Methods and compositions for the manufacture of insulin-producing cells

Abstract

The main objective of this invention is to provide a novel manufacturing method capable of efficiently and directly converting or inducing somatic cells into insulin-producing cells. As an example, a method for manufacturing insulin-producing cells is provided, which involves directly differentiating and inducing insulin production from somatic cells. This method includes a step of culturing somatic cells in a serum-free differentiation-induction medium; or a step of culturing somatic cells in a differentiation-induction medium containing 5 μg / mL or more of insulin. According to this invention, insulin-producing cells with high insulin secretion capacity can be efficiently and directly manufactured from somatic cells. The insulin-producing cells obtained by this invention are useful in regenerative medicine and the like.

Description

Technical Field

[0001] (Cross-reference to related applications)

[0002] This application claims priority to Japanese Application No. 2018-213449, filed with the Japan Patent Office on November 14, 2018. The entirety of that Japanese application (specification, scope of claims, drawings, abstract) is incorporated herein by reference, expressly for the purposes of this specification.

[0003] This invention belongs to the technical field of regenerative medicine. Specifically, it relates to a method for directly manufacturing insulin-producing cells from somatic cells using low-molecular-weight compounds, and to low-molecular-weight compound-induced insulin-producing cells (ciIPCs) manufactured by the method. The invention further relates to an induction culture medium composition that can be used in the method for manufacturing these insulin-producing cells. Background Technology

[0004] In recent years, research has been extensive on methods that directly convert somatic cells, such as fibroblasts, into other cell types without gene transfer (direct reprogramming). For example, in Patent Document 1, several low-molecular-weight compounds, such as ALK5 inhibitors and ALK6 inhibitors, are combined and somatic cells, particularly human fibroblasts, are cultured in their presence. These cells are then directly induced into brown adipocytes, osteoblasts, chondrocytes, nervous system cells, or cardiomyocytes. As with Patent Document 1, it would be highly beneficial to be able to directly induce common fibroblasts into cells such as brown adipocytes using readily available low-molecular-weight compounds. For example, since other cells can be easily created from one's own fibroblasts, its application in regenerative medicine can be expanded. Furthermore, cells can be easily prepared as experimental materials for the development of new pharmaceuticals.

[0005] Regarding pancreatic β cells that secrete insulin, besides reports of inducing human pancreatic β cells from differentiation of iPS cells or ES cells, there are also reports of directly inducing pancreatic β cells from endodermal cells such as pancreatic α cells, pancreatic acinar cells, pancreatic ductal gland cells, small intestinal crypt cells, hepatocytes, and bile duct cells using pancreatic β cell-specific transcription factors Pdx1, Ngn3, and MafA (Non-Patent Literature 1). Additionally, there are reports of directly inducing pancreatic β cells from mouse embryonic fibroblasts (MEFs) or human skin fibroblasts using factors such as Yamanaka 4 (Non-Patent Literature 2, 3). There are even reports of creating endodermal progenitor cells from mouse embryonic fibroblasts (MEFs) using low-molecular-weight compounds, and then differentiating these cells into pancreatic endocrine cells (Non-Patent Literature 4).

[0006] As described above, the induction of differentiation from somatic cells into pancreatic β cells, etc., must be carried out by culturing in a culture medium containing a variety of other compounds or nutrients. Examples of such culture media include, for instance, general media such as MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), DMEM / F12, or modified versions thereof, all of which are commercially available. Furthermore, these media are typically supplemented with appropriate components (serum, proteins, amino acids, sugars, vitamins, fatty acids, antibiotics, etc.) after considering the type of cells to be induced, to induce cell differentiation.

[0007] Existing technical documents

[0008] Patent documents

[0009] [Patent Document 1] International Publication No. 2018 / 062269

[0010] Non-patent literature

[0011] [Non-patent literature 1] Current Pathobiology Reports, 2015, Vol. 3, pp. 57-65

[0012] [Non-Patent Literature 2] Cell Stem Cell, 2014, Vol. 14, pp. 228-236

[0013] [Non-Patent Literature 3] Nature Communications, 2016, Vol. 7, 10080 pages

[0014] [Non-Patent Literature 4] Journal of Biological Chemistry, 2017, Vol. 292, pp. 19122-19132 Summary of the Invention

[0015] The technical problem to be solved by the present invention

[0016] One of the problems in cell differentiation induction is that even when differentiation into the desired cell type is successfully induced, achieving a sufficient quantity of differentiation induction to a usable level is very difficult. This problem also exists when inducing insulin-producing cells.

[0017] The main objective of this invention is to provide a novel manufacturing method that enables the efficient direct conversion or induction of insulin-producing cells from somatic cells using low-molecular-weight compounds without the need for artificial gene introduction.

[0018] Technical means to solve technical problems

[0019] Through diligent exploration, the inventors discovered that the problem is typically solved by culturing cells with bovine embryonic serum (FBS) removed from the culture medium or by culturing cells with an additional amount of insulin than usual, thus completing this invention.

[0020] As an example of the present invention, the following items can be cited:

[0021] [1] A method for manufacturing insulin-producing cells, wherein the insulin-producing cells are manufactured by direct differentiation induction from somatic cells, wherein the method comprises:

[0022] The process of culturing somatic cells in a serum-free differentiation-inducing medium.

[0023] [2] A method for manufacturing insulin-producing cells, wherein the insulin-producing cells are manufactured by direct differentiation induction from somatic cells, wherein the method comprises:

[0024] The process of culturing somatic cells by introducing insulin at a concentration of 5 μg / mL or higher into the culture medium for differentiation induction.

[0025] [3] A method for manufacturing insulin-producing cells, wherein the insulin-producing cells are manufactured by direct differentiation induction from somatic cells, wherein the method comprises:

[0026] The process of culturing somatic cells using a serum-free differentiation-inducing medium containing insulin at a concentration of 5 μg / mL or higher.

[0027] [4] The method for manufacturing insulin-producing cells as described in any one of items [1] to [3], wherein,

[0028] The process described is the process of culturing somatic cells in the presence of an RSK inhibitor.

[0029] [5], the method for manufacturing insulin-producing cells as described in item [4], wherein,

[0030] The process involves further culturing somatic cells in the presence of a GSK3 inhibitor and / or a cAMP inducer.

[0031] [6], the method for manufacturing insulin-producing cells as described in item [4] or [5], wherein,

[0032] RSK inhibitors are BRD7389 or BI-D1870.

[0033] [7], the method for manufacturing insulin-producing cells as described in item [5] or [6], wherein,

[0034] The GSK3 inhibitor is CHIR99021, or the cAMP inducer is salivain.

[0035] [8], the method for manufacturing insulin-producing cells according to any one of items [1] to [7], wherein,

[0036] The somatic cells are fibroblasts or mesenchymal stem cells.

[0037] [9] An insulin-producing cell manufactured by any one of the methods for manufacturing insulin-producing cells described in any one of items [1] to [8].

[0038]

[10] A differentiation-inducing composition for producing insulin-producing cells by inducing direct differentiation from somatic cells, wherein the composition is serum-free.

[0039]

[11] A differentiation-inducing composition for producing insulin-producing cells by inducing direct differentiation from somatic cells, wherein the composition contains more than 5 μg / mL of insulin.

[0040]

[12] A differentiation-inducing composition for producing insulin-producing cells by inducing direct differentiation from somatic cells, wherein the composition is serum-free and contains more than 5 μg / mL of insulin.

[0041] The differentiation-inducing composition described in any one of items

[13] and

[10] to

[12] contains an RSK inhibitor.

[0042]

[14] The differentiation-inducing composition described in item

[13] further contains a GSK3 inhibitor and / or a cAMP inducer.

[0043]

[15] , the differentiation-inducing composition described in item

[13] or

[14] , wherein,

[0044] RSK inhibitors are BRD7389 or BI-D1870.

[0045] The differentiation-inducing composition described in

[16] ,

[14] , or

[15] , wherein,

[0046] The GSK3 inhibitor is CHIR99021, or the cAMP inducer is salivain.

[0047] The differentiation-inducing composition described in any one of items

[17] and

[10] to

[16] , wherein,

[0048] The somatic cells are fibroblasts or mesenchymal stem cells.

[0049] The effects of the invention

[0050] According to the present invention, it is possible to efficiently convert or induce somatic cells into insulin-producing cells with high insulin secretion capacity without artificial gene introduction. The insulin-producing cells obtained by the present invention are useful in regenerative medicine and the like. Attached Figure Description

[0051] [ Figure 1 The vertical axis represents the amount of insulin secreted by insulin-producing cells induced from direct differentiation of human fibroblasts, under conditions with or without bovine embryonic serum (FBS). The vertical axis represents the amount of insulin secreted per 1 mg of total cellular protein (μU / mg). In each experimental result, the left column represents the result with bovine embryonic serum (FBS), and the right column represents the result without bovine embryonic serum (FBS).

[0052] [ Figure 2 The vertical axis represents the amount of insulin secreted by insulin-producing cells induced from direct differentiation of human fibroblasts under conditions containing 20 μg / mL or 120 μg / mL insulin. The vertical axis represents the amount of insulin secreted per 1 mg of total cellular protein (μU / mg). In each experimental result, the left column represents the result with 20 μg / mL insulin, and the right column represents the result with 120 μg / mL insulin.

[0053] [ Figure 3 The vertical axis represents the amount of insulin secreted by insulin-producing cells induced from direct differentiation of human fibroblasts. The vertical axis represents the amount of insulin secreted per 1 mg of total cellular protein (μU / mg).

[0054] [ Figure 4 The vertical axis represents the amount of insulin secreted by insulin-producing cells induced from direct differentiation of human fibroblasts. The vertical axis represents the amount of insulin secreted per 1 mg of total cellular protein (μU / mg).

[0055] [ Figure 5 The vertical axis represents the amount of insulin secreted by insulin-producing cells induced from direct differentiation of mesenchymal stem cells (AdMSCs) derived from human adipose tissue. The vertical axis represents the amount of insulin secreted per 1 mg of total cellular protein (μU / mg).

[0056] Specific embodiments of the present invention

[0057] The present invention will now be described in detail.

[0058] 1. Methods for manufacturing insulin-producing cells

[0059] The method for manufacturing insulin-producing cells disclosed in this invention (hereinafter referred to as "the manufacturing method of this invention") is a method for manufacturing insulin-producing cells by direct differentiation-induction from somatic cells, characterized by a step of culturing somatic cells in a serum-free differentiation-induction medium. Alternatively, the method for manufacturing insulin-producing cells by direct differentiation-induction from somatic cells is characterized by a step of culturing somatic cells in a differentiation-induction medium containing 5 μg / mL or more of insulin.

[0060] As described above, one of the key points of the present invention is to produce insulin-producing cells by culturing somatic cells in a serum-free differentiation-inducing medium that substantially does not contain serum such as bovine embryonic serum (FBS). Furthermore, as a key point of the present invention, it can be cited that insulin-producing cells are produced by containing a relatively high amount of insulin (5 μg / mL or more) in the differentiation-inducing medium.

[0061] Here, "serum-free" means that it does not actually contain unadjusted or unpurified serum. In this invention, even if the differentiation induction culture medium contains purified blood-derived components or animal tissue-derived components, as long as it does not actually contain unadjusted or unpurified serum, it can be called a serum-free differentiation induction culture medium.

[0062] The manufacturing method of the present invention preferably includes the following step: culturing somatic cells using a serum-free differentiation-inducing culture medium containing 5 μg / mL or more of insulin.

[0063] Furthermore, the aforementioned step is preferably a step of culturing somatic cells in the presence of an RSK inhibitor. More preferably, the step is a step of further culturing somatic cells in the presence of a GSK3 inhibitor and / or a cAMP inducer, in addition to an RSK inhibitor. In particular, the step is preferably a step of culturing somatic cells in the presence of both an RSK inhibitor and a GSK3 inhibitor, or in the presence of an RSK inhibitor, a GSK3 inhibitor, and a cAMP inducer. The step may, as needed, be a step of further culturing somatic cells in the presence of any other inhibitor, inducer, etc., such as a PI3K inhibitor.

[0064] The inhibitor or inducer may be used in one or in combination of two or more.

[0065] Specifically, among the inhibitors, some may have two or more inhibitory effects. In this case, the presence of one inhibitor can 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 cell can be used as the starting material. There are no particular limitations on somatic cells; they can be primary cells collected from living organisms or cells that have been lined. In the manufacturing method of the present invention, somatic cells at various stages of differentiation can be used, such as finally differentiated somatic cells (e.g., fibroblasts, umbilical vein endothelial cells (HUVECs), hepatocytes, bile duct cells, pancreatic α cells, pancreatic acinar cells, pancreatic ductal cells, intestinal crypt cells, etc.), somatic cells in the process of final differentiation (e.g., mesenchymal stem cells, neural stem cells, endodermal progenitor cells, etc.), or somatic cells that have been initialized to acquire pluripotency. Somatic cells can be used in the manufacturing method of this invention. Any type of somatic cell can be used, such as 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, myoblasts, cardiomyocytes, etc.), fibroblasts, nerve cells, osteoblasts, chondrocytes, endothelial cells, mesenchymal cells, adipocytes (white adipocytes, etc.), embryonic stem cells (ES cells), etc. Furthermore, progenitor cells and cancer cells of these cells can also be used in the manufacturing method of this invention. Fibroblasts or mesenchymal stem cells are preferred.

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

[0069] There are no particular limitations on the mesenchymal stem cells that can be used in this invention. Examples 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, and synovium-derived mesenchymal stem cells. Mesenchymal stem cells (MSCs) derived from dermal tissue and periodontal ligament.

[0070] Examples of sources of somatic cells include humans, mammals other than humans, and animals other than mammals (birds, reptiles, amphibians, fish, etc.), but are not limited thereto. Humans and mammals other than humans are preferred sources of somatic cells, with humans being particularly preferred. When manufacturing insulin-producing cells using the manufacturing method of the present invention for the purpose of administering drugs to humans, somatic cells collected from donors whose tissue suitability antigens are consistent with or similar to those of the recipient are preferred. Somatic cells collected from the recipient themselves may also be used for the manufacture of insulin-producing cells.

[0071] 1.2 Somatic cell culture

[0072] The somatic cell culture in the manufacturing method of the present invention can be carried out by conventional methods, except that the differentiation induction medium does not contain serum such as bovine embryonic serum (FBS) and / or contains insulin at a concentration of 5 μg / mL or higher. Furthermore, the culture medium, temperature, and other conditions can be selected according to the type of somatic cells used, and the process can be carried out in the presence of the various inhibitors (in some cases, inducers or activators).

[0073] In the manufacturing method of the present invention, by culturing somatic cells in a differentiation-inducing culture medium containing the various inhibitors mentioned above, it is possible to produce insulin-producing cells from somatic cells in a one-stage culture.

[0074] Furthermore, by selecting easily cultured somatic cells, somatic cells that have already increased in number and are almost confluent can be converted into insulin-producing cells from the outset. Therefore, it is also possible to easily manufacture insulin-producing cells on a large scale.

[0075] When implementing this invention, the culture medium used for differentiation induction or for somatic cell passage can be selected from known or commercially available culture media. For example, suitable components (serum, proteins, amino acids, sugars, vitamins, fatty acids, antibiotics, etc.) can be added to general culture media such as MEM (minimum essential medium), DMEM (Dulbecco's Modified Eagle Medium), DMEM / F12, or modified media.

[0076] In one embodiment of the manufacturing method of the present invention, the insulin content in the differentiation induction culture medium is 5 μg / mL or more, but preferably 20 μg / mL or more or 25 μg / mL or more, and more preferably in the range of 80 to 120 μg / mL.

[0077] When the basic known or commercially available induction culture medium contains a certain amount of insulin in advance, insulin can be added to adjust the insulin content to the stated amount.

[0078] As for culture conditions, general cell culture conditions can be selected, such as 37°C and 5% CO2. During culture, it is preferable to exchange the culture medium at appropriate intervals (preferably once every 1 to 5 days, more preferably once every 2 to 4 days). When using fibroblasts as material to implement the manufacturing method of the present invention, insulin-producing cells appear after 6 or 8 to 12 days under conditions of 37°C and 5% CO2. When using mesenchymal stem cells as material to implement the manufacturing method of the present invention, insulin-producing cells begin to appear after about one week under conditions of 37°C and 5% CO2.

[0079] Somatic cell culture can be performed using cell culture containers such as culture plates, culture dishes, cell culture flasks, and cell culture bags. It should be noted that air-permeable cell culture bags are preferred. Large culture tanks can be used when a large number of cells are required. Culture can be carried out in open or closed culture modes; closed culture is preferred when the purpose is to administer insulin-producing cells to humans.

[0080] 1.3 Regarding inhibitors, etc., that can be used in this invention

[0081] 1.3.1 RSK Inhibitors

[0082] Ribosomal S6 protein kinase (RSK) is widely expressed in cells and is one of the serine / threonine kinases that respond to a wide variety of growth factors. It is divided into subfamilies with molecular weights of 70 kDa and 90 kDa, particularly the 90 kDa RSK subfamily, which is activated by phosphorylation of ERK, a pathway belonging to the MAPK signaling pathway. Four genes of this subfamily exist in mammals. Activated 90 kDa RSK phosphorylates various proteins downstream of ribosomal protein S6, playing a diverse role in controlling cell survival, proliferation, and differentiation.

[0083] "In the presence of an RSK inhibitor" refers to culture conditions that can inhibit RSK. The method is not particularly limited; substances that inhibit RSK activity can be used, such as anti-RSK antibodies or RSK inhibitors. Furthermore, since RSK is activated when phosphorylated at a specific site, methods that inhibit phosphorylation can also be used to inhibit RSK signaling.

[0084] There are no particular limitations on the RSK inhibitors used in this invention; for example, the following compounds can be used. BRD7389 or BI-D1870 are preferred.

[0085] BRD7389(CAS No.: 376382-11-5)

[0086] [Chemical Formula 1]

[0087]

[0088] SL 0101-1 (CAS No.: 77307-50-7)

[0089] BI-D1870(CAS No.: 501437-28-1)

[0090] LJH685(CAS No.: 1627710-50-2)

[0091] LJI308(CAS No.:1627709-94-7)

[0092] FMK (CAS No.: 821794-92-7)

[0093] RMM46 (CAS No.: 1307896-46-3)

[0094] CMK (CAS No.: 821794-90-5)

[0095] Carnosol (CAS No.: 5957-80-2)

[0096] Bix 02565(CAS No.: 1311367-27-7)

[0097] The concentration of RSK inhibitors varies depending on the somatic cells used, and there are no particular restrictions. It can be determined appropriately, for example, it can be used in the range of 0.05 μmol / L to 50 μmol / L, preferably in the range of 0.1 μmol / L to 20 μmol / L.

[0098] 1.3.2 GSK3 Inhibitors

[0099] GSK3 (glycogen synthase kinase-3) was discovered as a protein kinase that phosphorylates and inactivates glycogen synthase. In mammals, GSK3 is classified into two isoforms: a 51 kDa α (GSK3α) and a 47 kDa β (GSK3β). GSK3 possesses phosphorylation activity on various proteins and participates not only in glycogen metabolism but also in physiological processes such as cell division and cell proliferation.

[0100] "In the presence of a GSK3 inhibitor" refers to culture conditions that can inhibit GSK3. The method is not particularly limited; substances that inhibit GSK3 activity can be used, such as anti-GSK3 antibodies or GSK3 inhibitors. Furthermore, since GSK3 loses its activity when phosphorylated at specific sites, methods that promote phosphorylation can also be used to inhibit GSK3 signaling.

[0101] There are no particular limitations in this invention, but as a GSK3 inhibitor, the following compounds can be used, for example. CHIR99021 is preferred.

[0102] CHIR99021 (CAS No.: 252917-06-9)

[0103] [Chemical Formula 2]

[0104]

[0105] BIO((2'Z,3'E)-6-bromoindorubin-3'-oxime)(CAS No.: 667463-62-9)

[0106] Kenpaullone (CAS No.: 142273-20-9)

[0107] A1070722(CAS No.: 1384424-80-9)

[0108] SB216763 (CAS No.: 280744-09-4)

[0109] CHIR98014(CAS No.:556813-39-9)

[0110] TWS119 (CAS No.: 601514-19-6)

[0111] Tideglusib (CAS No.: 865854-05-3)

[0112] SB415286(CAS No.: 264218-23-7)

[0113] Bikinin(CAS No.:188011-69-0)

[0114] IM-12 (CAS No.: 1129669-05-1)

[0115] 1-Azakenpaullone (CAS No.: 676596-65-9)

[0116] LY2090314(CAS No.: 603288-22-8)

[0117] AZD1080 (CAS No.: 612487-72-6)

[0118] AZD2858(CAS No.: 486424-20-8)

[0119] AR-A014418(CAS No.: 487021-52-3)

[0120] TDZD-8 (CAS No.: 327036-89-5)

[0121] Indigo Red (CAS No.: 479-41-4)

[0122] The concentration of GSK3 inhibitors varies depending on the somatic cells used, and there are no particular restrictions. It can be determined appropriately, for example, it can be used in the range of 0.05 μmol / L to 20 μmol / L, preferably in the range of 0.1 μmol / L to 10 μmol / L.

[0123] 1.3.3 cAMP inducers

[0124] cAMP (cyclic adenosine monophosphate) is a second messenger involved in various intracellular signaling processes. cAMP is produced intracellularly by the cyclization of adenosine triphosphate (ATP) by adenylate cyclase.

[0125] "In the presence of a cAMP inducer" means under culture conditions that can induce cAMP, and the means are not particularly limited. For example, any means that can increase the intracellular cAMP concentration can be used. Substances that can directly induce adenylate cyclase, an enzyme involved in cAMP production, substances that can promote the expression of adenylate cyclase, and substances that inhibit phosphodiesterase, an enzyme that breaks down cAMP, can all be used to increase the intracellular cAMP concentration. Dibutyryl cAMP, a structural analog of cAMP, can also be used, as it has the same effect as cAMP in cells.

[0126] While there are no particular limitations in this invention, examples of cAMP inducers (adenylate cyclase activators) include, for example, forskolin (CAS No.: 66575-29-9) and forskolin derivatives (e.g., Japanese Patent Application Publication No. 2002-348243) or the following compounds. Forskolin is preferred.

[0127] Trichosanthesin (CAS No.: 66428-89-5)

[0128] [Chemical Formula 3]

[0129]

[0130] Isoproterenol (CAS No.: 7683-59-2)

[0131] NKH477 (CAS No.: 138605-00-2)

[0132] PACAP1-27 (CAS No.: 127317-03-7)

[0133] PACAP1-38(CAS No.: 137061-48-4)

[0134] The concentration of cAMP inducer varies depending on the somatic cells used, and there are no particular restrictions. It can be determined appropriately, for example, it can be used in the range of 0.2 μmol / L to 50 μmol / L, preferably in the range of 1 μmol / L to 30 μmol / L.

[0135] 1.3.4 PI3K Inhibitors

[0136] Phosphoinositide 3-kinase (PI3K) is an enzyme that phosphorylates inositol phospholipids, and the resulting phosphoinositol activates PDK1. PDK1 further phosphorylates AKT, thereby activating the PDK1 / AKT signaling pathway. LY294002 is a selective inhibitor of PI3K that inhibits the activation of the PDK1 / AKT signaling pathway by inhibiting the production of phosphoinositol.

[0137] "In the presence of a PI3K inhibitor" refers to culture conditions that inhibit PI3K. The method used is not particularly limited; any means capable of inhibiting PI3K can be employed. In this 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. Furthermore, PI3K can also be inhibited by inhibiting upstream of the signal transduction pathway involved in PI3K.

[0138] While there are no particular limitations in this invention, the following compounds can be used as PI3K inhibitors, for example. LY294002 is preferred.

[0139] LY294002(CAS No.: 154447-36-6)

[0140] [Chemical Formula 4]

[0141]

[0142] Buparlisib (CAS No.: 944396-07-0)

[0143] TGR-1202 (CAS No.: 1532533-67-7)

[0144] PI-103 (CAS No.: 371935-74-9)

[0145] IC-87114(CAS No.: 371242-69-2)

[0146] Wrocman penicillin (CAS No.: 19545-26-7)

[0147] ZSTK474(CAS No.: 475110-96-4)

[0148] AS-605240(CAS No.: 648450-29-7)

[0149] PIK-90 (CAS No.: 677338-12-4)

[0150] AZD6482 (CAS No.: 1173900-33-8)

[0151] Duvelisib (CAS No.: 1201438-56-3)

[0152] TG100-115(CAS No.: 677297-51-7)

[0153] CH5132799(CAS No.:1007207-67-1)

[0154] CAY10505(CAS No.: 1218777-13-9)

[0155] PIK-293 (CAS No.: 900185-01-5)

[0156] CZC24832(CAS No.: 1159824-67-5)

[0157] Pilaralisib (CAS No.: 934526-89-3)

[0158] AZD8835(CAS No.: 1620576-64-8)

[0159] The concentration of PI3K inhibitors varies depending on the somatic cells used, and there are no particular restrictions. It can be determined appropriately, for example, it can be used in the range of 0.1 μmol / L to 20 μmol / L, preferably in the range of 0.5 μmol / L to 10 μmol / L.

[0160] 1.4 Insulin-producing cells

[0161] The manufacturing method of the present invention described above can yield cell clusters containing insulin-producing cells. Insulin-producing cells manufactured using the manufacturing method of the present invention are also within the scope of the present invention. Insulin-producing cells manufactured using the manufacturing method of the present invention can be either ultimately differentiated cells or progenitor cells destined to differentiate into insulin-producing cells.

[0162] The insulin-producing cells manufactured by the method of this invention are induced to differentiate directly from somatic cells by low-molecular-weight compounds, which are called low-molecular-weight compound-induced insulin-producing cells (ciIPCs), and are different from cells induced by differentiation through gene introduction.

[0163] Insulin-producing cells manufactured using the method of the present invention can, for example, be detected, identified, and isolated using changes in cell morphology, characteristic properties of insulin-producing cells, or specific markers (e.g., anti-insulin antibodies). Furthermore, the secretory capacity of the manufactured insulin-producing cells can be evaluated by quantifying the amount of insulin secreted using a sandwich ELISA.

[0164] Detection of specific markers can be performed using quarantine methods (detection via antibodies), and for protein molecules, detection can also be performed by quantifying their mRNA levels. Antibodies that recognize insulin-producing cell-specific markers are useful for the isolation and purification of insulin-producing cells obtained by the manufacturing method of this invention.

[0165] Insulin-producing cells manufactured using the method of this invention can, for example, be used for tissue repair or to improve blood insulin concentration. By transplanting insulin-producing cells manufactured using the method of this invention, pharmaceutical compositions for tissue repair and the like can be manufactured. For patients with type 1 diabetes who are congenitally unable to secrete insulin, pancreas transplantation or islet transplantation is a fundamental treatment to alleviate symptoms and achieve a cure. Furthermore, it is predicted that the number of patients with type 2 diabetes will further increase both domestically and internationally in the future, contributing to soaring medical costs. Transplantation of insulin-secreting pancreatic β-cells may be an effective treatment method. As a treatment for pancreatic diseases such as diabetes, the development of methods for manufacturing and transplanting insulin-producing cells is underway. For example, it is hoped that transplanting insulin-producing cells subcortical into the renal lining or via the portal vein to the liver can be used to treat severe pancreatic diseases (such as diabetes).

[0166] When preparing a pharmaceutical composition using insulin-producing cells manufactured by the method of the present invention, the insulin-producing cells are mixed with a pharmaceutically permissible carrier using conventional methods to prepare a formulation suitable for individual administration. Examples of carriers include physiological saline and isotonic distilled water for injection containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). Further mixing with buffers (e.g., phosphate buffer, sodium acetate buffer), analgesics (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc., is also possible.

[0167] The insulin-producing cells manufactured by the method of the present invention can be further prepared into compositions in combination with other cells or components that are effective in enhancing the function or adhesion of the insulin-producing cells.

[0168] Furthermore, the insulin-producing cells manufactured using the method of the present invention can be used for screening pharmaceutical candidate compounds that act on insulin-producing cells or for safety evaluation of pharmaceutical candidate compounds. Insulin-producing cells are an important tool for evaluating the toxicity of pharmaceutical candidate compounds. According to the manufacturing method of the present invention, since a large number of insulin-producing cells can be obtained in a single operation, reproducible research results can be obtained regardless of batch-to-batch variations in cells.

[0169] 2 Compositions

[0170] The induction composition involved in this invention (hereinafter referred to as "the composition of the invention") is a composition for inducing the production of insulin-producing cells by direct differentiation from somatic cells, and is serum-free. Alternatively, it is a composition for inducing the production of insulin-producing cells by direct differentiation from somatic cells, containing more than 5 μg / mL of insulin.

[0171] As described above, one of the key points of the present invention is that the composition for inducing insulin-producing cells from direct differentiation of somatic cells substantially does not contain serum such as bovine embryonic serum (FBS). Furthermore, as an example of the key points of the present invention, the composition for inducing insulin-producing cells from direct differentiation of somatic cells contains a relatively high amount of insulin, at 5 μg / mL or more.

[0172] In this context, the preferred composition of the present invention is serum-free and contains insulin at a concentration of 5 μg / mL or higher.

[0173] Furthermore, compositions of the present invention containing RSK inhibitors are preferred. More preferably, compositions of the present invention, in addition to RSK inhibitors, further contain GSK3 inhibitors and / or cAMP inducers. In particular, compositions of the present invention containing RSK inhibitors and GSK3 inhibitors, or RSK inhibitors, GSK3 inhibitors, and cAMP inducers are preferred. Compositions of the present invention may also contain other inhibitors, inducers, etc., such as PI3K inhibitors, as needed.

[0174] The inhibitors, inducers, etc., may be used in one or in combination of two or more.

[0175] Specifically, some of the inhibitors may have more than two inhibitory effects. In this case, containing one inhibitor can be regarded as containing multiple inhibitors.

[0176] Specific examples or preferred examples of the inhibitors, inducers, etc., are synonymous with the above.

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

[0178] As an example, the composition of the present invention may be a basal medium prepared by mixing the components necessary for cell culture, but without serum such as bovine embryonic serum (FBS), and / or containing insulin at a concentration of 5 μg / mL or higher. For example, it may further contain an RSK inhibitor as an active ingredient, and further, if necessary, a GSK3 inhibitor and / or a cAMP inducer and a PI3K inhibitor. The active ingredient is contained at a concentration effective for the production of insulin-producing cells, and the concentration can be appropriately determined by those skilled in the art. The basal medium can be selected from known or commercially available media. For example, common media such as MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), DMEM / F12, RPMI 1640, or modified versions thereof can be used as the basal medium.

[0179] In one embodiment of the composition of the present invention, it contains insulin at a concentration of 5 μg / mL or more, but preferably at a concentration of 20 μg / mL or more or 25 μg / mL or more, and more preferably in the range of 80 to 120 μg / mL.

[0180] When a known or commercially available differentiation induction culture medium pre-contains a certain amount of insulin, insulin can be added to adjust the insulin content to the stated amount in order to prepare the composition of the present invention.

[0181] The culture medium involved in the composition of the present invention may be further supplemented with known culture medium components as described in this specification, such as serum, proteins (albumin, transferrin, growth factors, etc.), amino acids, sugars, vitamins, fatty acids, antibiotics, etc.

[0182] The culture medium involved in the composition of the present invention may be further supplemented with substances that are effective in inducing differentiation of insulin-producing cells as described in this specification. Example

[0183] The present invention will be specifically described below through embodiments, but the present invention is not limited to the scope of the embodiments.

[0184] Example 1: Manufacturing of Insulin-Producing Cells

[0185] Direct induction from human fibroblasts to insulin-producing cells

[0186] (1) Human fibroblasts

[0187] The human fibroblasts used as the material were purchased from DS Pharma Biomedical Co., Ltd. They were fibroblasts derived from the skin of a 38-year-old human.

[0188] (2) Direct induction from human fibroblasts to insulin-producing cells

[0189] Human fibroblasts were cultured in 35 mm culture dishes coated with gelatin (Cat#: 190-15805, manufactured by Wako Pure Chemical Industries, Ltd.) at a density of 5 × 10⁻⁶. 4 Individuals were inoculated per dish with DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured at 37°C and 5% CO2 until confluence. It should be noted that DMEM stands for Dulbecco's Modified Eagle Medium.

[0190] The culture medium of the human fibroblasts in the culture dish was exchanged with the differentiation-inducing culture medium described below.

[0191] Differentiation induction medium: with or without 10% bovine embryonic serum (FBS, Hyclone), and supplemented with ITS-X (Cat#: 51500056, Gibco), non-essential amino acids (NEAA; Cat#: 11140050, Gibco), glutamine (Gibco; final concentration 2 mmol / L), nicotinamide (Cat#: 72340-100G, Sigma-Aldrich; final concentration 10 mmol / L), exendin-4 (Cat#: av120214, Abcam; final concentration 100 ng / mL), 100 U / mL penicillin, 100 μg / mL streptomycin, insulin, and a modified DMEM / F-12 (Cat#: 12634010, Gibco) of the following low molecular weight compounds.

[0192] Then, the culture medium was exchanged every 3 days with the same composition, and cultured at 37°C and 5% CO2.

[0193] It should be noted that for the culture medium used for differentiation induction, recombinant human insulin (Cat#: 093-06351; Wako) should be added as needed to adjust the concentration to 20-120 μg / mL.

[0194] <Low molecular weight compounds>

[0195] 3μM CHIR99021 (Cat#: 13122, Cayman Chemical)

[0196] 7.5 μM Trichoderma (Cat#: 063-02193, Wako)

[0197] 1.5μM BRD7389 (Cat#: ab146161, Abcam)

[0198] 5μM LY294002 (Cat#: 70920, Cayman Chemical)

[0199] (3) Results

[0200] As described in (2), the results after 14 days of cultivation are as follows: Figures 1-4 As shown.

[0201] In the figure, "4C" refers to the four low-molecular-weight compounds, and "3C" refers to CHIR99021, BRD7389, and trichodin among the four low-molecular-weight compounds. "+FBS" indicates the presence of FBS in the culture medium, and "-FBS" indicates the absence of FBS in the culture medium. Additionally, "NoC" or "No Compound" indicates the absence of the four low-molecular-weight compounds (4C) or 3C in the culture medium. Therefore, for example, "No C.-FBS" indicates the experimental result when both 4C and FBS are absent, and "4C-FBS" indicates the experimental result when 4C is present but FBS is absent.

[0202] Figure 4 In this context, "B" refers to BRD7389, and "3C-B" indicates the absence of BRD7389 in 3C, i.e., the experimental results under the two-factor condition of CH and F. "3C-B+BI-D1870" indicates the absence of BRD7389 in 3C, and the presence of the RSK inhibitor BI-D1870 (Cat#: 15264, Cayman Chemical) as its substitute at a final concentration of 5 μM or 10 μM.

[0203] like Figure 1 As shown, by inducing differentiation induction in a culture medium free of bovine embryonic serum (FBS), insulin-producing cells with a 20-50 fold increase in insulin secretion were directly and efficiently induced from human fibroblasts.

[0204] like Figure 2As shown, by inducing differentiation induction with a high concentration of insulin (120 μg / mL) in the culture medium, insulin-producing cells with a higher secretion capacity, which were obtained directly and efficiently from human fibroblasts, were obtained with an increase of about 3 times in insulin secretion.

[0205] Based on the above, it can be concluded that the effects of FBS and high concentrations of insulin on increased insulin secretion are independent.

[0206] like Figure 3 As shown, insulin-producing cells with high concentration-dependent secretion capacity were obtained directly from human fibroblasts, with insulin concentrations in the differentiation-inducing culture medium up to 100 μg / mL. The higher the concentration, the greater the insulin secretion.

[0207] from Figure 4 The results showed that, under the condition of removing LY294002 and further removing the RSK inhibitor (BRD7389) from the three factors of the RSK inhibitor (BRD7389), GSK inhibitor (CHIR99021), and cAMP inducer (trichoamne), insulin secretion decreased, indicating that the presence of RSK inhibitors is important in the induction of insulin-producing cells. On the other hand, under the three-factor condition of replacing BRD7389 and presenting another RSK inhibitor, BI-D1870, insulin-producing cells with high secretory capacity were also obtained.

[0208] Example 2: Manufacturing of Insulin-Producing Cells

[0209] Direct induction from human mesenchymal stem cells to 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 cultured in 35mm culture dishes coated with gelatin (Cat#: 190-15805, manufactured by Wako Pure Chemical Industries, Ltd.) at a density of 5 × 10⁻⁶. 4 Each stem cell was inoculated into a medium containing 100 U / mL penicillin and 100 μg / mL streptomycin (Mesenchymal Stem Cell Growth Medium 2, Cat#: C-28009; manufactured by TakaraBio) and cultured at 37°C and 5% CO2 until almost confluent.

[0214] The culture medium of the human mesenchymal stem cell culture dish was exchanged with the differentiation-inducing culture medium described below.

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

[0216] Then, the culture medium was exchanged every 3 days with the same composition, and cultured at 37°C and 5% CO2.

[0217] It should be noted that in the experiment where the induction culture medium contained 120 μg / mL insulin, recombinant human insulin (Cat#: 093-06351; Wako) was added to adjust the concentration to achieve 120 μg / mL insulin.

[0218] <Low molecular weight compounds>

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

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

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

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

[0223] (3) Results

[0224] As described in (2), the results after 14 days of cultivation are as follows: Figure 5 As shown.

[0225] In the figure, "4C" refers to the four low-molecular-weight compounds. "+FBS" indicates the presence of FBS in the culture medium, and "-FBS" indicates the absence of FBS in the culture medium. Additionally, "No compound" indicates the absence of the four low-molecular-weight compounds in the culture medium. Therefore, for example, "No Compound-FBS" represents the experimental result when neither 4C nor FBS is present, and "4C-FBS" represents the experimental result when 4C is present but FBS is absent.

[0226] like Figure 5 As shown, the same principle applies to the direct differentiation induction of human adipose tissue-derived mesenchymal stem cells (AdMSCs). Insulin secretion was readily confirmed even when the differentiation induction medium did not contain bovine embryonic serum (FBS). Furthermore, when the differentiation induction medium contained a high concentration (120 μg / mL) of insulin, insulin-producing cells with significantly increased insulin secretion and higher secretion capacity were efficiently induced directly from human mesenchymal stem cells.

Claims

1. A method for manufacturing insulin-producing cells, comprising a method for manufacturing insulin-producing cells by direct differentiation-induced differentiation from fibroblasts, wherein, The method includes: The process of culturing fibroblasts using a serum-free differentiation-inducing medium containing at least 20 μg / mL of insulin. The process described involves culturing fibroblasts in the presence of RSK inhibitors, GSK3 inhibitors, cAMP inducers, and PI3K inhibitors. The RSK inhibitor is either BRD7389 or BI-D1870. The GSK3 inhibitor is CHIR99021. The cAMP inducer is trichodin. The PI3K inhibitor is LY294002.

2. A differentiation-inducing composition for producing insulin-producing cells by direct differentiation induction from fibroblasts, wherein, The composition is serum-free and contains at least 20 μg / mL of insulin. The composition contains an RSK inhibitor, a GSK3 inhibitor, a cAMP inducer, and a PI3K inhibitor. The RSK inhibitor is either BRD7389 or BI-D1870. The GSK3 inhibitor is CHIR99021. The cAMP inducer is trichodin. The PI3K inhibitor is LY294002.

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