mixing cells derived from human pluripotent stem cells, fibrinogen, and thrombin and gelling the composition immediately prior to use. [4] Any of the compositions [1] to [3], in which cells derived from human pluripotent stem cells are present in the form of multiple spheroids. [5] Any of the compositions [1] to [4], in which the percentage of Ki67-positive cells in cells derived from human pluripotent stem cells is less than 3%. [6] Any of the compositions according to [1] to [5], wherein the cells derived from human pluripotent stem cells are insulin-producing cells. [7] Any of the compositions [1] to [6], wherein the method comprises transplanting the composition into a biological tissue of a host animal and inducing differentiation of cells derived from human pluripotent stem cells dispersed and disposed in a biocompatible material. [8] The composition of [7], wherein the living tissue of the host animal is subcutaneous tissue. [9] Any of the compositions of [1] to [8], wherein the biotissue-like structure comprises a plurality of clusters of differentiated cells obtained by inducing differentiation of cells derived from human pluripotent stem cells, connective tissue derived from a host animal, and blood vessels derived from the host animal, the plurality of clusters of differentiated cells being dispersed in the biotissue-like structure, the connective tissue surrounding the plurality of clusters of differentiated cells, and the blood vessels penetrating into the plurality of clusters of differentiated cells.

[10] The composition of [9], wherein the differentiated cells do not include exocrine cells.

[11] A biological tissue-like structure comprising a plurality of clusters of differentiated cells obtained by inducing differentiation from cells derived from human pluripotent stem cells, connective tissue derived from a host animal, and blood vessels derived from the host animal, wherein the plurality of clusters of differentiated cells are dispersed throughout the biological tissue-like structure, the connective tissue surrounds the plurality of clusters of differentiated cells, and the blood vessels invade the plurality of clusters of differentiated cells.

[12] The biological tissue-like structure of

[11] , wherein the differentiated cells include pancreatic beta cells. [12-A] The biological tissue-like structure of

[11] , wherein the differentiated cells include pancreatic beta cells and pancreatic alpha cells.

[13] The biological tissue-like structure according to any one of

[11] to [12-A], wherein the differentiated cells do not contain exocrine cells.

[14] The biological tissue-like structure according to any one of

[11] to

[14] , which is used for controlling blood glucose level of a subject into which the biological tissue-like structure is transplanted, to a normal level.

[15] A method for producing a biological tissue-like structure, comprising: A method comprising transplanting a composition comprising a biocompatible material and cells derived from human pluripotent stem cells, the cells being dispersed and disposed in the biocompatible material, into the biological tissue of a host animal and inducing differentiation.

[16] The method of

[15] , wherein the biocompatible material is fibrin gel.

[17] The method of

[16] , wherein the fibrin gel is gelled by mixing cells derived from human pluripotent stem cells, fibrinogen and thrombin immediately prior to use of the composition.

[18] Any of the methods

[15] –

[17] in which cells derived from human pluripotent stem cells exist in the form of multiple spheroids.

[19] Any of the methods

[15] to

[18] , in which the percentage of Ki67-positive cells in cells derived from human pluripotent stem cells is less than 3%.

[20] Any of the methods

[15] to

[19] , wherein the cells derived from human pluripotent stem cells are insulin-producing cells.

[21] Any of the methods

[15] to

[20] , wherein the living tissue of the host animal is subcutaneous tissue.

[22] Any of the methods of

[15] to

[21] , wherein the biotissue-like structure comprises a plurality of clusters of differentiated cells obtained by inducing differentiation of cells derived from dispersed human pluripotent stem cells, connective tissue derived from the host animal, and blood vessels derived from the host animal, the plurality of clusters of differentiated cells being dispersed in the biotissue-like structure, the connective tissue surrounding the plurality of clusters of differentiated cells, and the blood vessels penetrating into the plurality of clusters of differentiated cells.

[23] Any of the methods

[15] to

[22] , wherein the differentiated cells do not include exocrine cells.

[0008] This specification includes the contents disclosed in the specification and / or drawings of Japanese Patent Application No. 2019-075100, which is the priority basis of this application.

[0009] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Effect of the Invention

[0010] According to the present invention, a novel method for producing a biological tissue-like structure containing differentiated cells induced from pluripotent stem cells can be provided. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of flow cytometric analysis of protein expression in insulin-producing cells before transplantation. [Diagram 2] Figure 2 shows the results of measuring blood human C-peptide concentrations (A) and blood glucose levels (B) over time in immunodeficient NOD / SCID mice in which diabetes was induced with streptozotocin (STZ) and fibrin gel containing dispersed insulin-producing cells was implanted. [Diagram 3] Figure 3 shows the results of an analysis of the response to glucose loading of insulin-producing cell-derived tissue-like structures formed in immunodeficient NOD / SCID mice in which diabetes was induced by streptozotocin (STZ). The results show the time course of measurements of blood human C-peptide concentration (A) and plasma glucose concentration (B) after glucose loading. [Figure 4] FIG. 4 shows a photograph of a biological tissue-like construct derived from insulin-producing cells that was extracted from the body six months after transplantation. [Diagram 5] FIG. 5 shows an HE stained image of a biological tissue-like structure derived from insulin-producing cells that was extracted from the body 6 months after transplantation. [Figure 6] FIG. 6 shows a Masson's trichrome stained image of a biological tissue-like structure derived from insulin-producing cells that was extracted from the body 6 months after transplantation. [Figure 7] FIG. 7 shows immunohistochemical staining images of human nuclei (HuN) and PDX1 in a biological tissue-like structure derived from insulin-producing cells extracted from the body 6 months after transplantation. [Figure 8]FIG. 8 shows immunohistochemical staining images of insulin (INS) and glucagon (GCG) in a biological tissue-like structure derived from insulin-producing cells that was extracted from the body 6 months after transplantation. [Figure 9] FIG. 9 shows immunohistochemical staining images of mouse CD31 (mCD31) and chromogranin A (CHGA) of the insulin-producing cell-derived tissue-like structure extracted from the body 6 months after transplantation. [Figure 10] Figure 10 shows immunohistochemical staining images of human nuclei (HuN) and Ki67 in a tissue-like structure derived from insulin-producing cells extracted from the body 6 months after transplantation. Arrows: Ki67-positive cells. [Figure 11] Figure 11 shows the blood human C-peptide, glucagon and blood glucose levels measured over time after transplantation of insulin-producing cells dispersed in fibrin gel into immunodeficient NOD / SCID mice in which diabetes was induced by streptozotocin (STZ). [Figure 12] 12 shows the results of measuring insulin and glucagon contents in insulin-producing cell-derived tissue-like structures excised from the body 28 weeks after transplantation of insulin-producing cells dispersed in fibrin gel into immunodeficient NOD / SCID mice in which diabetes was induced by streptozotocin (STZ) and non-diabetic NOD / SCID mice. The upper row shows the results of measuring insulin content, the lower row shows the results of measuring glucagon content, and in each row, the left side shows the results of measuring each hormone content in the pancreas of each mouse (control), and the right side shows the results of measuring each hormone content in the excised insulin-producing cell-derived tissue-like structure. [Figure 13] Figure 13 shows the glucagon and glucagon-like peptide-1 measured over time after glucose loading in NOD / SCID mice with Akita gene mutation in which hyperglycemia was completely improved by transplantation of insulin-producing cells dispersed in fibrin gel. [Figure 14]FIG. 14 shows blood glucose levels and blood human C-peptide concentrations measured over time in STZ-diabetic NOD / SCID mice in which hyperglycemia was completely improved by transplantation of insulin-producing cells dispersed in fibrin gel, and then treated with the glucagon receptor antagonist MK-0893 and glucose loading. [Figure 15] Figure 15 shows the human C-peptide concentration in the blood during fasting measured before and after treatment of Akita diabetic NOD / SCID mice in which hyperglycemia was completely improved by transplantation of insulin-producing cells dispersed in fibrin gel, with the glucagon-like peptide-1 receptor antagonist Exendin-9 for three days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1. Terminology As used herein, "about" 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.

[0013] As used herein, "comprise(s)" or "comprising" means the inclusion, but not limitation, of the elements that follow the phrase, and thus implies the inclusion of the elements that follow the phrase, but not the exclusion of any other elements.

[0014] As used herein, the term "consist(s) of" means inclusive and limited to all elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that other elements are substantially absent.

[0015] As used herein, "not using feeder cells" means that feeder cells are not basically included, 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 from feeder cells.

[0016] The term "feeder cells" or "feeders" refers to cells that are co-cultured with other types of cells to provide an environment in which the cells can grow and support them. Feeder cells may be from the same species as the cells they support or from a different species. 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 may be inactivated by irradiation or mitomycin C treatment, for example.

[0017] 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 medium. Some cells cannot be maintained or grown in culture unless attached to a cell culture container. In contrast, non-adherent cells can be maintained and grown in culture without being attached to a container.

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

[0019] 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, such that the proportion of the target cell type is increased compared to the proportion of target cells present in the cell population prior to enrichment. A cell population can also be enriched for a target cell type by cell selection and sorting methods known in the art. A cell population can also be enriched by certain sorting or selection processes described herein. In certain embodiments of the 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.

[0020] As used herein, "deplete" and "depletion" refer to reducing the amount of a particular component in a composition, such as a cell or composition of cells, and "depleted," when used to describe a cell or 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 for a target cell type, and thus the proportion of the target cell type is reduced compared to the proportion of target cells present in the cell population prior to depletion. A cell population can also be depleted for a target cell type by cell selection and sorting methods known in the art. A cell population can also be depleted by certain sorting or selection processes described herein. In certain embodiments of the 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) for the target cell population.

[0021] As used herein, "purify" and "purification" refer to removing impurities in a composition, such as a composition of cells, to render it pure for a particular component, and "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 for a target cell type, and thus the proportion of the target cell type is increased 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 certain 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 has no detectable impurities (including contaminating cells).

[0022] In this specification, "factor having CDK8 / 19 inhibitory activity" means any substance having CDK8 / 19 inhibitory activity.In contrast to other proteins of 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.

[0023] A "growth factor" is an endogenous protein that promotes the differentiation and / or proliferation of a particular cell. 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 beta (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).

[0024] 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), 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-1-zenecarboxamide (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-oxadiazol-3-yl )-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide (GSK269962A), N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A). ROCK inhibitors are not limited to these, and antisense oligonucleotides and siRNAs against ROCK mRNA, antibodies that bind to ROCK, dominant negative ROCK mutants, etc. can also be used as ROCK inhibitors, and are commercially available or can be synthesized according to known methods.

[0025] In the present specification, the term "GSK3β inhibitor" refers to a substance having inhibitory activity against GSK3β (glycogen synthase kinase 3β). GSK3 (glycogen synthase kinase 3) is a type of serine / threonine protein kinase, and is involved in many signal pathways involved in glycogen production, apoptosis, stem cell maintenance, and the like. GSK3 has two isoforms, α and β. The "GSK3β inhibitor" used in the present invention is not particularly limited as long as it has GSK3β inhibitory activity, and may be a substance having both GSK3α inhibitory activity and GSK3β inhibitory activity.

[0026] 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), (3-(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, 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, lithium, and the like. 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.

[0027] As used herein, the term "serum replacement" refers to, for example, Knockout Serum Replacement (KSR: Invitrogen), StemSure Serum Replacement (Wako), B-27 supplement, N2-supplement, albumin (e.g., lipid-rich albumin), insulin, transferrin, fatty acid, collagen precursor, trace elements (e.g., zinc, selenium (e.g., sodium selenite)), 2-mercaptoethanol, 3'-thiolglycerol, or a mixture thereof (e.g., ITS-G). As serum replacement, B-27 supplement, KSR, StemSure Serum Replacement, and ITS-G are preferred. When a serum replacement is added to a medium, the concentration in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight. In the present invention, it is preferred to use a "serum replacement" instead of serum.

[0028] As used herein, the term "FGFR1 inhibitor" refers to a substance having inhibitory activity against fibroblast growth factor receptor (FGFR) 1. FGFR1 is a member of the four-transmembrane tyrosine kinase family (FGFR1, 2, 3, 4) as a receptor having high affinity for growth factors FGF1 to FGF17. The FGFR1 inhibitor is not particularly limited as long as it has FGFR1 inhibitory activity, and may be a substance having FGFR1 inhibitory activity as well as inhibitory activity against other FGFRs. As used herein, the term "FGFR1 inhibitor" includes substances that have even a small amount of FGFR1 inhibitory activity, but preferably refers to a substance that inhibits FGFR1 by 50% or more, and more preferably refers to a substance that inhibits FGFR1 by 50% or more, and more preferably refers to a substance that inhibits FGFR1 by 50% or more (IC 50 ) is 1 μM or less, more preferably 100 nM or less. The method for determining FGFR1 inhibitory activity may be selected from known methods, such as a method using EnzyChrom Kinase Assay Kit (BioAssaySystems). FGFR1 inhibitors that are known in the art can be used and can be found in patent literature or non-patent literature.

[0029] Specifically, FGFR1 inhibitors that can be used in the present invention include PD-166866 (1-[2-amino-6-(3,5-dimethoxyphenyl)-pyrido(2,3-d)pyrimidin-7-yl]-3-tert-butylurea: CAS No.: 192705-79-6), E-3810 (CAS No.: 1058137-23-7), PD-173074 (CAS No.: 219580-11-7), FGFR4-IN-1 (CAS No.: 1708971-72-5), FGFR-IN-1 (CAS No.: 1448169-71-8), FIIN-2 (CAS No.: 1633044-56-0), AZD4547 (CAS No.: 1035270-39-3), FIIN-3 (CAS No.: 1633044-56-0), AZD4547 (CAS No.: 1035270-39-3), and FIIN-3 (CAS No.: 1633044-56-0). No.:1637735-84-2), NVP-BGJ398(CAS No.:1310746-10-1), NVP-BGJ398(CAS No.:872511-34-7), CH5183284(CAS No.:1265229-25-1), Derazantinib(CAS No.:1234356-69-4), Derazantinib Racemate, Ferulic acid(CAS No.:1135-24-6), SSR128129E(CAS No.:848318-25-2), SSR128129E free acid(CAS No.:848463-13-8), Erdafitinib(CAS No.:1346242-81-6), BLU9931(CAS No.:1538604-68-0), PRN1371(CAS No.:1802929-43-6), S49076(CAS No.:1265965-22-7), LY2874455(CAS No.:1254473-64-7), Linsitinib(CAS No.:867160-71-2), Dovitinib (CAS No.:405169-16-6), Anlotinib (CAS No.:1058156-90-3), Brivanib (CAS No.:649735-46-6), Derazantinib (CAS No.:1234356-69-4), Anlotinib Dihydrochloride (CAS No.:1360460-82-7), ACTB-1003(CAS No.:939805-30-8)、BLU-554(CAS No.:1707289-21-1)、Rogaratinib(CAS No.:1443530-05-9)、BIBF 1120 esylate(CAS No.:656247-18-6)、TG 100572 Hydrochloride(CAS No.:867331-64-4)、ENMD-2076(CAS No.:934353-76-1)、Brivanib alaninate(CAS No.:649735-63-7)、TG 100572(CAS No.:867334-05-2)、BIBF 1120(CAS No.:656247-17-5)、ENMD-2076 Tartrate(CAS No.:1291074-87-7)、TSU-68(CAS No.:252916-29-3)、Ponatinib(CAS No.:943319-70-8)、Sulfatinib(CAS No.:1308672-74-3)、LY2784544(CAS No.:1229236-86-5)、Dovitinib lactate(CAS No.:692737-80-7)、SU 5402(CAS No.:215543-92-3)、FGF-401(CAS No.:1708971-55-4)、Tyrosine kinase-IN-1(CAS No.:705946-27-6)、PP58(CAS No.:212391-58-7)、TG 100801 Hydrochloride(CAS No.:1018069-81-2)、Crenolanib(CAS No.:670220-88-9)、TG 100801(CAS No.:867331-82-6)、Pazopanib Hydrochloride(CAS No.:635702-64-6)、Pazopanib(CAS No.:444731-52-6)、PD168393(CAS No.:194423-15-9)、Apatinib(CAS No.:1218779-75-9)、Palbociclibisethionate(CAS No.:827022-33-3)、Foretinib(CAS No.:849217-64-7)、Lenvatinib(CAS No.:417716-92-8)、Tandutinib(CAS No.:387867-13-2), etc. or salts thereof (these compounds are referred to as compound group D). In addition, as long as these compounds have FGFR1 inhibitory activity, they are preferably used in an FGFR1 50% inhibitory concentration (IC. 50 ) is 100 nM or less, it may have one or more substituents selected from the above. In addition, as long as these compounds have FGFR1 inhibitory activity, they are preferably used in combination with a 50% inhibitory concentration (IC 50 As long as the concentration of the compound is 100 nM or less, some of the partial structures (substituents, rings, etc.) may be transformed.

[0030] Preferably, the FGFR1 inhibitor in the present invention is CAS192705-79-6 (1-[2-amino-6-(3,5-dimethoxyphenyl)-pyrido(2,3-d)pyrimidin-7-yl]-3-tert-butylurea: CAS No.: 192705-79-6), E-3810 (CAS No.: 1058137-23-7), or PD173074 (CAS No.: 219580-11-7).

[0031] FGFR1 inhibitors are not limited to the compounds listed above, and antisense oligonucleotides and siRNAs against FGFR1 mRNA, antibodies that bind to FGFR1, dominant-negative FGFR1 mutants, etc. can also be used as FGFR1 inhibitors, and are commercially available or can be synthesized according to known methods.

[0032] As used herein, "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.

[0033] The detection of a marker protein can be carried out by immunological assays using an antibody specific to the marker protein, such as ELISA, immunostaining, and flow cytometry. The detection of a marker gene can be carried out by using a nucleic acid amplification method and / or a nucleic acid detection method known in the art, such as RT-PCR, microarrays, and biochips. In this specification, the term "positive" for a marker protein 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, the term "positive" for a marker gene means that it is detected by RT-PCR, and "negative" means that it is below the detection limit by RT-PCR.

[0034] As used herein, "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter in a cell.

[0035] As used herein, unless otherwise specified, "cells" refers to a composition of cells, i.e., a cell population. Thus, "cells" can include not only a particular cell, but also one or more other cells. A particular cell in "cells" can be enriched or purified, or can be increased by depleting one or more other cells. In addition, as used herein, the term "cells" is preferably human cells.

[0036] 2. Composition containing cells derived from pluripotent stem cells and a biocompatible material 2-1. Cells derived from pluripotent stem cells As used herein, "pluripotency" refers to the ability to differentiate into tissues and cells with various 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 has no ability to form an individual, and is therefore distinct from "totipotency," which can differentiate into any tissue of the body, including the germ disc.

[0037] As used herein, "multipotency" refers to the ability to differentiate into cells of a limited number of lineages. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent but not pluripotent.

[0038] As used herein, the term "pluripotent stem cell" refers to embryonic stem cells (ES cells) and cells that have the same differentiation pluripotency, i.e., the potential ability to differentiate into various tissues of the body (all of the endoderm, mesoderm, and ectoderm). Cells that have the same differentiation pluripotency as ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in the present specification). Preferably, in the present invention, the pluripotent stem cells are human pluripotent stem cells.

[0039] As for "ES cells", in the case of mouse ES cells, various mouse ES cell lines established by inGenious, Inc., RIKEN, etc. can be used, and in the case of human ES cells, various human ES cell lines established by NIH, RIKEN, Kyoto University, and Cellartis can be used. For example, as ES cell lines, NIH's CHB-1 to CHB-12 strains, RUES1 strain, RUES2 strain, HUES1 to HUES28 strains, etc., WisCell Research's H1 strain, H9 strain, RIKEN's KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. can be used.

[0040] The term "induced pluripotent stem cells" refers 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 the expression of Nanog as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.); and iPS cells created by 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), induced pluripotent stem cells created by Sakurada et al. (JP Patent Publication No. 2008-307007), etc. can also be used.

[0041] In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, 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 Application Laid-Open Nos. 7,795-797, or patents (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008 / 2336610, US 2009 / 047263, WO 2007 / 069666, WO 2008 / 118220, WO 2008 / 124133, WO 2008 / 151058, WO 2009 / 006930, WO 2009 / 006997, WO 2009 / 007852) can be used.

[0042] As artificial pluripotent cell lines, various iPS cell lines established by NIH, RIKEN, Kyoto University, etc. are available. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A line, HiPS-RIKEN-2A line, HiPS-RIKEN-12A line, and Nips-B2 line, and Kyoto University's Ff-WJ-18 line, Ff-I01s01 line, Ff-I01s02 line, Ff-I01s04 line, Ff-I01s06 line, and Ff-I14s line. 03 strain, Ff-I14s04 strain, QHJI01s01 strain, QHJI01s04 strain, QHJI14s03 strain, QHJI14s04 strain, RWMH15s02 strain, Ff-MH15s02 strain, 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, CDI's MyCell iPS Cells (21525.102.10A) strain, and MyCell iPS Cells (21526.101.10A) strain.

[0043] As used herein, the term "cells derived from pluripotent stem cells" refers to cells obtained by inducing differentiation from pluripotent stem cells, and examples of such cells include ectodermal lineage cells, mesodermal lineage cells, or endodermal lineage cells, or cells consisting of a combination thereof, which are induced to differentiate from pluripotent stem cells. More specifically, the term refers to cells that have functions equivalent to or similar to cells constituting organs or tissues such as (but not limited to) the epidermis, nerves, brain, spinal cord, esophagus, stomach, small intestine, large intestine, bladder, urethra, lung, thyroid, pancreas, liver, muscle, skeleton, heart, blood vessels, spleen, and kidney, which are induced to differentiate from pluripotent stem cells, or their precursor cells.

[0044] Preferably, in the present invention, "cells derived from pluripotent stem cells" have a state of cell aggregates in which cells are aggregated and coagulated, that is, a spheroid form. Spheroids of cells derived from pluripotent stem cells can be produced by a conventionally known method such as suspension culture, or a culture plate for forming spheroids having a microspace arranged on the culture bottom surface (for example, trade name: Elplacia (Kuraray Co., Ltd.), trade name: EZSPHERE (AGC Technoglass Co., Ltd.), trade name: Corning Spheroid Microplate (Corning International Inc.)) may be used. The spheroids have a diameter of about 10 μm to 1000 μm, preferably about 50 μm to 500 μm, more preferably about 50 μm to 300 μm, and even more preferably about 50 μm to 200 μm. And / or, the spheroids consist of about 100 to about 1000 cells, preferably about 200 to about 800 cells, and more preferably about 300 to about 500 cells.

[0045] In one embodiment of the present invention, the "cells derived from pluripotent stem cells" are definitive endoderm cells, primitive gut cells, posterior foregut cells, pancreatic progenitor cells, endocrine precursor cells, or insulin-producing cells that appear during the process of differentiation of pluripotent stem cells into pancreatic β cells, and are particularly preferably insulin-producing cells.

[0046] It is known that in the process of differentiation from pluripotent stem cells to pancreatic β cells, cells with different characteristics emerge depending on the differentiation stage (WO2009 / 012428, WO2016 / 021734). For example, this differentiation stage can be broadly classified in order of relative immaturity into pluripotent stem cells, definitive endoderm cells, primitive gut cells, posterior foregut cells, pancreatic progenitor cells, endocrine precursor cells, insulin-producing cells, and pancreatic β cells.

[0047] By "definitive endoderm cell" is meant a cell characterized by the expression of at least one of the following markers: SOX17, FOXA2, BMP2, CER, and CXCR4.

[0048] By "primitive gut cell" is meant a cell characterized by the expression of at least one of the markers HNF1B and HNF4A.

[0049] By "posterior foregut cell" is meant a cell characterized by the expression of at least one of the markers PDX-1, HNF6, and HLXB9.

[0050] By "pancreatic progenitor cell" is meant a cell characterized by the expression of at least one of the following markers: PDX-1, NKX6.1, PTF-1α, GATA4, and SOX9.

[0051] "Endocrine precursor cells" refers to cells characterized by the expression of at least one of the markers Chromogranin A, NeuroD, and NGN3, and the absence of expression of markers of the pancreatic-related hormone system (e.g., insulin, etc.). Endocrine precursor cells may express markers such as PAX-4, NKX2-2, Islet-1, PDX-1, and PTF-1α.

[0052] "Insulin-producing cells" refers to cells characterized by the expression of insulin markers. More specifically, "insulin-producing cells" are characterized by containing cells expressing both insulin and NKX6.1 markers (i.e., insulin-positive and NKX6.1-positive cells, hereinafter referred to as "Ins+NKX+ cells") at a rate of about 30% or more, and cells expressing only insulin among insulin and NKX6.1 markers (i.e., insulin-positive and NKX6.1-negative cells, hereinafter referred to as "Ins+NKX- cells") at a rate of more than about 15%. The upper limit of the rate of Ins+NKX+ cells in insulin-producing cells is not particularly limited, but can be preferably about 50% or less.

[0053] The ratio of Ins+NKX- cells in insulin-producing cells can be preferably about 20% or more, more preferably about 25% or more, and even more preferably about 30% or more. The upper limit of the ratio of Ins+NKX- cells in insulin-producing cells is not particularly limited, but can be preferably about 40% or less.

[0054] For example, insulin-producing cells contain Ins+NKX+ cells in a proportion of about 30% or more and about 50% or less, and Ins+NKX- cells in a proportion of more than about 15% and about 40% or less, preferably about 20% or more and about 40% or less, more preferably about 25% or more and about 40% or less, and even more preferably about 30% or more and about 40% or less.

[0055] In this specification, the percentage of a given cell in insulin-producing cells means the percentage relative to the total number of cells contained in the insulin-producing cells. The percentage of each cell type is the value in insulin-producing cells that are subjected to differentiation induction into pancreatic islet-like cells (i.e., to transplantation into a living body).

[0056] Since Ins+NKX- cells are found in large numbers among immature (early differentiated) insulin-producing cells, a higher proportion of Ins+NKX- cells indicates less mature (early differentiated) insulin-producing cells.

[0057] Insulin-producing cells further include the following a. to f.: a. The expression level of the MAFA gene or its protein is low; b. The percentage of Ki67 positive cells is low. c. A low percentage of glucagon-positive and insulin-negative cells; d. exhibiting a glucose-stimulated insulin secretion response; e. A high percentage of chromogranin A positive cells. f. A low percentage of alkaline phosphatase-positive pluripotent stem cells; wherein "multiple" means 2, 3, 4, 5, or 6.

[0058] a. Low expression of the MAFA gene or its gene product The insulin-producing cells of the present invention are characterized in that the expression level of the MAFA gene or its protein is lower than the expression level of the MAFA gene or its protein in pancreatic islets.

[0059] The term "pancreatic islet" refers to a cell that is more differentiated than an insulin-producing cell, including a mature pancreatic β cell, and characterized by the expression of at least one of MAFA, UCN3, and IAPP, which are markers of mature pancreatic β cells. The pancreatic islet may be isolated from a healthy subject.

[0060] The expression levels of the MAFA gene or its protein between insulin-producing cells and pancreatic islets can be compared using techniques known in the art, for example, by comparing the relative values ​​obtained by correcting the expression levels of the MAFA gene or its protein detected and quantified using techniques such as RT-PCR, microarrays, biochips, Western blots, ELISA, immunostaining, flow cytometry, etc., with the expression levels of an internal standard gene or its protein. The "internal standard gene" is not particularly limited, and examples that can be used include GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-actin, β2-microglobulin, HPRT 1 (hypoxanthine phosphoribosyltransferase 1), etc.

[0061] The expression level of the MAFA gene or its protein in insulin-producing cells is about 20% or less, preferably about 15% or less, more preferably about 10% or less, even more preferably about 5% or less, and particularly preferably about 1% or less, of the expression level of the MAFA gene or its protein in pancreatic islets.

[0062] Since the MAFA gene or its protein is a marker for mature pancreatic β cells, this characteristic indicates that the insulin-producing cells of the present invention are at a differentiation stage where they contain almost no or a low proportion of mature pancreatic β cells.

[0063] b. Low percentage of Ki67 positive cells The insulin-producing cells of the present invention are characterized in that they contain Ki67-positive cells at a rate of less than 3%.

[0064] "Ki67-positive cells" refer to highly proliferative cells that are present among insulin-producing cells induced to differentiate from pluripotent stem cells and are characterized by the expression of Ki67 as a marker. "Ki67" is known as a cell cycle-related nuclear protein, and is also known as a marker for cell proliferation and cell cycle, since its expression is observed in the G1, S, G2, and M phases of proliferating cells and not in the G0 phase in which proliferation is quiescent.

[0065] Hereinafter, in the present specification, "Ki67 positive" may be referred to as "Ki67+". Furthermore, "Ki67 positive cells" may be referred to as "Ki67+ cells".

[0066] The percentage of Ki67+ cells among insulin-producing cells is less than about 3%, preferably less than about 1%, more preferably less than about 0.8%, and even more preferably less than about 0.5%.

[0067] This characteristic indicates that there is little or a low proportion of contaminating or remaining Ki67+ cells in the insulin-producing cells. Ki67+ cells have a high proliferation rate and may adversely affect the final tissue-like construct and its survival, so their contamination or remaining may be undesirable.

[0068] c. A low percentage of glucagon-positive and insulin-negative cells The insulin-producing cells of the present invention are characterized by containing glucagon-positive and Ins- cells at a ratio of less than 3%. Hereinafter, in this specification, "glucagon-positive" may be referred to as "Gcg+". Also, "glucagon-positive and insulin-negative cells" may be referred to as "Gcg+Ins- cells".

[0069] The proportion of Gcg+Ins- cells among insulin-producing cells is about 2.5% or less, preferably about 2% or less, more preferably about 1% or less, and even more preferably about 0.5% or less.

[0070] Since Gcg+Ins- is a marker for mature pancreatic alpha cells, this characteristic indicates that the insulin-producing cells of the present invention are at a differentiation stage where they contain almost no mature pancreatic alpha cells or only a low proportion of them.

[0071] d. Demonstrate glucose-stimulated insulin secretion response The insulin-producing cells of the present invention exhibit a glucose stimulated insulin secretion (GSIS) response. The GSIS response in insulin-producing cells can be evaluated according to a conventional method (e.g., U.S. Application No. 11 / 773,944), for example, by measuring the amount of C-peptide secreted into the medium. C-peptide is a degradation product produced in equimolar amounts to insulin during the maturation of proinsulin. The amount of C-peptide can be measured, for example, by ELISA using anti-C-peptide monoclonal antibody.

[0072] e. A high percentage of chromogranin A positive cells The insulin-producing cells of the present invention are characterized by containing chromogranin A-positive cells at a rate of more than about 45%.

[0073] Hereinafter, in this specification, "chromogranin A positive" may be referred to as "Chga+." Furthermore, "chromogranin A positive cells" may be referred to as "Chga+ cells."

[0074] The ratio of Chga+ cells in insulin-producing cells is preferably about 50% or more (e.g., about 55% or more), more preferably about 60% or more, even more preferably about 70% or more, even more preferably about 80% or more, and particularly preferably about 90% or more. The upper limit of the ratio of Chga+ cells in insulin-producing cells is not particularly limited, but can be, for example, about 99% or less.

[0075] Chga+ cells include cells that secrete hormones such as insulin (endocrine cells), including the above-mentioned Ins+NKX+ cells and Ins+NKX- cells. Therefore, this characteristic indicates that the insulin-producing cells of the present invention contain a high proportion of endocrine cells.

[0076] f. Low percentage of alkaline phosphatase-positive pluripotent stem cells The insulin-producing cells of the present invention are characterized in that they contain alkaline phosphatase-positive pluripotent stem cells at a ratio of less than about 0.01%.

[0077] The ratio of alkaline phosphatase-positive pluripotent stem cells among insulin-producing cells can be preferably about 0.008% or less, more preferably about 0.005% or less, and even more preferably about 0.001% or less.

[0078] Since alkaline phosphatase is a marker that indicates the undifferentiated state of pluripotent stem cells, this characteristic indicates that the insulin-producing cells of the present invention contain almost no, or only a low proportion of, unintended pluripotent stem cells that have not been induced to differentiate.

[0079] Alkaline phosphatase-positive pluripotent stem cells may further express other markers indicative of pluripotency, such as at least one selected from NANOG, SOX2, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0080] Differentiation of pluripotent stem cells into definitive endoderm cells, primitive gut cells, posterior foregut cells, pancreatic precursor cells, endocrine precursor cells, and insulin-producing cells can be achieved by 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 tube cells; Step 3) Induce differentiation of primitive gut cells into posterior foregut cells; Step 4) Inducing 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 the endocrine precursor cells into insulin-producing cells. Each step will be explained below, but the differentiation induction into each cell is not limited to these techniques.

[0081] Step 1) Differentiation into definitive endoderm cells Pluripotent stem cells are cultured in medium containing low doses of activin A to differentiate into definitive endoderm cells.

[0082] The medium used in this process may be a basic medium used for culturing mammalian cells, such as RPMI medium, MEM medium, iMEM medium, DMEM (Dulbecco's modified Eagle's medium), Improved MEM Zinc Option medium, Improved MEM / 1% B-27 supplement / Penisilin Streptomycin medium, or MCDB131 / 10 mM Glucose / 20 mM Glucose / NaHCO3 / FAF-BSA / ITS-X / Glutamax / ascorbic acid / Penisilin Streptomycin medium.

[0083] Activin A can be included in the medium at low doses, for example, at an amount of 5 to 10 ng / mL. In 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.

[0084] The medium can further contain a ROCK inhibitor and a GSK3β inhibitor.

[0085] The concentration of the GSK3β inhibitor in the medium is appropriately set 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.

[0086] 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.

[0087] Insulin may be further added to the medium. Insulin may be contained in the medium in an amount of 0.01 to 20 μM, preferably 0.1 to 10 μM, more preferably 0.5 to 5 μM. The concentration of insulin in the medium may be, but is not limited to, the concentration of insulin contained in the added B-27 supplement.

[0088] The culture may be performed in either two-dimensional or three-dimensional culture. The number of cells at the start of culture is not particularly limited, but in the case of two-dimensional culture, it is about 50,000 to 1,000,000 cells / cm. 2 , preferably about 100,000 to 800,000 cells / cm 2 , and more preferably about 100,000 to 300,000 cells / cm 2 The number of cells at the start of the culture is not particularly limited, but in the case of three-dimensional culture, the number of cells can be about 10,000 to 1,000,000 cells / mL, preferably about 100,000 to 800,000 cells / mL, and more preferably about 300,000 to 600,000 cells / mL. The culture period is 1 to 4 days, preferably 1 to 3 days, and particularly preferably 3 days.

[0089] 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%.

[0090] Alternatively, in the present invention, definitive endoderm cells can be produced by first culturing pluripotent stem cells in a medium under conditions in which insulin is active in the presence of a low dose of activin A, followed by a second culturing in a medium under conditions in which insulin is not active.

[0091] (1) First culture "Conditions under which insulin acts" refers to conditions under which insulin activates the insulin signaling pathway in cells. Normally, insulin binds to the insulin receptor present on the surface of the cell membrane, activating the tyrosine kinase present in the receptor and tyrosine phosphorylating the insulin receptor substrate protein family (IRS: IRS-1, 2, 3). In this specification, the occurrence of this series of reactions initiated by the binding of insulin to the insulin receptor is referred to as "activating the insulin signaling pathway."

[0092] An example of the condition under which insulin acts is when the medium contains insulin. The insulin may be any insulin that can activate the insulin signaling pathway in pluripotent stem cells, and may be produced by recombinant methods or by synthesis using solid-phase synthesis. Insulin may be derived from humans, non-human primates, pigs, cows, horses, sheep, goats, llamas, dogs, cats, rabbits, mice, guinea pigs, etc., and is preferably human insulin.

[0093] In the present invention, an insulin mutant, an insulin derivative, or an insulin agonist can also be used as "insulin" as long as it activates the insulin signaling pathway in pluripotent stem cells. The "insulin mutant" includes a polypeptide that has an amino acid sequence in which 1 to 20, preferably 1 to 10, and more preferably 1 to 5 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of insulin and can activate the insulin signaling pathway, and a polypeptide that has an amino acid sequence that has 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more sequence identity with the amino acid sequence of insulin and can activate the insulin signaling pathway. Comparison of amino acid sequences can be performed by known methods, for example, using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like, for example, with default settings. "Insulin derivative" refers to a polypeptide or a substance having a similar action, which consists of an amino acid sequence in which some groups of amino acid residues of insulin or an insulin mutant are chemically substituted (e.g., α-methylation, α-hydroxylation), deleted (e.g., deamination), or modified (e.g., N-methylation), and which can cause activation of the insulin signaling pathway. "Insulin agonist" refers to a polypeptide or a substance having a similar action, which can cause activation of the insulin signaling pathway by binding to the insulin receptor, regardless of the structure of insulin.

[0094] The medium for the first culture may contain insulin in an amount of 0.01 to 20 μM, preferably 0.1 to 10 μM, more preferably 0.5 to 5 μM. The concentration of insulin in the medium may be, but is not limited to, the concentration of insulin contained in the added B-27 supplement.

[0095] The medium may further contain a ROCK inhibitor and / or a GSK3β inhibitor. The concentration of the ROCK inhibitor in the medium is appropriately set depending on the type of ROCK inhibitor used. For example, when Y-27632 is used as the ROCK inhibitor, the concentration can be usually 5 to 20 μM, preferably 5 to 15 μM, and particularly preferably about 10 μM. The concentration of the GSK3β inhibitor in the medium is appropriately set depending on the type of GSK3β inhibitor used. For example, when CHIR99021 is used as the GSK3β inhibitor, the concentration can be usually 2 to 5 μM, preferably 2 to 4 μM, and particularly preferably about 3 μM.

[0096] The medium may further contain one or more selected from the group consisting of pyruvate (sodium salt, etc.), L-alanyl L-glutamine, and glucose. Pyruvate may be contained in the medium in an amount of 10 to 1000 mg / L, preferably 30 to 500 mg / L, more preferably 50 to 200 mg / L, and particularly preferably about 110 mg / L. L-alanyl L-glutamine may be contained in the medium in an amount of 50 to 2000 mg / L, preferably 100 to 1500 mg / L, more preferably 500 to 1000 mg / L, and particularly preferably about 860 mg / L. Glucose may be contained in the medium in an amount of 15 mM or more, preferably 15 to 30 mM, more preferably 15 to 25, and particularly preferably about 25 mM. The concentrations of pyruvate, L-alanyl L-glutamine, and glucose in the medium may be, but are not limited to, the concentrations of pyruvate, L-alanyl L-glutamine, and glucose contained in DMEM medium (DMEM, high glucose, GlutaMAX™, pyruvate (Thermo Fisher Scientific)) or other DMEM medium.

[0097] The medium may be one based on the above-mentioned basal medium and supplemented with one or more of the above-mentioned components. The basal medium is preferably a DMEM medium, more preferably a DMEM medium containing pyruvate, L-alanyl-L-glutamine, and glucose in the amounts described above.

[0098] The culture period of the first culture can be in the range of 6 to 48 hours, preferably 12 to 24 hours. The culture temperature is not particularly limited, but is performed at 30 to 40°C (e.g., 37°C). The carbon dioxide concentration in the culture vessel is, for example, about 5%. Culture may be performed by either two-dimensional culture or three-dimensional culture. The number of cells at the start of culture is not particularly limited, but is about 50,000 to 1,000,000 cells / cm in the case of two-dimensional culture. 2 , preferably about 100,000 to 800,000 cells / cm 2 , and more preferably about 100,000 to 300,000 cells / cm 2 The number of cells at the start of the culture is not particularly limited, but in the case of three-dimensional culture, the number of cells can be about 10,000 to 1,000,000 cells / mL, preferably about 100,000 to 800,000 cells / mL, and more preferably about 300,000 to 600,000 cells / mL.

[0099] (2) Second culture "Conditions in which insulin does not act" refers to conditions in which insulin does not activate the insulin signaling pathway in cells. "Does not activate the insulin signaling pathway in cells" refers not only to no activation of the insulin signaling pathway at all, but also to only slight activation to the extent that no significant difference is observed compared to activation of the insulin signaling pathway in the absence of insulin. Therefore, "conditions in which insulin does not act" refers to, for example, conditions in which insulin is not contained in the medium, or, even if insulin is contained in the medium, the amount of insulin contained in the medium is such that only slight activation is observed to the extent that no significant difference is observed. Alternatively, even if insulin is contained in the medium, it also refers to cases in which the insulin signaling pathway is not activated by containing an insulin signal inhibitor together. "Insulin signal inhibitor" refers to a component capable of blocking the insulin signaling pathway at any position. Examples of such insulin signal inhibitors include polypeptides and compounds that bind to or compete with insulin, insulin receptors, various proteins that act as signaling substances, and inhibit the interaction between molecules involving these factors. For example, such insulin signal inhibitors include LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], which competitively inhibits ATP binding to the catalytic subunit of PI3 kinase. Insulin signal inhibitors are not limited to these, and antibodies that bind to insulin, insulin receptors, and various proteins that act as signal transduction substances, and dominant negative mutants thereof, as well as antisense oligonucleotides and siRNAs against the mRNAs of insulin receptors and various proteins that act as signal transduction substances, can also be used as insulin signal inhibitors. Insulin signal inhibitors are commercially available or can be synthesized according to known methods.

[0100] The medium may further contain a ROCK inhibitor and / or a GSK3β inhibitor. The amount of the ROCK inhibitor and / or the GSK3β inhibitor in the medium may be selected from the ranges described in the above-mentioned first culture, and may be the same as or different from the amount used in the first culture.

[0101] The medium may further contain one or more selected from the group consisting of pyruvate, L-alanyl L-glutamine, and glucose. The amounts of pyruvate, L-alanyl L-glutamine, and glucose in the medium may be selected from the ranges described above in the first culture, and may be the same as or different from those used in the first culture.

[0102] The medium used in the second culture may be a medium based on a basal medium used for culturing mammalian cells and supplemented with one or more of the above-mentioned components. The basal medium may be the same as that used in the first culture, or may be different. A DMEM medium is preferable, and a DMEM medium containing pyruvate, L-alanyl-L-glutamine, and glucose in the amounts described above is more preferable.

[0103] The culture period of the second culture can be at least 6 hours, preferably 6 to 72 hours, and more preferably 24 to 72 hours. The culture temperature is not particularly limited, but is performed at 30 to 40°C (for example, 37°C). The culture may be performed by either two-dimensional culture or three-dimensional culture. The carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0104] The medium for the first culture and the second culture may contain the above-mentioned low dose of activin A. The amount of activin A contained in the medium for the first culture and the second culture may be the same or different.

[0105] The medium for the first culture and the second culture may further contain dimethyl sulfoxide.

[0106] The proportion of endocrine cells obtained from step 6) onwards can be increased by culturing pluripotent stem cells in the presence of a low dose of activin A, or by first culturing the pluripotent stem cells in a medium under conditions in which insulin is active in the presence of a low dose of activin A, and then second culturing the pluripotent stem cells in a medium under conditions in which insulin is not active.

[0107] Step 2) Differentiation into primitive gut cells The definitive endoderm cells obtained in step 1) are further cultured in a medium containing a growth factor to induce differentiation into primitive gut cells. The culture period is 2 to 8 days, preferably about 4 days. The culture temperature is not particularly limited, but is performed at 30 to 40° C. (for example, 37° C.). The carbon dioxide concentration in the culture vessel is, for example, about 5%. The culture may be performed by either two-dimensional culture or three-dimensional culture.

[0108] The medium may be a basal medium used for culturing mammalian cells as described in step 1) above. In addition to growth factors, serum substitutes, vitamins, antibiotics, etc. may be appropriately added to the medium.

[0109] As the growth factor, EGF, KGF and FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is even more preferred.

[0110] The concentration of the growth factor in the medium is appropriately set depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / ml (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / ml (i.e., about 0.8 to 160 nM), more preferably about 10 to 1000 ng / ml (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / ml (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM), more 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.

[0111] 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., and induced to differentiate into posterior foregut cells. The culture period is 1 to 5 days, preferably about 2 days. The culture may be performed by either two-dimensional culture or three-dimensional culture.

[0112] 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%.

[0113] The medium may be a basal medium used for culturing mammalian cells as described in step 1) above. In addition to growth factors, serum substitutes, vitamins, antibiotics, etc. may be appropriately added to the medium.

[0114] As the growth factor, EGF, KGF and FGF10 are preferred, EGF and / or KGF are more preferred, and KGF is even more preferred.

[0115] The concentration of the growth factor in the medium is appropriately set depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / ml (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / ml (i.e., about 0.8 to 160 nM), more preferably about 10 to 1000 ng / ml (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / ml (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM), more 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.

[0116] The concentration of cyclopamine in the medium is not particularly limited, but is usually 0.5 to 1.5 μM, preferably 0.3 to 1.0 μM, and particularly preferably about 0.5 μM.

[0117] The concentration of Noggin in the medium is not particularly limited, but is usually 10 to 200 ng / mL, preferably 50 to 150 ng / mL, and particularly preferably about 100 ng / mL. The medium may also contain dimethyl sulfoxide.

[0118] Step 4) Differentiation into pancreatic progenitor cells The posterior foregut cells obtained in step 3) may be 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, and induced to differentiate into pancreatic progenitor cells. The culture period is 2 to 10 days, preferably about 5 days. The culture may be performed in either two-dimensional culture or three-dimensional culture. In the case of two-dimensional culture, the posterior foregut cells obtained in step 3) are dispersed by pipetting after treatment with 0.25% trypsin-EDTA according to a previous report (Toyoda et al., Stem cell Research (2015) 14, 185-197), and the 0.25% trypsin-EDTA is centrifuged to suspend the cells, and then reseeded in a new medium in step 4).

[0119] 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.

[0120] The factor having CDK8 / 19 inhibitory activity can be any of the above-mentioned various compounds or salts thereof, and the amount added to the medium is appropriately determined 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.

[0121] As the growth factor, EGF, KGF, and FGF10 are preferable, KGF and / or EGF are more preferable, and KGF and EGF are even more preferable.

[0122] The concentration of the growth factor in the medium is appropriately set depending on the type of growth factor used, but is usually about 0.1 nM to 1000 μM, preferably about 0.1 nM to 100 μM. In the case of EGF, the concentration is about 5 to 2000 ng / ml (i.e., about 0.8 to 320 nM), preferably about 5 to 1000 ng / ml (i.e., about 0.8 to 160 nM), more preferably about 10 to 1000 ng / ml (i.e., about 1.6 to 160 nM). In the case of FGF10, the concentration is about 5 to 2000 ng / ml (i.e., about 0.3 to 116 nM), preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM), more 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 usually 5 to 150 ng / mL, preferably 30 to 100 ng / mL, and particularly preferably about 50 ng / mL, and KGF are usually 10 to 200 ng / mL, preferably 50 to 150 ng / mL, and particularly preferably about 100 ng / mL.

[0123] 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 not containing a ROCK inhibitor.

[0124] The medium may also contain a 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. In addition, dimethyl sulfoxide and activin (1 to 50 ng / ml) may be added to the medium.

[0125] In any step, 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 (sometimes referred to as AA in the present specification), penicillin, streptomycin, or a mixture thereof), antibacterial agents (e.g., amphotericin B), antioxidants, pyruvic acid, buffers, inorganic salts, and the like may be added. When an antibiotic is added to the medium, the concentration of the antibiotic in the medium is usually 0.01 to 20% by weight, preferably 0.1 to 10% by weight. The culture may be performed by either two-dimensional culture or three-dimensional culture. The culture temperature is not particularly limited, but is performed at 30 to 40° C. (for example, 37° C.). The carbon dioxide concentration in the culture vessel is, for example, about 5%.

[0126] Step 5) Differentiation into endocrine precursor cells The pancreatic precursor cells obtained in step 4) are further cultured in a medium containing a growth factor to induce differentiation into endocrine precursor cells. The culture may be performed in either 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 to remove the 0.25% trypsin-EDTA, and then re-seeded in a new medium in step 5). The culture period is 2 to 3 days, preferably about 2 days.

[0127] The medium may be a basal medium used for culturing mammalian cells, as described in step 1) above. 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 appropriately added. Dimethyl sulfoxide may also be added to the medium.

[0128] The culture is performed in a non-adherent manner without using feeder cells. For the culture, dishes, flasks, microplates, multi-hole plates (Nunc), or bioreactors are used. The culture vessels are preferably surface-treated to reduce adhesion to the cells. 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%.

[0129] Step 6) Differentiation into insulin-producing cells The endocrine precursor cells obtained in step 5) are further cultured in a medium containing an FGFR1 inhibitor to induce differentiation into insulin-producing cells. The culture period is 10 to 30 days, preferably about 10 to 20 days.

[0130] The medium may be a basal medium used for culturing mammalian cells, as described in step 1) above. 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 may be added to the medium, and further, Wnt inhibitors, ROCK inhibitors, FGF (preferably FGF2), serum substitutes, vitamins, antibiotics, and the like may be appropriately added. For example, the medium may be supplemented with ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, and ascorbic acid, or T3, ALK5 inhibitor II, ZnSO4, heparin, N-acetylcysteine, Trolox, and R428.

[0131] The culture may be performed by either two-dimensional culture or three-dimensional culture. Preferably, the culture is performed by non-adherent culture without using feeder cells. For the culture, a dish, a flask, a microplate, a multi-hole plate (Nunc), or a bioreactor is used. The culture vessel is preferably surface-treated to reduce adhesion to the cells.

[0132] 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%.

[0133] The medium may contain an FGFR1 inhibitor in any amount capable of inhibiting FGFR1 activity, for example, 10 μM or less, or 5 μM or less, preferably less than 5 μM, less than 4 μM, less than 3 μM, or less than 2 μM. The lower limit of the amount of the FGFR1 inhibitor to be added is not particularly limited, but may be 0.1 μM or more, preferably 0.5 μM or more. The amount of the FGFR1 inhibitor to be added is preferably less than 5 μM and 0.1 μM or more, more preferably less than 5 μM and 0.5 μM or more. The culture in the presence of the FGFR1 inhibitor may 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. The culture in the presence of the FGFR1 inhibitor is preferably performed for 4 days or more. For example, the culture in the presence of the FGFR1 inhibitor may be performed for about the last 4 to 15 days, preferably about the last 4 to 7 days, of step 6). The medium can be replaced even during the treatment with an FGFR1 inhibitor, and can be replaced with a medium having the same composition as that before the replacement to which the FGFR1 inhibitor was added, or with a medium having a different composition, according to the culture schedule. By culturing cells in a medium containing an FGFR1 inhibitor, the proliferation of Ki67-positive cells in the resulting insulin-producing cells can be suppressed.

[0134] The insulin-producing cells obtained in step 6) can be dissociated and collected using an enzyme such as trypsin. The collected insulin-producing cells can be cryopreserved until use. Next, the collected insulin-producing cells are seeded in the above-mentioned medium at about 500,000 to 5 million cells, preferably about 1 to 4 million cells, more preferably about 2 to 3 million cells per culture vessel or well, and three-dimensional culture is performed to obtain insulin-producing cells in the form of spheroids. Each spheroid consists of about 100 to about 1,000 cells, preferably about 200 to about 800 cells, more preferably about 300 to about 500 cells.

[0135] 2-2.Biocompatible materials As used herein, the term "biocompatible material" refers to any material that, when implanted in a living body and left in place for a short or long period of time, does not induce a significant immune response or adverse biological reaction (e.g., toxic reaction, blood coagulation, etc.). Preferably, the "biocompatible material" promotes angiogenesis and connective tissue production in the host after implantation. In addition, 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 acid-co-glycolic acid (PLGA), poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), poly(ethylene-co-vinyl acetate) (PEVA), polyacrylamide, polyethylene oxide, polyethyleneamine, polyhydroxybutyric acid, poly(N-vinylpyrrolidone), polyvinyl alcohol, polypropylene fumarate, polypropylene glycol, polypropylene glycolate ... Examples of the polyimide-containing polymers include 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, alginic acid, alginate esters, collagen, cellulose, silk fibroin, keratin, gelatin, fibrin, pullulan, laminin, gellan, silicone, urethane, elastin, and modified forms thereof, as well as combinations thereof. If necessary, the surface of the "biocompatible material" may be subjected to a surface modification that enables cell adhesion (for example, coating with a cell adhesion substrate (collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, matrigel, vitronectin, etc.)) or modification with a functional group known to control cell proliferation, differentiation or function (for example, amino group, carboxyl group, hydroxyl group, methacrylic acid group, acrylic acid group, etc.). The "biocompatible material" may have any shape, such as a block, bead, pellet, sphere, sheet, gel, etc., and may be solid or porous.The shape of the "biocompatible material" is preferably a three-dimensional shape, and may be, for example, a sphere, a tetrahedron, a hexahedron, or other polyhedron, a cylinder, a prism, a cone, a truncated cone, a pyramid, a truncated pyramid, a torus, a disk, an ellipsoid, or a modified solid thereof, as long as the cells can be dispersed in the biocompatible material. The "biocompatible material" may be of any size that allows transplantation and placement in a living body, and may have, for example, a side length at the longest point (diameter in the case of a circle) of about 0.1 to 3.0 cm, preferably about 0.5 to 2.0 cm, and a thickness of about 0.1 to 2.0 cm, preferably about 0.3 to 1.5 cm. The cells contained in the biocompatible material consist of about 500,000 to 5 million, preferably about 1 million to 3 million.

[0136] In another embodiment, for example, the size of the "biocompatible material" can be about 1 to 30 cm, preferably about 1 to 15 cm, and more preferably about 2 to 10 cm, at the longest side (diameter if circular).

[0137] The "biocompatible material" may be multiple implanted and placed.

[0138] In the present invention, the "biocompatible material" is preferably in the form of a gel (hydrogel). The gelation of the biocompatible material can be carried out according to a known means appropriate for each biocompatible material, for example, by adding a crosslinker (e.g., a metal cation such as calcium ion, magnesium ion, strontium ion, barium ion, or a salt form thereof) to an aqueous solution of the biocompatible material, or by reacting fibrinogen with thrombin in water. In the present invention, the "biocompatible material" is particularly preferably a fibrin gel. The gel can contain the biocompatible material in an appropriate solvent (e.g., water, physiological saline, culture medium, etc.) in an arbitrary amount of about 0.01 to 10% by weight.

[0139] In the present invention, the cells derived from pluripotent stem cells are dispersed and disposed in a biocompatible material. "Dispersed and disposed" means that the cells derived from pluripotent stem cells are widely distributed and disposed in a biocompatible material without being localized to any site. For example, when the cells derived from pluripotent stem cells have a spheroid form, it means that adjacent spheroids are present with a biocompatible material interposed therebetween. For example, when the cells derived from pluripotent stem cells have a spheroid form, it means that adjacent spheroids are disposed with a distance of about 1 μm or more, preferably about 5 μm or more.

[0140] In another embodiment, "dispersed arrangement" means that, when the cells derived from pluripotent stem cells have a spheroid morphology, a certain percentage or more of the spheroids in the biocompatible material (30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more) are arranged so that they do not come into contact with each other, and preferably 95% or more of the spheroids in the biocompatible material are arranged so that they do not come into contact with each other.

[0141] In another embodiment, "dispersed arrangement" means that, when the cells derived from pluripotent stem cells have a spheroid morphology, a certain percentage or more (30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more) of the spheroids in the biocompatible material are spheroids with a diameter of 50 to 500 μm (meaning that they are not in contact with each other to form larger spheroids), and preferably 95% or more of the spheroids in the biocompatible material are spheroids with a diameter of 50 to 500 μm.

[0142] In another embodiment, "distributed" means that when the cells derived from pluripotent stem cells have a spheroid form, the spheroids are three-dimensionally arranged in a pin dot shape in the biocompatible material. "Distributed three-dimensionally in a pin dot shape" means that each spheroid exists not only three-dimensionally with a space between the spheroids around it, but also in contact with the spheroids around it. Each spheroid may be the same size or different sizes. Here, "distributed" does not mean that each spheroid is arranged in a biocompatible material and then aggregates or fuses to form a larger spheroid.

[0143] "Dispersed arrangement" can be achieved by any means, but can be achieved by thoroughly stirring and mixing a cell solution derived from pluripotent stem cells and seeding it on a biocompatible material, and / or by adjusting the concentration of cells derived from pluripotent stem cells to be seeded on a biocompatible material, and / or by adding cells derived from pluripotent stem cells to a solution of a biocompatible material and thoroughly stirring and mixing. For example, when the biocompatible material is in a gel state, cells derived from pluripotent stem cells are mixed into a solution of the biocompatible material before gelation so that the amount of cells derived from pluripotent stem cells is 1 to 6 million cells, preferably 2 to 5 million cells per 50 to 200 μL, and stirred, and gelation occurs before the cells settle, whereby the cells derived from pluripotent stem cells can be dispersed and arranged in the biocompatible material.

[0144] In another embodiment, for example, when the biocompatible material is in a gel state, 1 × 10 cells derived from pluripotent stem cells are added to a solution of the biocompatible material before gelation at a concentration of 1 × 10 per 15 to 50 mL. 7 ~1×10 10 Cells, preferably 3 x 10 8 ~1×10 9 By mixing and stirring to obtain a volume of 1 cell, and allowing the cells to gel before they settle, the cells derived from pluripotent stem cells can be dispersed and placed in the biocompatible material.

[0145] 3. Biotissue-like structures As used herein, the term "biotissue-like structure" refers to a structure having the same or similar functions as biological organs or tissues, such as (but not limited to) the epidermis, nerves, brain, spinal cord, esophagus, stomach, small intestine, large intestine, bladder, urethra, lung, thyroid, pancreas, liver, muscle, skeleton, heart, blood vessels, spleen, kidney, etc. A biotissue-like structure can be obtained by transplanting and retaining a composition in which cells derived from pluripotent stem cells are dispersed and arranged in a biocompatible material into the living body of an animal, and inducing differentiation of the cells derived from pluripotent stem cells.

[0146] The "animal" is preferably a mammal, and examples thereof include humans, non-human primates, pigs, cows, horses, sheep, goats, llamas, dogs, cats, rabbits, mice, guinea pigs, etc., with humans being preferred.

[0147] The transplantation is preferably carried out in an area of ​​the body where the composition 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.

[0148] After transplantation, the cells derived from the pluripotent stem cells in the composition are induced to differentiate and mature in the in vivo environment, and depending on the "cells derived from the pluripotent stem cells" used, it is possible to obtain structures with functions equivalent to or similar to those of organs or tissues such as (but not limited to) the epidermis, nerves, brain, spinal cord, esophagus, stomach, small intestine, large intestine, bladder, urethra, lungs, thyroid gland, pancreas, liver, muscles, skeleton, heart, blood vessels, spleen, kidneys, etc.

[0149] The obtained biological tissue-like structure may then be recovered or may be left in the body as it is.

[0150] The biological tissue-like structure may contain multiple clusters of differentiated cells obtained by inducing differentiation of cells derived from pluripotent stem cells, connective tissue derived from the host animal, and blood vessels derived from the host animal.

[0151] Differentiated cells obtained by inducing differentiation from cells derived from pluripotent stem cells form multiple clusters and are present in a distributed manner in the biological tissue-like structure. Each cluster has a diameter of about 10 μm to 1000 μm, preferably about 50 μm to 500 μm, more preferably about 50 μm to 300 μm. And / or each cluster is composed of about 100 to about 1000 cells, preferably about 200 to about 800 cells, more preferably about 300 to about 500 cells. In the biological tissue-like structure, about 50 to 2000 clusters, preferably about 100 to 500 clusters, may be present in a distributed manner, but this number may vary depending on the number of cells derived from pluripotent stem cells distributed in the biocompatible material. "Present in a distributed manner" means that each cluster of differentiated cells is not limited to any one site but is widely distributed in the biological tissue-like structure. "Present in a distributed manner" also means that adjacent clusters are present with the host animal-derived connective tissue intervening.

[0152] "Connective tissue derived from a host animal" refers to an extracellular matrix produced by cells derived from an animal into which the composition is transplanted, and includes elastin, fibrillin, type I collagen, type III collagen, etc. In the connective tissue derived from a host animal, differentiated cells exist so as to surround multiple clusters, and fill the spaces between each cluster in the biological tissue-like structure.

[0153] "Blood vessels derived from a host animal" refers to blood vessels formed by cells derived from an animal to which the composition is transplanted, and connected to blood vessels of the host animal outside the biological tissue-like structure. The blood vessels derived from the host animal penetrate into each cluster in the biological tissue-like structure, circulate blood to each cluster, supply oxygen, nutrients, etc., and discharge waste products.

[0154] In one embodiment of the present invention, when definitive endoderm cells, primitive gut cells, posterior foregut cells, pancreatic progenitor cells, endocrine precursor cells, or insulin-producing cells, preferably insulin-producing cells, are used as cells derived from pluripotent stem cells, which appear during the process of differentiation from pluripotent stem cells to pancreatic β cells, these cells are induced to differentiate and mature within the aforementioned composition transplanted into a living body, and a biological tissue-like structure containing differentiated cells (hereinafter referred to as "pancreatic islet-like cells") having functions equivalent or similar to those of pancreatic islets can be obtained.

[0155] In the thus obtained biological tissue-like structure, the pancreatic islet-like cells are cultured in the following manner (a) to (g): (a) expressing at least one marker of MAFA, UCN3, and IAPP; (b) a high proportion of chromogranin A-positive cells (Chga+ cells); (c) The percentage of Ki67-positive cells (Ki67+ cells) is low. (d) a high proportion of glucagon-positive and insulin-negative cells (Gcg+Ins- cells); (e) having an insulin secreting effect in response to hypoglycemia; (f) containing no exocrine cells; (g) a high proportion of insulin-positive and glucagon-negative cells (Ins+Gcg- cells); wherein "multiple" means 2, 3, 4, 5, 6, or 7.

[0156] The presence or absence of the characteristics (a) to (g) can be evaluated 4 weeks or more, preferably 8 weeks or more, after the differentiation of the insulin-producing cells is induced (i.e., after the above-mentioned composition is transplanted into a living body). The ratio of a given cell in the pancreatic islet-like cells means the ratio to the total number of cells in the cell mass derived from the graft.

[0157] (a) expressing at least one marker of MAFA, UCN3, and IAPP; The islet-like cells include pancreatic β cells, which express at least one of the maturation markers MAFA, UCN3, and IAPP, and can also be characterized by an increased insulin secretion response to glucose stimulation.

[0158] (b) A high percentage of Chga+ cells Pancreatic islet-like cells are characterized by comprising Chga+ cells at a ratio of about 50% or more.Preferably, the ratio of Chga+ cells in pancreatic islet-like cells is about 60% or more, more preferably about 70% or more, even more preferably about 90% or more, particularly preferably about 95% or more (for example, about 97% or more, about 98% or more).

[0159] Chga+ cells include cells that secrete hormones such as insulin and glucagon (endocrine cells), and this characteristic indicates that the pancreatic islet-like cells contain a high proportion of endocrine cells.

[0160] (c) Low percentage of Ki67+ cells The pancreatic islet-like cells are characterized by containing Ki67-positive cells at a rate of less than about 3%. Preferably, the rate of Ki67-positive cells in the pancreatic islet-like cells is less than about 1%, more preferably less than about 0.8%, and even more preferably less than about 0.5%.

[0161] This characteristic indicates that there are almost no Ki67+ cells or a very low percentage of Ki67+ cells in the islet-like cells, the inclusion or persistence of which may be undesirable.

[0162] (d) A high percentage of Gcg+Ins- cells The islet-like cells are characterized by containing Gcg+Ins- cells at a ratio of about 10% or more. Preferably, the ratio of Gcg+Ins- cells in the islet-like cells is about 15% or more, more preferably about 20% or more, and even more preferably about 25% or more. The upper limit of the ratio of Gcg+Ins- cells in the islet-like cells is not particularly limited, but can be, for example, about 50% or less, preferably about 45% or less, and more preferably about 40% or less.

[0163] Because Gcg+Ins- is a marker for mature pancreatic alpha cells, this indicates that the islet-like cells contain a higher proportion of mature pancreatic alpha cells than insulin-producing cells, indicating that the islet-like cells are at a more mature differentiation stage than insulin-producing cells.

[0164] (e) Having an insulin secreting effect in response to hypoglycemia The islet-like cells are characterized by having insulin secretion in response to hypoglycemia. "Insulin secretion in response to hypoglycemia" means that the amount of insulin secretion increases within 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours from the time of hypoglycemia. "Hypoglycemia" means that the glucose concentration in blood or in the medium is about 70 mg / dL or less. The amount of insulin secretion can be measured by any conventionally known means, and can be measured by measuring the amount of C-peptide in blood or in the medium, although this is not particularly limited.

[0165] Normally, when hypoglycemia occurs in a living body, the secretion of glucagon and the like promotes an increase in blood glucose level, and insulin is secreted in opposition to glucagon to control the blood glucose level. The islet-like cells of the present invention similarly enable control of blood glucose level in response to hypoglycemia, and have the effect of promoting an increase in blood glucose level in response to hypoglycemia and secreting insulin in opposition to this.

[0166] (f) No exocrine cells The islet-like cells are characterized by not containing exocrine cells. In other words, the exocrine cells that secrete pancreatic juice containing digestive enzymes such as amylase and lipase, which usually constitute the majority (about 95%) of pancreatic islets in an animal's living body, are not contained in the islet-like cells. The term "not containing exocrine cells" may include not only the case where the islet-like cells do not contain any exocrine cells, but also the case where the islet-like cells contain exocrine cells at a low rate of 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less.

[0167] (g) A high percentage of Ins+Gcg- cells Islet-like cells are characterized by a high ratio of Ins+Gcg- cells.Preferably, the ratio of Ins+Gcg- cells in islet-like cells is about 20% or more, more preferably about 30% or more, and even more preferably about 40% or more.The upper limit of the ratio of Ins+Gcg- cells in islet-like cells is not particularly limited, but can be, for example, about 80% or less, preferably about 70% or less, and more preferably about 60% or less.

[0168] Since Ins+Gcg- is a marker for mature pancreatic β cells, this indicates that the islet-like cells contain a higher proportion of mature pancreatic β cells than insulin-producing cells, and that the islet-like cells are at a more mature differentiation stage than insulin-producing cells.

[0169] Furthermore, when the biological tissue-like structure contains pancreatic islet-like cells, the pancreatic islet-like cells can form clusters in which insulin-positive cells are located on the inside and glucagon-positive cells are located on the outside.

[0170] 5.Applications The biological tissue-like structures obtained by this method have functions equivalent to or similar to those of organs and tissues such as the epidermis, nerves, brain, spinal cord, esophagus, stomach, small intestine, large intestine, bladder, urethra, lungs, thyroid gland, pancreas, liver, muscle, skeleton, heart, blood vessels, spleen, and kidneys (but are not limited to these), depending on the "pluripotent stem cell-derived cells" used, and can be used to enhance or maintain the functions of these organs and tissues, or to support or supplement the functions of these organs and tissues that have been reduced or lost due to disease, disorder, etc. As one embodiment of the present invention, when the biological tissue-like structure contains the aforementioned pancreatic islet-like cells, the biological tissue-like structure can improve and / or maintain blood sugar (glucose) levels in a patient through the action of insulin and glucagon secreted by the pancreatic islet-like cells, and can control blood sugar levels to normal levels.

[0171] Therefore, the biological tissue-like structure can be used to treat or prevent diseases, disorders, or conditions that require improvement and / or maintenance of blood glucose levels. Such diseases, disorders, or conditions include, but are not limited to, diabetes (type 1 diabetes, type 2 diabetes), altered fasting and postprandial glucose levels, hypoglycemia (e.g., hypoglycemia due to insulin administration in diabetic patients), and the like. Note that "treatment" refers to treating, curing, preventing, or improving remission of a disease, disorder, or condition, or reducing the rate of progression of a disease, disorder, or condition. Also, "prevention" refers to reducing the possibility or risk of onset of a disease, disorder, or condition, or delaying onset of a disease, disorder, or condition.

[0172] The patient is a mammal (eg, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human), preferably a human.

[0173] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0174] Example 1: Generation, transplantation, and functional and morphological evaluation of insulin-producing cells (STZ-Nod-scid mice) 1. Method (1) Creation of insulin-producing cells Differentiation of the Ff-I14s04 iPS cell line into insulin-producing cells was performed by three-dimensional culture using a bioreactor, following steps 1) to 6) above and a previous report (Nature Biotechnology 2014;32:1121-1133). That is, iPS cells were first cultured in a medium (DMEM / 1% B27 / penisilin streptomycin / dimethyl sulfoxide) containing differentiation inducers (GSK3β inhibitor, ROCK inhibitor and low dose of activin A) under conditions in which insulin is active, and then second cultured in a medium (DMEM / 1% B27 / penisilin streptomycin / dimethyl sulfoxide) containing differentiation inducers (low dose of activin A) under conditions in which insulin is not active, to obtain definitive endoderm cells.

[0175] The endocrine precursor cell population obtained by differentiation induction from the definitive endoderm cells was cultured for 8 days in a medium (Improved MEM / 1% B27 / Penisilin Streptomycin) containing differentiation inducers (ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, ascorbic acid) and an FGF receptor 1 inhibitor (PD-166866).Furthermore, the cells were cultured for 4 days in a medium (MCDB / ITS-X / 2% BSA / 20 mM glucose / NaHCO3 / Glutamax / Penisilin Streptomycin) containing some different differentiation inducers (ALK5 inhibitor II, T3, ZnSO4, heparin, N-acetylcysteine, Trolox, R428, Y-27632) and an FGF receptor 1 inhibitor (PD-166866), to obtain insulin-producing cells in the form of spheroids.

[0176] (2) Evaluation of protein expression in insulin-producing cells before transplantation Protein expression (insulin, NKX6.1, chromogranin, Ki67) of the insulin-producing cells prepared in (1) was measured by flow cytometry.

[0177] (3) Dispersion and gelation of insulin-producing cells into fibrin gel Fibrinogen (Merck Millipore, 341576-100MG) for preparing fibrin gel was dissolved in basal medium (MCDB) to a concentration of 10 mg / mL and stored at -80°C until use. Thrombin (Sigma, 10602400001) was dissolved in PBS(-) to a concentration of 50 U / mL and stored at -80°C until use. The fibrinogen solution and thrombin solution were dissolved immediately before use. The insulin-producing cell spheroids (approximately 50-500 μm in diameter, containing a total of 3 million cells) prepared in (1) were collected in a 1.5 mL tube and allowed to settle. After settling, the culture medium was removed as much as possible, and 100 μL of fibrinogen solution was added and thoroughly stirred. Next, before the aggregates had settled, 1 / 2 the amount (50 μL) of thrombin solution was added to the fibrinogen solution. After adding the thrombin solution, the mixture was left at room temperature for 5 minutes or more to gel it.

[0178] (4) Transplantation of insulin-producing cells into the body and functional evaluation of the process of forming a tissue-like structure The fibrin gel prepared in (3) containing dispersed insulin-producing cells was subcutaneously transplanted into immunodeficient NOD / SCID mice in which diabetes was induced by streptozotocin (STZ) (iPIC group). After transplantation, the concentration of human C-peptide (an indicator of insulin derived from insulin-producing cells) and blood glucose level in the blood were measured to evaluate the engraftment and function of the insulin-producing cells. As controls, non-transplanted individuals (sham group) and non-diabetic NOD / SCID mice (control group) were set up, and the same indicators were measured and evaluated.

[0179] (5) Evaluation of glucose response of insulin-producing cell-derived tissue-like constructs Fibrin gels containing dispersed insulin-producing cells were implanted subcutaneously, and after six months, glucose was forcefully administered orally to transiently increase blood glucose levels. The subsequent response of the insulin-producing cells was evaluated by measuring plasma glucose and human C-peptide concentrations.

[0180] (6) Evaluation of general staining and protein expression in tissue-like structures derived from insulin-producing cells The insulin-producing cell-derived tissue-like constructs were extracted 6 months after transplantation and fixed with paraformaldehyde. Paraffin sections and frozen sections were prepared from the fixed tissue-like constructs. HE staining and Masson's trichrome staining were performed on the paraffin sections. In addition, expression of target proteins (human nucleus (HuN), PDX1, insulin (INS), glucagon (GCG), mouse CD31 (mCD31), chromogranin A (CHGA), and Ki67) was evaluated by immunohistochemical staining using frozen sections.

[0181] 2.Results (1) Protein expression assessment of insulin-producing cells before transplantation The results of flow cytometry measurements of insulin-producing cells before transplantation are shown in Figure 1, and the rates of insulin-positive / NKX6.1-positive cells, chromogranin A-positive cells, and Ki67-positive cells are shown in Table 1. More than 95% of the insulin-producing cells before transplantation were chromogranin A-positive endocrine cells, and after transplantation, 42.6% of the cell population were insulin-positive / NKX6.1-positive cells that would mature into β cells, and 30.3% were insulin-positive / NKX6.1-negative cells that would mature into α cells. In addition, Ki67-positive cells, a proliferation marker, accounted for only a very small proportion (0.4%).

[0182] [Table 1]

[0183] (2) Functional evaluation of the process in which insulin-producing cells transplanted into the body form a tissue-like structure The results of measuring the blood human C-peptide concentration and blood glucose level after transplantation are shown in Figure 2 and Table 2. In animals transplanted with fibrin gel containing dispersed insulin-producing cells, high concentrations of human C-peptide were detected in the blood three months after transplantation, and hyperglycemia improved accordingly. Blood glucose levels remained at normal levels for up to six months. These results suggest that the fibrin gel containing dispersed transplanted insulin-producing cells differentiates and matures while mixing with host cells subcutaneously, forming a functional biological tissue-like structure.

[0184] [Table 2]

[0185] (3) Evaluation of the response of insulin-producing cell-derived tissue-like constructs to glucose loading Figure 3 shows the blood human C-peptide concentration and plasma glucose concentration after glucose loading. The tissue-like construct formed by implanting fibrin gel with dispersed insulin-producing cells secreted human C-peptide in the blood in a sensitive glucose response six months after implantation. Despite the implantation site being subcutaneous, a clear increase in human C-peptide in the blood was observed 15 minutes after glucose was added, confirming a biological response similar to that of actual pancreatic islets mediated by an abundant blood flow. Furthermore, blood human C-peptide also decreased as blood glucose decreased, suggesting that there is little concern about hypoglycemia.

[0186] (4) Evaluation of general staining and protein expression in tissue-like structures derived from insulin-producing cells Figure 4 shows the macroscopic appearance of the insulin-producing cell-derived tissue-like construct 6 months after transplantation, Figure 5 shows an HE stained image of the excised tissue-like construct, Figure 6 shows a Masson's trichrome stained image, and Figures 7-10 show the results of immunohistochemical staining of the target protein expression. The macroscopic findings of the insulin-producing cell-derived biotissue-like construct (Figure 4) were that it was a rice grain-sized biotissue-like construct without any noticeable hypertrophy and could be easily isolated from the subcutaneous tissue. A total of four animals were dissected at the same time, and all had similar macroscopic findings. Histological evaluation showed that the biotissue-like construct was composed of 1) insulin-producing cell-derived pancreatic islet-like cell clusters with a diameter of 50 to 500 μm, 2) host-derived fibrous tissue surrounding the clusters, and 3) host-derived vascular structures (Figures 5-10).

[0187] 3) The host-derived vascular structure (=mouse CD31 positive, Figure 9) mainly penetrated into the endocrine cell cluster 1), and many red blood cells were observed within the islet-like cell cluster 1) (Figures 5 and 6). This strongly suggests that this tissue-like structure, like an actual pancreatic islet, has abundant blood flow and a sensitive biological response mediated by the blood flow. In addition, since it contains a large amount of fibrous tissue (Figure 6), it also has high physical strength. When an attempt was made to actually cut the insulin-producing cell-derived tissue-like structure, it had the strength of cartilage and could not be cut easily.

[0188] The majority of cells derived from HuN-positive insulin-producing cells were pancreatic islet-like cells (Figures 7-10), and no proliferation of non-target cells was observed. Cells positive for both proliferation markers Ki67 and HuN were also minimal, at less than 0.5% (Figure 10). Insulin-positive cells were distributed inside the pancreatic islet-like cell clusters in 1), while glucagon-positive cells were distributed outside the pancreatic islet-like cell clusters in 1), an arrangement observed in prenatal fetal human pancreas or rodent pancreatic islets. This suggests that this tissue-like structure can function for decades, similar to fetal human islets.

[0189] These results show that when insulin-producing cells are dispersed in fibrin gel and transplanted into the body, they survive for a long time in the subcutaneous area, which is poor in blood flow and nutrients, and furthermore, they mature while mixing with the host cells to form a new tissue-like structure. The formed subcutaneous tissue-like structure was shown to have a blood glucose control function accompanied by physiological and sensitive insulin secretion regulation, similar to pancreatic islets in the body.

[0190] Example 2: Formation and functional evaluation of tissue-like structures derived from insulin-producing cells in vivo with diabetic pathology 1. Method The fibrin gel containing dispersed insulin-producing cells prepared in (3) of Example 1 above was subcutaneously transplanted into immunodeficient NOD / SCID mice in which diabetes was induced by streptozotocin (STZ) or into non-diabetic NOD / SCID mice. Human C-peptide, glucagon and blood glucose levels in the blood were measured under a state of fasting up to 28 weeks after transplantation. After 28 weeks, the insulin-producing cell-derived tissue-like structures were excised from all individuals. The weight of the excised graft tissue-like structures was measured, and the insulin and glucagon contents in the graft tissue-like structures were measured after crushing and treating with acid ethanol. As controls, the pancreases of each transplanted mouse and non-transplanted non-diabetic NOD / SCID mice were collected and the hormone contents were measured in the same manner.

[0191] 2.Results Figure 11 shows the changes in blood levels of human C-peptide and glucagon, as well as blood glucose levels. Human C-peptide was continuously detected in the blood up to 28 weeks after transplantation of fibrin gels containing dispersed insulin-producing cells. Blood glucagon levels were also elevated, and were higher than those of diabetic and non-diabetic non-transplanted mice. It was suggested that the fibrin gels containing dispersed insulin-producing cells survived for a long time in the subcutaneous environment, and that the formed tissue-like structures released multiple islet hormones. Hyperglycemia in diabetic mice improved by 12 weeks after transplantation, and blood glucose levels remained in the normal range up to 28 weeks after transplantation. When the tissue-like structures were removed, the hyperglycemic state before transplantation was completely restored, indicating that the sustained and stable hyperglycemia-improving effect was derived from the tissue-like structures. The hormone content in the graft tissue-like structure and the pancreas is shown in Figure 12. Compared to the pancreas (left), the graft tissue-like structure (right) had a higher insulin and glucagon content per unit weight. The graft tissue-like structure was shown to contain a high density of pancreatic endocrine cells, similar to the islets in the pancreas.

[0192] Example 3: Blood glucose control function of a biological tissue-like structure derived from insulin-producing cells 1. Method The fibrin gel containing dispersed insulin-producing cells prepared in (3) of Example 1 above was subcutaneously transplanted into immunodeficient NOD / SCID mice in which diabetes was induced by STZ, or into NOD / SCID mice with Akita gene mutations that spontaneously develop diabetes. After 14 weeks from transplantation, the following test was performed using mice in which hyperglycemia was completely improved.

[0193] Test (1): Glucose was administered orally to transiently increase blood glucose levels, and the response of the insulin-producing cell-derived tissue-like construct was evaluated by measuring the blood levels of human C-peptide, glucagon, and glucagon-like peptide-1. As a control, non-transplanted, non-diabetic NOD / SCID mice were used to measure the blood levels of endogenous hormones.

[0194] Test (2): To evaluate the involvement of glucagon in the response of the insulin-producing cell-derived biological tissue-like structure, the glucagon receptor antagonist MK-0893 was administered orally before glucose administration, and a test similar to that in Test (1) was performed. MK-0893 shows stronger antagonist activity against the human glucagon receptor than that in mice (J Med Chem. 2012 Jul 12; 55(13): 6137-48).

[0195] Study (3): To evaluate the involvement of glucagon-like peptide-1 in insulin release by insulin-producing cell-derived tissue-like structures, the glucagon-like peptide-1 receptor antagonist Exendin-9 was continuously administered subcutaneously for three days using an osmotic pump, and blood human C-peptide concentrations were measured before and after administration.

[0196] Study (4): To induce hypoglycemia, the insulin preparation glargine was administered subcutaneously, and the response of the insulin-producing cell-derived tissue-like construct was evaluated by measuring human C-peptide in the blood. As a control, non-transplanted, non-diabetic NOD / SCID mice were used, and the blood concentration of mouse C-peptide derived from endogenous pancreatic islets was measured.

[0197] 2.Results Test (1): The blood glucagon and glucagon-like peptide-1 concentrations after glucose loading are shown in Figure 13. Blood human C-peptide and glucagon-like peptide-1 concentrations transiently increased with glucose loading, while blood glucagon concentrations tended to decrease. Transplanted mice showed higher blood glucagon-like peptide-1 and glucagon concentrations than non-transplanted, non-diabetic mice, suggesting that the biological tissue-like structure releases these hormones.

[0198] Test (2): Blood glucose levels and blood human C-peptide concentrations after glucose loading are shown in Figure 14. Pretreatment with MK-0893 attenuated the transient rise in blood glucose levels after glucose loading, as well as the rise in blood human C-peptide concentrations. On the other hand, MK-0893 did not affect blood glucose levels or blood concentrations of endogenous mouse C-peptide in non-transplant, non-diabetic mice. These results indicate that glucagon contributes to the blood glucose control effect of the biological tissue-like structure.

[0199] Test (3): Figure 15 shows the human C-peptide concentration in blood during fasting before and after 3-day administration of Exendin-9. The human C-peptide concentration in blood decreased with continuous administration of Exendin-9, and returned to the level before administration when administration was stopped. It was shown that the release of human C-peptide by the biological tissue-like structure is controlled by glucagon-like peptide-1.

[0200] Study (4): Blood human C-peptide concentrations were significantly reduced when hypoglycemia was induced by administration of glargine. This change was similar to the changes in blood concentrations of endogenous mouse C-peptide in non-transplant, non-diabetic mice. This demonstrated that the tissue-like structures exhibited insulin secretion regulation functions similar to endogenous pancreatic islets when hypoglycemia was induced.

[0201] These results demonstrate that the insulin-producing cell-derived tissue-like construct formed by transplanting fibrin gel containing dispersed insulin-producing cells exerts a physiological blood glucose control function similar to that of endogenous pancreatic islets, with multiple islet hormones contributing to it.

Claims

1. A composition for use in a method for producing a biological tissue-like structure in a biological tissue of a host animal, comprising a biocompatible material and cells derived from human pluripotent stem cells, the cells derived from human pluripotent stem cells being dispersed and disposed in the biocompatible material; the cells derived from human pluripotent stem cells are insulin-producing cells, and a rate of Ki67-positive cells in the insulin-producing cells is less than 3%; A composition, wherein the cells derived from human pluripotent stem cells are present in the form of multiple spheroids.

2. The composition of claim 1 , wherein the biocompatible material is a fibrin gel.

3. The composition according to claim 2 , wherein the fibrin gel is gelled by mixing cells derived from human pluripotent stem cells, fibrinogen and thrombin immediately prior to use of the composition.

4. The composition according to any one of claims 1 to 3, wherein the method comprises transplanting the composition into a biological tissue of a host animal and inducing differentiation of cells derived from human pluripotent stem cells dispersed and disposed in a biocompatible material.

5. The composition according to claim 4 , wherein the living tissue of the host animal is subcutaneous tissue.

6. The composition according to any one of claims 1 to 5, wherein the biological tissue-like structure comprises a plurality of clusters of differentiated cells obtained by inducing differentiation of cells derived from human pluripotent stem cells, connective tissue derived from a host animal, and blood vessels derived from the host animal, the plurality of clusters of the differentiated cells being dispersed in the biological tissue-like structure, the connective tissue surrounding the plurality of clusters of the differentiated cells, and the blood vessels penetrating into the plurality of clusters of the differentiated cells.

7. The composition of claim 6 , wherein the differentiated cells do not include exocrine cells.

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