Method for producing insulin-producing cells

Abstract

The main subject of the present invention is to provide a method for producing insulin-producing cells from somatic cells without artificial gene introduction, insulin-producing cells obtained by the production method, or a composition containing a combination of chemical substances that can be used in the production method. As examples of the present invention, there can be mentioned: a method for producing insulin-producing cells by direct differentiation induction from somatic cells, characterized in that it includes a step of culturing somatic cells in the presence of an RSK inhibitor; insulin-producing cells obtained by the production method; a composition for producing insulin-producing cells by direct differentiation induction from somatic cells, characterized in that it contains an RSK inhibitor. The insulin-producing cells obtained by the present invention are useful in regenerative medicine, etc.

Description

Technical Field

[0001] (Cross-reference to related applications)

[0002] This application claims the priority of Japanese Application No. 2018-213448 filed with the Japan Patent Office on November 14, 2018. All application documents (specification, claims, drawings, abstract) of this Japanese application are expressly incorporated herein by reference for all purposes of this specification.

[0003] The present invention belongs to the technical field of regenerative medicine, or direct reprogramming from somatic cells. The present invention relates to: in this technical field, a method for directly producing insulin-producing cells from somatic cells using low molecular weight compounds, and low molecular weight compound-induced insulin-producing cells (ciIPCs: chemical compound-induced Insulin-producing cells) produced by the production method. The present invention further relates to the insulin-producing cells, and a composition that can be used in the method for producing the insulin-producing cells. Background Art

[0004] There have been reports of methods for combining several low molecular weight compounds such as ALK5 inhibitors and ALK6 inhibitors, culturing somatic cells, especially human fibroblasts, in the presence of these compounds, and then inducing them to differentiate directly into brown fat cells, osteoblasts, chondrocytes, nervous system cells or cardiomyocytes without gene introduction (e.g., Patent Document 1). As in the invention of Patent Document 1, it would be very beneficial if common fibroblasts could be directly induced into cells such as brown fat cells by easily available low molecular weight compounds. For example, since other cells of the body can be easily made from its own fibroblasts, its application in regenerative medicine can be expanded. In addition, cells that are used as experimental materials for the development of new pharmaceuticals can also be easily made.

[0005] Mesenchymal cells form various organs of organisms such as muscle, bone, cartilage, bone marrow, fat, and connective tissue, and are expected to be used as materials for regenerative medicine. Mesenchymal stem cells (MSC) are undifferentiated cells found in tissues such as bone marrow, adipose tissue, blood, placenta, and umbilical cord. Since they have the ability to differentiate into cells belonging to the mesenchymal system, mesenchymal stem cells have attracted attention as starting materials for the production of these cells. In addition, regenerative medicine that uses mesenchymal stem cells themselves to rebuild bones, cartilage, myocardium, etc. is also being explored.

[0006] In fact, there are also reports on differentiation of mesenchymal stem cells into neural cells or insulin-producing cells (Non-Patent Documents 1 and 2). In Non-Patent Document 1, mesenchymal stem cells are differentiated into neural cells by combining SB431542 and a low-molecular-weight inhibitor such as dolmorphine.

[0007] For the pancreatic β cells that secrete insulin, in addition to the reports of differentiation induction into human pancreatic β cells from iPS cells or ES cells, there are also endodermal cells from pancreatic α cells, pancreatic acinar cells, pancreatic ductal gland cells, small intestinal crypt cells, hepatocytes, bile duct cells and the like, using pancreatic β cell-specific transcription factors Pdx1, Ngn3 and MafA, directly induced into reports of pancreatic β cells (non-patent literature 3). In addition, there are also reports of direct induction into pancreatic β cells from mouse embryonic fibroblasts (MEFs) or human skin fibroblasts, using Yamanaka 4 factors, etc. (non-patent literature 4, 5). There is even a report (non-patent literature 6) from mouse embryonic fibroblasts (MEFs), by low molecular weight compounds, making endodermal progenitor cells, and then differentiating into pancreatic endocrine cells from the cells.

[0008] Regarding BRD7389, a low molecular weight compound that is an RSK inhibitor, it has been reported to have the ability to activate the expression of the insulin gene in pancreatic α cells (Non-Patent Document 7).

[0009] Prior art literature

[0010] Patent Literature

[0011] [Patent Document 1] International Publication No. 2018 / 062269

[0012] Non-patent literature

[0013] [Non-patent document 1] Stem Cells International, Volume 2016, Article ID 1035374

[0014] [Non-patent document 2] BioMed Research International, Volume 2015, Article ID 575837

[0015] [Non-patent document 3] Current Pathobiology Reports, 2015, Vol. 3, pp. 57-65

[0016] [Non-patent document 4] Cell Stem Cell, 2014, Vol. 14, pp. 228-236

[0017] [Non-patent document 5] Nature Communications, 2016, Vol. 7, p. 10080

[0018] [Non-patent document 6] Journal of Biological Chemistry, 2017, Vol. 292, pp. 19122-19132

[0019] [Non-patent document 7] PNAS, August 24 (2010), Vol. 107, no. 34, 15099-15104 Summary of the invention

[0020] Technical Problems to be Solved by the Invention

[0021] As described in Patent Document 1, a method of directly converting somatic cells into desired cells without gene introduction is an effective option as a means of obtaining therapeutic cells. As described above, methods of directly converting pancreatic β cells from somatic cells have also been reported, and these inventions induce by introducing specific genes into cells of the endoderm class that are similar in embryology.

[0022] The main object of the present invention is to provide a method for efficiently and directly inducing differentiation of insulin-producing cells from somatic cells by combining low molecular weight compounds without artificial gene introduction, that is, a new production method capable of directly producing insulin-producing cells from somatic cells by using certain low molecular weight compounds.

[0023] Technical means to solve technical problems

[0024] As a result of intensive studies, the present inventors have found that somatic cells can be efficiently and directly converted into insulin-producing cells by culturing them in the presence of certain low-molecular-weight inhibitors and the like, thereby completing the present invention.

[0025] As the present invention, for example, the following items can be cited:

[0026] [1] A method for producing insulin-producing cells, which is a method for producing insulin-producing cells by direct differentiation induction from somatic cells, wherein the method comprises:

[0027] A step of culturing somatic cells in the presence of an RSK inhibitor.

[0028] [2] The method for producing insulin-producing cells according to item [1], wherein

[0029] The step is a step of further culturing somatic cells in the presence of a GSK3 inhibitor.

[0030] [3] The method for producing insulin-producing cells according to item [1] or [2], wherein

[0031] The step is a step of further culturing the somatic cells in the presence of a cAMP inducer and / or a PI3K inhibitor.

[0032] [4] The method for producing insulin-producing cells according to any one of items [1] to [3], wherein

[0033] The RSK inhibitors were BRD7389 or BI-D1870.

[0034] [5] The method for producing insulin-producing cells according to any one of items [2] to [4], wherein

[0035] The GSK3 inhibitor was CHIR99021.

[0036] [6] The method for producing insulin-producing cells according to any one of items [3] to [5], wherein the cAMP inducer is forskolin or the PI3K inhibitor is LY294002.

[0037] [7] The method for producing insulin-producing cells according to any one of items [1] to [6], wherein

[0038] The somatic cells are fibroblasts or mesenchymal stem cells.

[0039] [8] An insulin-producing cell produced by the method for producing an insulin-producing cell according to any one of [1] to [7].

[0040] [9] A composition for producing insulin-producing cells from somatic cells, which is a composition for producing insulin-producing cells by direct differentiation induction from somatic cells, wherein:

[0041] The composition comprises a RSK inhibitor.

[0042]

[10] The composition described in item [9], further comprising a GSK3 inhibitor.

[0043]

[11] The composition of item [9] or

[10] , further comprising a cAMP inducer and / or a PI3K inhibitor.

[0044]

[12] The composition according to any one of items [9] to

[11] , wherein

[0045] The RSK inhibitors were BRD7389 or BI-D1870.

[0046]

[13] The composition according to any one of items

[10] to

[12] , wherein the GSK3 inhibitor is CHIR99021.

[0047]

[14] The composition according to any one of items

[11] to

[13] , wherein the cAMP inducer is forskolin or the PI3K inhibitor is LY294002.

[0048]

[15] The composition according to any one of items [9] to

[14] , wherein

[0049] The somatic cells are fibroblasts or mesenchymal stem cells.

[0050] Effects of the Invention

[0051] According to the present invention, insulin-producing cells with high secretion capacity can be efficiently produced directly from somatic cells without artificial gene introduction. The insulin-producing cells obtained by the present invention are useful in regenerative medicine and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] [ Figure 1 ] represents the amount of insulin secreted by insulin-producing cells directly differentiated from human fibroblasts. The vertical axis represents the amount of insulin secreted per 1 mg of total protein of the cells (μU / mg).

[0053] [ Figure 2 ] represents the amount of insulin secreted by insulin-producing cells directly differentiated from human fibroblasts. The vertical axis represents the amount of insulin secreted per 1 mg of total protein of the cells (μU / mg).

[0054] [ Figure 3 ] represents the amount of insulin secreted by insulin-producing cells directly differentiated from human fibroblasts. The vertical axis represents the amount of insulin secreted per 1 mg of total protein of the cells (μU / mg).

[0055] [ Figure 4 ] represents the amount of insulin secreted by insulin-producing cells directly differentiated from human fibroblasts. The vertical axis represents the amount of insulin secreted per 1 mg of total protein of the cells (μU / mg).

[0056] [ Figure 5 ] represents the amount of insulin secreted by insulin-producing cells directly differentiated and induced from human adipose tissue-derived mesenchymal stem cells (AdMSC). The vertical axis represents the amount of insulin secreted per 1 mg of total protein of the cells (μU / mg).

[0057] [ Figure 6] represents the amount of insulin secreted by insulin-producing cells directly differentiated and induced from human adipose tissue-derived mesenchymal stem cells (AdMSC). The vertical axis represents the amount of insulin secreted per 1 mg of total protein of the cells (μU / mg).

[0058] Specific embodiments of the present invention

[0059] Hereinafter, the present invention will be described in detail.

[0060] 1. Method for producing insulin-producing cells

[0061] The method for producing insulin-producing cells according to the present invention (hereinafter referred to as "the production method of the present invention") is a method for producing insulin-producing cells by direct differentiation induction from somatic cells, wherein the method comprises the step of culturing somatic cells in the presence of an RSK inhibitor.

[0062] It is preferred that the production method of the present invention further comprises a step of culturing somatic cells in the presence of a GSK3 inhibitor. It is more preferred that the production method of the present invention further comprises a step of culturing somatic cells in the presence of a cAMP inducer and / or a PI3K inhibitor. In the production method of the present invention, it is particularly preferred that the step of culturing somatic cells in the presence of an RSK inhibitor and a GSK3 inhibitor or further in the presence of a cAMP inducer is included. The step may also be a step of culturing somatic cells in the presence of any other inhibitor, inducer, etc. as required.

[0063] In the production method of the present invention, it is sufficient to culture somatic cells at least in the presence of the above-mentioned inhibitor or the like, and if necessary, other inhibitors, inducers, or the like may be further arbitrarily added to culture the somatic cells to produce insulin-producing cells.

[0064] The above inhibitors and inducers may be used alone or in combination of two or more.

[0065] Specifically, some of the inhibitors may have two or more inhibitory effects, and in this case, the presence of one inhibitor may be regarded as the presence of multiple inhibitors.

[0066] 1.1 About somatic cells

[0067] Biological cells can be classified into somatic cells and germ cells. In the manufacturing method of the present invention, any somatic cells can be used as its starting material. Somatic cells are not particularly limited and can be primary cells collected from a living body or cells of cell lines. In the manufacturing method of the present invention, somatic cells in various stages of differentiation can be used, for example, terminally differentiated somatic cells (e.g., fibroblasts, umbilical vein endothelial cells (HUVEC), hepatocytes (Hepatocytes), bile duct cells (Biliary cells), pancreatic α cells (Pancreaticαcells), pancreatic acinar cells (Acinar cells), pancreatic ductal gland cells (Ductal cells), intestinal crypt cells (Intestinal crypt cells), etc.), somatic cells in the process of terminal differentiation (e.g., mesenchymal stem cells, neural stem cells, endodermal progenitor cells, etc.) or somatic cells that have been initialized and obtained pluripotency. Somatic cells can be used as the manufacturing method of the present invention, and any somatic cells can be enumerated, for example, hematopoietic cells (various lymphocytes, macrophages, dendritic cells, bone marrow cells, etc.), organ-derived cells (hepatocytes, spleen cells, pancreatic cells, kidney cells, lung cells, etc.), muscle tissue cells (skeletal muscle cells, smooth muscle cells, muscle bud cells, cardiomyocytes, etc.), fibroblasts, nerve cells, osteoblasts, chondrocytes, endothelial cells, interstitial cells, adipocytes (white adipocytes, etc.), embryonic stem cells (ES cells), etc. In addition, the progenitor cells and cancer cells of these cells can also be applied to the manufacturing method of the present invention. Fibroblasts or mesenchymal stem cells can be preferably used.

[0068] There are no particular limitations on the fibroblasts that can be used in the present invention, and examples thereof include dermal fibroblasts, adventitial fibroblasts, cardiac fibroblasts, pulmonary fibroblasts, uterine fibroblasts, and villous mesenchymal fibroblasts, which are the main cell components that constitute connective tissue in various tissues or organs and produce collagen fibers.

[0069] The mesenchymal stem cells that can be used in the present invention are not particularly limited, and examples thereof include adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, endometrium-derived mesenchymal stem cells, synovium-derived mesenchymal stem cells, and mesenchymal stem cells derived from the uterus. cells), mesenchymal stem cells derived from dermis tissue, and mesenchymal stem cells derived from dental ligament.

[0070] As the supply source of the somatic cells, humans, mammals other than humans, and animals other than mammals (birds, reptiles, amphibians, fish, etc.) can be cited, but are not limited thereto. As the supply source of the somatic cells, humans and mammals other than humans are preferred, and humans are particularly preferred. When insulin-producing cells are manufactured by the manufacturing method of the present invention for the purpose of administering to humans, somatic cells collected from donors with the same or similar types of tissue compatibility antigens as the recipient can be preferably used. Somatic cells collected from the recipient himself can also be used for the manufacture of insulin-producing cells.

[0071] 1.2 Inhibitors and the like involved in the present invention

[0072] 1.2.1 RSK Inhibitor

[0073] Ribosomal S6 Protein Kinase (RSK) is widely expressed in cells and is one of the serine / threonine kinases that respond to various growth factors. It is divided into subfamilies with molecular weights of 70kDa and 90kDa. In particular, the 90kDa RSK subfamily is activated by phosphorylation by ERK, which belongs to the MAPK signaling pathway, and there are four genes in mammals. The activated 90kDa RSK phosphorylates various proteins including downstream of ribosomal protein S6, and controls various aspects of cell survival, cell proliferation, differentiation, etc.

[0074] "In the presence of an RSK inhibitor" means that the culture conditions can inhibit RSK, and the means are not particularly limited, and substances that inhibit RSK activity can be used, for example, RSK signal inhibition means such as anti-RSK antibodies or RSK inhibitors can be used. In addition, since RSK is activated when phosphorylated at a specific site of itself, the means of inhibiting the phosphorylation can also be used to inhibit RSK signaling.

[0075] The RSK inhibitor is not particularly limited in the present invention, and for example, the following compounds can be used: BRD7389 or BI-D1870 can be preferably used.

[0076] BRD7389(CAS No.: 376382-11-5)

[0077] [Chemical formula 1]

[0078]

[0079] SL 0101-1 (CAS No.: 77307-50-7)

[0080] BI-D1870(CAS No.: 501437-28-1)

[0081] LJH685(CAS No.:1627710-50-2)

[0082] LJI308(CAS No.:1627709-94-7)

[0083] FMK(CAS No.:821794-92-7)

[0084] RMM46 (CAS No.: 1307896-46-3)

[0085] CMK(CAS No.:821794-90-5)

[0086] Carnosol (CAS No.: 5957-80-2)

[0087] Bix 02565(CAS No.: 1311367-27-7)

[0088] The concentration of the RSK inhibitor varies depending on the somatic cells used, etc., and is not particularly limited and may be appropriately determined. For example, it can be used in the range of 0.05 μmol / L to 50 μmol / L, preferably 0.1 μmol / L to 20 μmol / L.

[0089] 1.2.2 GSK3 inhibitors

[0090] GSK3 (glycogen synthase kinase-3) was discovered as a protein kinase that phosphorylates glycogen synthase to inactivate it. In mammals, GSK3 is classified into two isoforms: 51kDa α (GSK3α) and 47kDa β (GSK3β). GSK3 has the activity of phosphorylating various proteins and is involved not only in glycogen metabolism but also in physiological phenomena such as cell division and cell proliferation.

[0091] "In the presence of a GSK3 inhibitor" means culture conditions that can inhibit GSK3, and the means are not particularly limited, and substances that inhibit the activity of GSK3, such as anti-GSK3 antibodies or GSK3 inhibitors, can be used to inhibit GSK3 signals. In addition, since GSK3 loses its activity when phosphorylated at a specific site of itself, means that promote the phosphorylation can also be used to inhibit GSK3 signals.

[0092] In the present invention, there is no particular limitation, but as the GSK3 inhibitor, for example, the following compounds can be used: Preferably, CHIR99021 can be used.

[0093] CHIR99021(CAS No.: 252917-06-9)

[0094] [Chemical formula 2]

[0095]

[0096] BIO((2'Z,3'E)-6-bromoindigorubin-3'-oxime)(CAS No.:667463-62-9)

[0097] Kenpaullone (CAS No.: 142273-20-9)

[0098] A1070722 (CAS No.: 1384424-80-9)

[0099] SB216763(CAS No.: 280744-09-4)

[0100] CHIR98014(CAS No.:556813-39-9)

[0101] TWS119 (CAS No.: 601514-19-6)

[0102] Tideglusib (CAS No.: 865854-05-3)

[0103] SB415286(CAS No.: 264218-23-7)

[0104] Bikinin(CAS No.:188011-69-0)

[0105] IM-12 (CAS No.: 1129669-05-1)

[0106] 1-Azakenpaullone (CAS No.: 676596-65-9)

[0107] LY2090314(CAS No.: 603288-22-8)

[0108] AZD1080 (CAS No.: 612487-72-6)

[0109] AZD2858(CAS No.: 486424-20-8)

[0110] AR-A014418(CAS No.: 487021-52-3)

[0111] TDZD-8(CAS No.:327036-89-5)

[0112] Indirubin (CAS No.: 479-41-4)

[0113] The concentration of the GSK3 inhibitor varies depending on the somatic cells used, etc., and is not particularly limited and may be appropriately determined. For example, it can be used in the range of 0.05 μmol / L to 20 μmol / L, preferably 0.1 μmol / L to 10 μmol / L.

[0114] 1.2.3 cAMP inducers

[0115] cAMP (cyclic adenosine monophosphate) is a substance that acts as a second messenger and participates in the transmission of various intracellular signals. cAMP is generated in cells by cyclizing adenosine triphosphate (ATP) by adenylate cyclase.

[0116] "In the presence of a cAMP inducer" means that the culture is carried out under conditions that can induce cAMP. The means are not particularly limited, and for example, any means that can increase the intracellular cAMP concentration can be used. Substances that can directly act on adenylate cyclase, an enzyme involved in the production of cAMP, to induce it, substances that can promote the expression of adenylate cyclase, and substances that inhibit phosphodiesterase, an enzyme that decomposes cAMP, can all be used as means for increasing the intracellular cAMP concentration. Dibutyryl cAMP, a structural analog of cAMP that has the same effect as cAMP in cells, can also be used.

[0117] Although not particularly limited in the present invention, examples of cAMP inducers (adenylate cyclase activators) include forskolin (CAS No.: 66575-29-9) and forskolin derivatives (eg, Japanese Patent Application Laid-Open No. 2002-348243) and the following compounds. Preferably, forskolin can be used.

[0118] Forskolin(CAS No.:66428-89-5)

[0119] [Chemical formula 3]

[0120]

[0121] Isoproterenol (CAS No.: 7683-59-2)

[0122] NKH477 (CAS No.: 138605-00-2)

[0123] PACAP1-27 (CAS No.: 127317-03-7)

[0124] PACAP1-38(CAS No.: 137061-48-4)

[0125] The concentration of the cAMP inducer varies depending on the somatic cells used, etc., and is not particularly limited and may be appropriately determined. For example, it can be used in the range of 0.2 μmol / L to 50 μmol / L, preferably 1 μmol / L to 30 μmol / L.

[0126] 1.2.4 PI3K inhibitors

[0127] Phosphoinositide 3-kinase (PI3K) is an enzyme that phosphorylates inositol phospholipids. The generated phosphoinositides activate PDK1. PDK1 further phosphorylates AKT, which activates the PDK1 / AKT signaling pathway. LY294002 is a selective inhibitor of PI3K, which inhibits the activation of the PDK1 / AKT signaling pathway by inhibiting the production of phosphoinositides.

[0128] "In the presence of a PI3K inhibitor" means that under culture conditions that can inhibit PI3K, the means are not particularly limited, and any means that can inhibit PI3K can be used. In the present invention, substances that directly act on PI3K to inhibit its function (e.g., anti-PI3K antibodies, other agents), agents that inhibit the production of PI3K itself, etc. can be used. In addition, PI3K can also be inhibited by inhibiting the upstream of the signal transmission involved in PI3K.

[0129] Although not particularly limited in the present invention, as PI3K inhibitors, for example, the following compounds can be used. Preferably, LY294002 can be used.

[0130] LY294002(CAS No.: 154447-36-6)

[0131] [Chemical formula 4]

[0132]

[0133] Buparlisib (CAS No.: 944396-07-0)

[0134] TGR-1202 (CAS No.: 1532533-67-7)

[0135] PI-103 (CAS No.: 371935-74-9)

[0136] IC-87114(CAS No.: 371242-69-2)

[0137] Wortmannin (CAS No.: 19545-26-7)

[0138] ZSTK474(CAS No.: 475110-96-4)

[0139] AS-605240(CAS No.: 648450-29-7)

[0140] PIK-90(CAS No.:677338-12-4)

[0141] AZD6482 (CAS No.: 1173900-33-8)

[0142] Duvelisib (CAS No.: 1201438-56-3)

[0143] TG100-115(CAS No.: 677297-51-7)

[0144] CH5132799(CAS No.:1007207-67-1)

[0145] CAY10505(CAS No.: 1218777-13-9)

[0146] PIK-293 (CAS No.: 900185-01-5)

[0147] CZC24832(CAS No.: 1159824-67-5)

[0148] Pilaralisib (CAS No.: 934526-89-3)

[0149] AZD8835(CAS No.: 1620576-64-8)

[0150] The concentration of the PI3K inhibitor varies depending on the somatic cells used, etc., and is not particularly limited and may be appropriately determined. For example, it can be used in the range of 0.1 μmol / L to 20 μmol / L, preferably 0.5 μmol / L to 10 μmol / L.

[0151] 1.3 Culture of somatic cells

[0152] In the production method of the present invention, the culture of somatic cells can be carried out in the presence of the various inhibitors (inducers or activators in some cases) by selecting a medium, temperature, and other conditions depending on the type of somatic cells used.

[0153] In the production method of the present invention, by culturing somatic cells in an induction medium containing the above-mentioned various inhibitors and the like, insulin-producing cells can be produced from somatic cells in a single-step culture.

[0154] In addition, by selecting somatic cells that are easy to culture, somatic cells that have increased in number from the beginning and have reached a nearly confluent state can be converted into insulin-producing cells. Therefore, large-scale production of insulin-producing cells can be easily performed.

[0155] The differentiation induction medium or the medium for subculture of somatic cells used as the basis for the implementation of the present invention can be selected from known mediums or commercially available mediums. For example, suitable components (serum, protein, amino acid, sugar, vitamins, fatty acids, antibiotics, etc.) can be added to MEM (minimum essential medium), DMEM (Dulbecco's Modified Eagle Medium), DMEM / F12 or a medium modified therefrom, which are common mediums, and used.

[0156] In the manufacturing method of the present invention, the culture of somatic cells is preferably carried out in a culture medium for differentiation induction without containing serum such as fetal bovine serum (FBS). It is also preferably carried out in a culture medium for differentiation induction without containing serum such as fetal bovine serum (FBS), and containing more insulin. It is further more preferred that the culture medium for differentiation induction does not contain serum such as fetal bovine serum (FBS), and contains more insulin.

[0157] In one embodiment of the production method of the present invention, it is preferred to culture somatic cells with a differentiation induction medium containing a large amount of insulin. The amount thereof may be, for example, 5 μg / mL or more, preferably 20 μg / mL or more or 25 μg / mL, and more preferably in the range of 80 to 120 μg / mL.

[0158] When a known or commercially available induction medium as a basis contains a certain amount of insulin in advance, insulin may be added so as to adjust the insulin content to the aforementioned amount.

[0159] As culture conditions, general cell culture conditions can be selected. For example, conditions of 37°C and 5% CO2. During the culture, it is preferred to exchange the culture medium at appropriate intervals (preferably once every 1 to 5 days, more preferably once every 2 to 4 days). When the manufacturing method of the present invention is implemented using fibroblasts as materials, insulin-producing cells will appear after 6 or 8 to 12 days at 37°C and 5% CO2. When the manufacturing method of the present invention is implemented using mesenchymal stem cells as materials, insulin-producing cells will begin to appear after about a week at 37°C and 5% CO2.

[0160] The culture of somatic cells can be carried out using a cell culture container such as a culture plate, a culture dish, a cell culture flask, a cell culture bag, etc. It should be noted that the cell culture bag preferably has air permeability. When a large amount of cells are required, a large culture tank can be used. The culture can be carried out in an open culture or a closed culture. When the purpose is to administer the obtained insulin-producing cells to humans, a closed culture is preferred.

[0161] 1.4 Insulin-producing cells

[0162] By the manufacturing method of the present invention, a cell group containing insulin-producing cells can be obtained. Insulin-producing cells manufactured by the manufacturing method of the present invention are also within the scope of the present invention. Insulin-producing cells manufactured by the manufacturing method of the present invention may be progenitor cells destined to differentiate into insulin-producing cells in addition to terminally differentiated cells.

[0163] The insulin-producing cells produced by the production method of the present invention are directly induced to differentiate from somatic cells by low molecular weight compounds, that is, so-called low molecular weight compound-induced insulin-producing cells (ciIPCs), and are distinguished from cells induced to differentiate by gene introduction.

[0164] Insulin-producing cells produced by the production method of the present invention can be detected, confirmed and separated, for example, by using changes in cell morphology, characteristic properties of insulin-producing cells or specific markers (e.g., anti-insulin antibodies). In addition, the secretory capacity of the produced insulin-producing cells can also be evaluated by quantifying the amount of insulin secreted by the sandwich ELISA.

[0165] Specific markers can be detected by quarantine methods (detection by antibodies), and for protein molecules, detection can also be performed by quantifying the amount of mRNA. Antibodies that recognize insulin-producing cell-specific markers are useful for separating and purifying insulin-producing cells obtained by the production method of the present invention.

[0166] The insulin-producing cells manufactured by the manufacture method of the present invention, for example, can be used for tissue repair or improvement of insulin concentration in the blood, etc. By transplanting the insulin-producing cells manufactured by the manufacture method of the present invention, it is possible to manufacture a pharmaceutical composition for tissue repair, etc. For patients with type 1 diabetes who are congenitally almost unable to secrete insulin, in order to alleviate their symptoms and to cure them radically, pancreatic transplantation or transplantation of pancreatic islets is a fundamental treatment method. In addition, it is predicted that the patients with type 2 diabetes will further increase in the future at home and abroad, and become the reason for the high medical expenses. The transplantation of pancreatic β cells that secrete insulin may be an effective treatment method. As a treatment method for pancreatic diseases such as such diabetes, the development of the manufacture method of insulin-producing cells and the transplantation method of insulin-producing cells is in progress. For example, it is expected that by transplanting insulin-producing cells under the renal epithelium or transplanting to the liver via the portal vein, it is used for the treatment of severe pancreatic diseases (diabetes, etc.).

[0167] When using the insulin-producing cells produced by the manufacturing method of the present invention to prepare a pharmaceutical composition, the insulin-producing cells can be mixed with a pharmaceutically acceptable carrier by conventional methods, etc., and a preparation in a form suitable for administration to an individual can be prepared. As a carrier, for example, physiological saline, isotonic distilled water for injection with the addition of glucose or other auxiliary drugs (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) can be cited. Buffers (for example, phosphate buffer, sodium acetate buffer), pain-relieving agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. can also be further mixed.

[0168] The insulin-producing cells produced by the production method of the present invention can be further prepared as a composition in combination with other cells or components that are effective for the expression of the functions of the insulin-producing cells or the improvement of the adhesion.

[0169] Furthermore, the insulin-producing cells produced by the production method of the present invention can be used for screening of candidate pharmaceutical compounds acting on insulin-producing cells or for safety evaluation of candidate pharmaceutical compounds. Insulin-producing cells are an important tool for evaluating the toxicity of candidate pharmaceutical compounds. According to the production method of the present invention, since a large number of insulin-producing cells can be obtained by a single operation, it is possible to obtain reproducible research results without being affected by differences in cell batches.

[0170] 2 Composition

[0171] The composition according to the present invention (hereinafter referred to as "the composition of the present invention") is a composition for producing insulin-producing cells by direct differentiation induction from somatic cells, and contains an RSK inhibitor.

[0172] Preferably, the composition of the present invention further contains a GSK3 inhibitor. More preferably, the composition of the present invention further contains a cAMP inducer and / or a PI3K inhibitor. The composition of the present invention particularly preferably contains an RSK inhibitor and a GSK3 inhibitor or further contains a cAMP inducer.

[0173] The composition of the present invention may contain at least the above-mentioned inhibitor and the like, and may further contain other inhibitors, inducers and the like as necessary.

[0174] The above inhibitors, inducers, and the like may be used alone or in combination of two or more.

[0175] Some of the specific inhibitors may have two or more inhibitory effects. In this case, the presence of one inhibitor may be regarded as the presence of a plurality of inhibitors.

[0176] Specific examples or preferred examples of the inhibitor, inducer, etc. are synonymous with those described above.

[0177] The composition of the present invention can be used as a composition for producing insulin-producing cells from somatic cells. The composition of the present invention can also be used as a culture medium for producing insulin-producing cells from somatic cells.

[0178] As a culture medium for producing insulin-producing cells from somatic cells, a basal culture medium produced by mixing the necessary components of cell culture can be cited, which contains an RSK inhibitor as an effective ingredient, and further contains a GSK3 inhibitor and / or a cAMP inducer, a PI3K inhibitor as required. The effective ingredient is contained in a concentration effective for the production of insulin-producing cells, and the concentration can be appropriately determined by personnel in the industry. The basal culture medium can be selected from a known culture medium or a commercially available culture medium. For example, a general culture medium MEM (minimum essential medium), DMEM (Dulbecco's Modified Eagle Medium), DMEM / F12, RPMI1640 or a culture medium adjusted thereto can be used as a basal culture medium.

[0179] The medium involved in the composition of the present invention preferably does not contain serum such as fetal bovine serum (FBS). In addition, it preferably contains more insulin. More preferably, it does not contain serum such as fetal bovine serum (FBS) and contains more insulin.

[0180] In one embodiment of the culture medium involved in the composition of the present invention, it preferably contains more insulin. The amount thereof is, for example, 5 μg / mL or more, preferably 20 μg / mL or more or 25 μg / mL or more, and more preferably in the range of 80 to 120 μg / mL.

[0181] When a known or commercially available induction medium as a basis contains a certain amount of insulin in advance, the composition of the present invention can be prepared by adding insulin to adjust the content of insulin to the aforementioned amount.

[0182] The culture medium involved in the composition of the present invention can be further supplemented with known culture medium components described in this specification, for example, serum, protein (albumin, transferrin, growth factors, etc.), amino acids, sugars, vitamins, fatty acids, antibiotics, etc.

[0183] The medium according to the composition of the present invention may be further supplemented with a substance effective for inducing differentiation into insulin-producing cells as described in the present specification.

[0184] Further, in the present invention, for example, by administering an RSK inhibitor, and further administering a GSK3 inhibitor and / or a cAMP inducer, a PI3K inhibitor to a living body as needed, insulin-producing cells can be produced from somatic cells in vivo. That is, the present invention provides, for example, a method for producing insulin-producing cells from somatic cells in vivo, comprising a step of administering an RSK inhibitor, and further administering a GSK3 inhibitor and / or a cAMP inducer, a PI3K inhibitor to a living body as needed. The preferred combination of administering the inhibitor to a living body is as described in this specification. In addition, as a living body, humans, mammals other than humans, and animals other than mammals (birds, reptiles, amphibians, fish, etc.) can be cited, and humans are particularly preferred. For example, by administering an RSK inhibitor, and further administering a GSK3 inhibitor and / or a cAMP inducer, a PI3K inhibitor to a specific site in the living body as needed, insulin-producing cells can be produced from somatic cells in the specific site. Example

[0185] Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to the scope of the examples.

[0186] Example 1 Preparation of insulin-producing cells

[0187] ~Direct induction of human fibroblasts into insulin-producing cells~

[0188] (1) Human fibroblasts

[0189] Human fibroblasts used as materials were purchased from DS Pharma Biomedical Co., Ltd. They were fibroblasts derived from the skin of a 38-year-old human.

[0190] (2) Direct induction of human fibroblasts into insulin-producing cells

[0191] Human fibroblasts were cultured at 5×10 cells / mL in a 35 mm culture dish coated with gelatin (Cat#: 190-15805, Wako Pure Chemical Industries, Ltd.). 4 Each cell was inoculated with 100 cells / dish and cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2 until confluence. DMEM stands for Dulbecco's Modified Eagle Medium.

[0192] The culture medium of the human fibroblast culture dish was replaced with the following differentiation induction medium.

[0193] Differentiation induction medium: medium with or without 10% fetal bovine serum (FBS, manufactured by Hyclone), ITS-X (Cat#: 51500056, manufactured by Gibco), non-essential amino acids (NEAA) aminoacids; Cat#: 11140050, manufactured by Gibco), glutamine (manufactured by Gibco; final concentration 2 mmol / L), nicotinamide (Cat#: 72340-100G, manufactured by Sigma-Aldrich; final concentration 10 mmol / L), Exendin-4 (Cat#: av120214, manufactured by Abcam; final concentration 100 ng / mL), 100 U / mL penicillin, 100 μg / mL streptomycin, insulin (Cat#: 093-06351; manufactured by Wako), and modified DMEM / F-12 (Cat#: 12634010; manufactured by Gibco) containing the following low molecular weight compounds.

[0194] Then, the medium was replaced with a medium of the same composition every 3 days, and the cells were cultured at 37°C and 5% CO2.

[0195] <Low molecular weight compounds>

[0196] 3μM CHIR99021 (Cat#: 13122, Cayman Chemical)

[0197] 7.5 μM Forskolin (Cat#: 063-02193, Wako)

[0198] 1.5μM BRD7389 (Cat#: ab146161, Abcam)

[0199] 5μM LY294002 (Cat#: 70920, Cayman Chemical)

[0200] (3) Results

[0201] The results of culturing for 14 days as described (2) are as follows Figures 1 to 4 shown.

[0202] In the figure, "4C" refers to the four low-molecular compounds, and "3C" refers to three of the four low-molecular compounds, CHIR99021, BRD7389 and forskolin. "+FBS" indicates the presence of FBS in the culture medium, and "-FBS" indicates the absence of FBS in the culture medium. "+LY294002 (20 μM)" indicates the presence of LY294002 in the culture medium at a final concentration of 20 μM, and "-LY294002" indicates that LY294002 is not present in the culture medium (i.e., "4C-LY294002" is synonymous with 3C). In addition, "No Compound" indicates that the four low-molecular compounds are not present in the culture medium. Therefore, for example, "No Compound-FBS" indicates the experimental results when 4C and FBS are not present, and "4C-FBS-LY294002" indicates the experimental results when LY294002 and FBS are not present in 4C.

[0203] Figure 2 In 3, "CH" refers to CHIR99021, indicating the experimental results when only CHIR99021 is present in 4C or 3C. "B" refers to BRD7389, indicating the experimental results when only BRD7389 is present in 4C or 3C. "F" refers to forskolin, indicating the experimental results when only forskolin is present in 4C or 3C. The "+" in "CH+B" and so on indicates the experimental results when both are present, and "3C" indicates the experimental results when all 3C are present. "+Nicotinamide / Exendin4" indicates that nicotinamide and Exendin4 are added as culture medium components, and "-Nicotinamide / Exendin4" indicates that nicotinamide and Exendin4 are not added as culture medium components.

[0204] Figure 4 In the table, "B" refers to BRD7389, and "3C-B" refers to the case where BRD7389 is not present in 3C, that is, the experimental results under the 2-factor conditions of CH and F. "3C-B+BI-D1870" refers to the experimental results when BRD7389 is not present in 3C, and RSK inhibitor BI-D1870 (Cat#: 15264, Cayman Chemical) is present at a final concentration of 5 μM or 10 μM as a replacement.

[0205] like Figure 1As shown, by allowing the low molecular weight compounds (RSK inhibitor, GSK inhibitor, cAMP inducer, PI3K inhibitor) to exist in the differentiation induction medium, insulin-producing cells with high secretion capacity can be directly induced from human fibroblasts efficiently, and insulin-producing cells with high secretion capacity can be obtained even in the absence of the PI3K inhibitor (LY294002). Figure 2 as well as Figure 3 As shown in the results, the secretion of insulin increased synergistically under the combination of three factors, namely, RSK inhibitor (BRD7389), GSK inhibitor (CHIR99021), and cAMP inducer (forskolin), but a considerable amount of insulin secretion was also observed under the combination of two factors, namely, RSK inhibitor (BRD7389) and GSK inhibitor (CHIR99021), or under the condition of only one factor, namely, RSK inhibitor (BRD7389), confirming the induction of insulin-producing cells. This tendency was also observed when nicotinamide and exendin-4 were not added to the culture medium (refer to Figure 3 ).

[0206] from Figure 4 The results showed that the secretion of insulin decreased when LY294002 was removed and two factors, RSK inhibitor (BRD7389), GSK inhibitor (CHIR99021) and cAMP inducer (forskolin) were removed, indicating that the presence of RSK inhibitor is important in the induction of insulin-producing cells. On the other hand, under the three-factor condition in which BRD7389 was replaced with another RSK inhibitor BI-D1870, insulin-producing cells with high secretion capacity were also obtained.

[0207] In addition, in the group in which the differentiation induction medium did not contain fetal bovine serum (FBS), insulin-producing cells with a high secretion capacity were obtained from human fibroblasts.

[0208] Example 2 Preparation of insulin-producing cells

[0209] ~Direct induction of human mesenchymal stem cells into insulin-producing cells~

[0210] (1) Human Mesenchymal Stem Cells

[0211] Human mesenchymal stem cells isolated from adipose tissue were purchased from Takara Bio Co., Ltd.

[0212] (2) Direct induction of human mesenchymal stem cells into insulin-producing cells

[0213] Human mesenchymal stem cells were placed in a 35 mm culture dish coated with gelatin (Cat#: 190-15805, Wako Pure Chemical Industries, Ltd.) at a rate of 5×10 4 Each cell was inoculated with mesenchymal stem cell growth medium 2 (Cat#: C-28009; manufactured by TakaraBio) supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured at 37°C and 5% CO2 until almost confluent.

[0214] The culture medium of the human mesenchymal stem cell culture dish was replaced with the following differentiation induction medium.

[0215] Differentiation induction medium: modified DMEM / F12 (Cat#: 12634010; manufactured by Gibco) supplemented with or without 10% fetal bovine serum (FBS, manufactured by Hyclone), ITS-X (Cat#: 51500056, manufactured by Gibco), non-essential amino acids (NEAA; Cat#: 11140050, manufactured by Gibco), glutamine (manufactured by Gibco; final concentration 2 mmol / L), nicotinamide (Cat#: 72340-100G, manufactured by Sigma-Aldrich; final concentration 5 mmol / L), Exendin-4 (Cat#: av120214, manufactured by Abcam; final concentration 50 ng / mL), 100 U / mL penicillin, 100 μg / mL streptomycin, and the following low molecular weight compounds.

[0216] Then, the medium was replaced with a medium of the same composition every 3 days, and the cells were cultured at 37°C and 5% CO2.

[0217] In the experiment in which the induction medium contained 120 μg / mL of insulin, human recombinant insulin (Cat#: 093-06351; Wako) was added to adjust the medium to contain 120 μg / mL of insulin.

[0218] <Low molecular weight compounds>

[0219] 0.5μM CHIR99021 (Cat#: 13122, Cayman Chemical)

[0220] 3.75 μM Forskolin (Cat#: 063-02193, Wako)

[0221] 0.2μM BRD7389 (Cat#: ab146161, Abcam)

[0222] 2.5μM LY294002 (Cat#: 70920, Cayman Chemical)

[0223] (3) Results

[0224] The results of culturing for 14 days as described (2) are as follows Figure 5 as well as Figure 6 shown.

[0225] In the figure, "4C" refers to the four low-molecular compounds. "+FBS" indicates the presence of FBS in the culture medium, and "-FBS" indicates the absence of FBS in the culture medium. "-LY294002" indicates that LY294002 is not present in the culture medium (i.e., "4C-LY294002" is synonymous with 3C). In addition, "No compound" indicates that the four low-molecular compounds are not present in the culture medium. Therefore, for example, "NoCompound-FBS" indicates the experimental results when there is no 4C and no FBS, and "4C-FBS-LY294002" indicates the experimental results when there is no LY294002 in 4C and no FBS.

[0226] like Figure 5 as well as Figure 6 As shown, by making the low molecular weight compound present in the differentiation induction medium, insulin-producing cells with high secretion capacity can be efficiently induced directly from human adipose tissue-derived mesenchymal stem cells (AdMSC). It should be noted that insulin-producing cells with high secretion capacity were also obtained in the absence of LY294002.

[0227] In addition, the group without fetal bovine serum (FBS) in the differentiation induction medium was able to directly induce insulin-producing cells with high secretion capacity from human mesenchymal stem cells. Furthermore, when the differentiation induction medium contained a high concentration (120 μg / mL) of insulin for induction, insulin-producing cells with significantly increased insulin secretion and higher secretion capacity were directly and efficiently induced from human mesenchymal stem cells.

Claims

1. A method for producing insulin-producing cells in vitro, which is a method for producing insulin-producing cells by direct differentiation induction from fibroblasts or mesenchymal stem cells, wherein: The method comprises: a step of culturing fibroblasts or mesenchymal stem cells in the presence of an RSK inhibitor, The RSK inhibitor is BRD7389, The differentiation induction medium does not contain serum. The manufacturing method is a non-disease treatment method.

2. The in vitro method for producing insulin-producing cells according to claim 1, wherein: The step is a step of further culturing fibroblasts or mesenchymal stem cells in the presence of a GSK3 inhibitor.

3. The in vitro method for producing insulin-producing cells according to claim 1 or 2, wherein: The step is a step of further culturing fibroblasts or mesenchymal stem cells in the presence of a cAMP inducer and / or a PI3K inhibitor.

4. The in vitro method for producing insulin-producing cells according to claim 2, wherein: The GSK3 inhibitor was CHIR99021.

5. The in vitro method for producing insulin-producing cells according to claim 3, wherein: The cAMP inducer was forskolin or the PI3K inhibitor was LY294002.

6. A composition for producing insulin-producing cells from fibroblasts or mesenchymal stem cells, which is a composition for producing insulin-producing cells by direct differentiation induction from fibroblasts or mesenchymal stem cells, the composition comprising an RSK inhibitor, wherein: The RSK inhibitor is BRD7389. The composition contains a differentiation induction medium which does not contain serum.

7. The composition of claim 6, further comprising a GSK3 inhibitor.

8. The composition of claim 6 or 7, further comprising a cAMP inducer and / or a PI 3K inhibitor.

9. The composition of claim 7, wherein The GSK3 inhibitor was CHIR99021.

10. The composition of claim 8, wherein the cAMP inducer is forskolin or the PI3K inhibitor is LY294002.

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