KR102717835B1 - Process and compositions for the direct differentiation of somatic cell into pancreatic beta cells using microRNA - Google Patents

Abstract

The present invention relates to a method for directly differentiating somatic cells into pancreatic beta cells using micro RNA and a low-molecular-weight substance. The inventors of the present invention attempted direct differentiation by co-treatment with various differentiation-inducing substances and low-molecular-weight substances and micro RNA. As a result, the expression level of PDX1 in pancreatic beta cells was significantly increased, and it was confirmed that direct differentiation was achieved at a very high yield when pancreatic beta cells-like cells were induced using this method. In addition, since the present invention uses autologous cells, it has the advantage of not causing an immune rejection response and having a low possibility of cancer development, and thus it is expected to be usefully used in the development of safer cell therapy agents. In addition, it is expected that the pancreatic beta cells produced by the present invention can be usefully used as a cell composition for preventing, treating, and improving diabetes or pancreatic cancer.

Description

Process and compositions for the direct differentiation of somatic cells into pancreatic beta cells using microRNA

The present invention relates to a method for directly differentiating pancreatic beta cells from somatic cells and a differentiation composition, and more specifically, to a composition for inducing direct differentiation of somatic cells into pancreatic beta cells, the composition comprising at least one selected from the group consisting of micro RNA (miRNA) miR-127 and miR-709 as an effective ingredient, and a method for directly differentiating pancreatic beta cells using the same.

In diabetes, glucose toxicity induces apoptosis of β-cells and affects various organ systems, including the pancreas. However, the underlying mechanisms are not completely known. β-cell dysfunction and impaired insulin production are typical features of diabetes, but despite the rapid spread of diabetes, the precise molecular mechanisms of glucotoxicity that induces β-cell apoptosis are still unknown.

In particular, diseases such as type 1 diabetes and pancreatic cancer are caused by dysfunction due to damage or loss of beta cells, so pancreatic beta cell transplantation is the only treatment.

As stem cell research has advanced recently, not only has the time and efficiency issues been reduced in the process of directly differentiating somatic cells into various lineages (redifferentiation or reprogramming) without going through intermediate differentiation, but a new path has been opened for autologous transplantation. However, pancreatic beta cells derived from induced pluripotent stem cells (iPSCs) may cause immune rejection because they use cells from others, and there may be safety issues due to the possibility of stem cells causing cancer.

Accordingly, direct differentiation methods that convert one's own somatic cells into pancreatic beta cells are gaining attention. Methods for converting somatic cells into pancreatic beta cells include methods for forcibly expressing beta cell marker genes and methods using small molecule substances, but methods for introducing external genes have the disadvantage of affecting the genome and causing cancer, and when using small molecule substances, the conversion efficiency into pancreatic beta cells is low.

In addition, the period required to convert into pancreatic beta cells was more than 27 days, which was a long period for producing pancreatic beta cells, and there was a need to shorten this period (Cell Stem Cell. 2014 Feb 6; 14(2):228-36. doi:10.1016/j.stem.2014.01.006.)).

To overcome these problems, the inventors of the present invention developed a technology to effectively convert somatic cells into pancreatic beta cells in a short period of time by treating the somatic cells with microRNA alone or together with small molecule substances.

Publication Patent No. 10-2015-0117532

The present invention has been made to solve the problems of the prior art as described above, and as a result of the present inventors' efforts to find a method for directly converting somatic cells into pancreatic beta cells, the present inventors have completed the present invention by confirming that the microRNA of the present invention alone; or a histone methyltransferase inhibitor, a retinoic acid agonist, an ALK-5 kinase inhibitor, a hedgehog inhibitor, a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, a supplement; and a composition including microRNA can be treated to induce conversion of somatic cells into pancreatic beta cells.

Accordingly, the purpose of the present invention is to provide a composition for inducing direct differentiation of somatic cells into pancreatic beta cells, which comprises at least one miRNA selected from the group consisting of miR-127 and miR-709.

Another object of the present invention is to provide a method for direct differentiation of somatic cells into pancreatic beta cells, comprising the step of culturing somatic cells in the presence of a composition comprising a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes or pancreatic cancer, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-127 and miR-709.

Another object of the present invention is to provide a method for producing a cell therapy agent for treating diabetes or pancreatic cancer, comprising a step of mixing pancreatic beta cells directly differentiated by the above method with at least one pharmaceutically acceptable carrier and excipient selected from the group consisting of pharmaceutically acceptable carriers and excipients.

Another object of the present invention is to provide a method for preventing or treating diabetes or pancreatic cancer, comprising a step of delivering into a living body a composition comprising as an active ingredient at least one miRNA selected from the group consisting of miR-127 and miR-709, thereby inducing direct differentiation of somatic cells into beta cells in a living body.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

To achieve the above purpose, the present invention provides a composition for inducing direct differentiation of somatic cells into pancreatic beta cells, comprising micro RNA or a low-molecular-weight substance as an effective ingredient.

In addition, the present invention provides a composition for inducing direct differentiation of somatic cells into pancreatic beta cells, comprising one or more miRNAs selected from the group consisting of miR-127 and miR-709.

In one embodiment of the present invention, the somatic cell may be at least one selected from the group consisting of fibroblasts, pancreatic duct cells, and exocrine cells, but is not limited thereto.

In another embodiment of the present invention, the composition may additionally comprise, but is not limited to, miR-19b.

In another embodiment of the present invention, the composition may further comprise one or more small molecule substances selected from the group consisting of, but not limited to, a histone methyltransferase inhibitor, a retinoic acid agonist, an ALK-5 kinase inhibitor, a hedgehog inhibitor, a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, and a supplement.

In another embodiment of the present invention, the histone methyltransferase inhibitor is BIX01294 (2-(Hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-6,7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinamine), 5-AZA-2'-deoxycytidine (5-aza-2'-deoxycytidine:DAC), Zebularine, 3'-Deazaneplanocin A hydrochloride, Lomeguatrib, Chaetocin, may be at least one selected from the group consisting of, 2,2',3S,3'S,5aR,5'aR,6,6'-octahydro-3,3'-bis(hydroxymethyl)-2,2'-dimethyl-[10bR,10'bR(11aS,11'aS)-bi-3,11a-epidithio-11aH-pyrazino[1',2':1,5]pyrrolo[2,3-b]indole]-1,1',4,4'-tetrone), and decitabine (5-aza-2'-deoxycytidine), but is not limited thereto.

In another embodiment of the present invention, the supplement may be one or more selected from the group consisting of, but not limited to, 2-phospho-L-ascorbic acid, B27, laminin, nicotinamide, and N2.

In another embodiment of the present invention, the retinoic acid agonist may be at least one selected from the group consisting of TTNPB, phytanic acid, and retinoic acid, but is not limited thereto.

In another embodiment of the present invention, the hedgehog inhibitor may be at least one selected from the group consisting of cyclopamine, mifepristone, GDC-0449 (Vismodegib), XL139 (BMS-833923), IPI926, IPI609 (IPI269609), LDE225, zervin, GANT61, firmopamine, SAG, SANT-2, tomatidine, SANT74, SANT75, zerumbone and derivatives thereof, but is not limited thereto.

In another embodiment of the present invention, the MAPK inhibitor may be at least one selected from the group consisting of, but not limited to, 1-Pyridinyl-2-phenylazole, SB 203580, SKF 86002, SKF 86096, SKF 104351, 1-Aryl-2-pyridinyl/pyrimidinyl heterocycles, SB 242235, RO-32001195, SX-011, and BIRB-796.

In another embodiment of the present invention, the ALK-5 kinase inhibitor is RepSox (1,5-Naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]); SB525334 (6-(2-tert-butyl-4-(6-methylpyridin-2-yl)-1H-imidazol-5-yl)quinoxaline); GW788388 (4-(4-(3)-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide); SD-208 (2-(5-chloro-2-fluorophenyl)-N-(pyridin-4-yl)pteridin-4-amine); Galunisertib (LY2157299, 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide); EW-7197 (N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methanamine); LY2109761 (7-(2-morpholinoethoxy)-4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline); SB505124 (2-(4-(benzo[d][1,3]dioxol-5-yl)-2-tert-butyl-1H-imidazol-5-yl)-6-methylpyridine); LY364947 (Quinoline, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]); SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide); K02288 (3)-[(6-Amino-5-(3),4,5-trimethoxyphenyl)-3-pyridinyl]phenol]; and LDN-212854 (Quinoline, 5-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]), but is not limited thereto.

In another embodiment of the present invention, the calcium channel agonist may be one or more selected from the group consisting of, but not limited to, Bay K-8644, FPL 64179, and CGP28392.

In another embodiment of the present invention, the GLP receptor agonist may be at least one selected from the group consisting of, but not limited to, Exendin-4, Dulaglutide, Exenatide, Semaglutide, Liraglutide, Lixisenatide, and Albiglutide.

In addition, the present invention provides a method for direct differentiation of somatic cells into pancreatic beta cells, comprising the step of culturing somatic cells in the presence of a composition comprising a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709.

In one embodiment of the present invention, the method may comprise or consist of, but is not limited to, the steps of (1) inducing somatic cells into pancreatic endoderm cells; (2) inducing pancreatic endoderm cells into pancreatic progenitor cells; and (3) inducing pancreatic progenitor cells into pancreatic beta cells.

In another embodiment of the present invention, the method may comprise, or consist of, but is not limited to, the step of (3) inducing pancreatic progenitor cells into pancreatic beta cells.

In another embodiment of the present invention, when the somatic cells of the method are fibroblasts, differentiation through three steps is required, but when the somatic cells are pancreatic ductal cells and exocrine cells having the properties of pancreatic progenitor cells, direct differentiation into pancreatic beta cells from the somatic cells is possible in the presence of the composition.

In another embodiment of the present invention, the direct differentiation method may include, but is not limited to, the following steps:

(1) a step of inducing a somatic cell into a pancreatic endoderm cell in the presence of a composition comprising a histone methyltransferase inhibitor; activin A, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709;

(2) a step of inducing pancreatic endoderm cells into pancreatic progenitor cells in the presence of a composition comprising a retinoic acid agonist, an ALK-5 kinase inhibitor, a hedgehog inhibitor, a supplement; and one or more miRNAs selected from the group consisting of miR-127 and miR-709; and

(3) A step of culturing somatic cells in the presence of a composition comprising a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes or pancreatic cancer, comprising as an active ingredient one or more miRNAs selected from the group consisting of miR-127 and miR-709.

In one embodiment of the present invention, the diabetes may be selected from the group consisting of type 1 diabetes, type 2 diabetes, and gestational diabetes, but is not limited thereto.

In addition, the present invention provides a method for producing a cell therapy agent for treating diabetes or pancreatic cancer, comprising a step of mixing pancreatic beta cells directly differentiated by the direct differentiation method with at least one selected from the group consisting of pharmaceutically acceptable carriers and excipients.

In addition, the present invention provides a cell therapeutic agent for treating diabetes or pancreatic cancer, which comprises pancreatic beta cells directly differentiated by the direct differentiation method as an active ingredient.

In addition, the present invention provides a method for preventing or treating diabetes or pancreatic cancer, comprising a step of delivering into a living body a composition comprising at least one miRNA selected from the group consisting of miR-127 and miR-709, thereby inducing direct differentiation of somatic cells into beta cells in a living body.

In addition, the present invention provides a method for preventing or treating diabetes or pancreatic cancer, comprising a step of administering to a subject in need thereof a pharmaceutical composition comprising, as an active ingredient, at least one miRNA selected from the group consisting of miR-127 and miR-709.

In addition, the present invention provides a pharmaceutical composition comprising at least one miRNA selected from the group consisting of miR-127 and miR-709 as an active ingredient for the prevention or treatment of diabetes or pancreatic cancer.

The present invention also provides a use for preparing one or more diabetes or pancreatic cancer treatment agents selected from the group consisting of miR-127 and miR-709.

The present invention also provides a use of a composition comprising one or more miRNAs selected from the group consisting of miR-127 and miR-709 for inducing direct differentiation of somatic cells into pancreatic beta cells.

The present invention relates to a method for directly differentiating somatic cells into pancreatic beta cells using micro RNA and a low-molecular-weight substance. The inventors of the present invention attempted direct differentiation by concurrently treating various differentiation-inducing substances and low-molecular-weight substances with micro RNA, and as a result, the expression level of PDX1 in pancreatic beta cells was significantly increased, and it was confirmed that direct differentiation was achieved with a very high yield when pseudo-pancreatic beta cells were induced using this method. In addition, the present invention has the advantage of using autologous cells when transplanting converted pancreatic beta cells into patients with diabetes or pancreatic cancer, so there is no immune rejection response and the possibility of cancer occurrence is low, and thus it is expected to be usefully used in the development of safer cell therapy agents. In addition, it is expected that the pancreatic beta cells produced by the present invention can be usefully used as a cell composition for preventing, treating, and improving diabetes or pancreatic cancer.

Figure 1a is a diagram showing a method for converting somatic cells into beta cells using a low-molecular substance, Figure 1b shows a method for additionally adding micro RNA to the conversion conditions using a low-molecular substance, and Figure 1c is a diagram showing that beta cell markers Pdx1 and Ins-2 are overexpressed in beta cells converted using a low-molecular substance.
Figure 2 shows the expression of major beta cell markers during the differentiation of fibroblasts into beta cell-like cells. * P < 0.05, ** P < 0.01, and *** P < 0.001. Statistical significance was determined by a two-tailed, paired Student's t-test. Data shown represent the mean ± SEM from three independent experiments.
Figure 2a shows the results of comparing Pdx1 gene expression in stage 1 cells after treatment with various types of miRNA.
Figure 2b shows the effect of inducing Pdx1 expression according to the combination of different miR-127 and miR-709 concentrations in stage 1 cells.
Figure 2c shows the Ngn3 transcript level in stage 1 cells after transfection with miR-127 and miR-709 using combination-3.
Figure 2d shows the results of qRT-PCR to determine the pancreatic gene expression profile of stage 2 cells transfected with miR-127 and miR-709 (combination-3).
Figure 2e shows the results of qRT-PCR for pancreatic β-cell markers in stage 3 cells transfected with miR-127 and miR-709 (combination-3).
Figure 3 shows the proliferation ability of mouse embryonic fibroblasts (MEFs) following transfection with a combination of miRNA mimics and the miRNAs of the present invention (miR-127, miR-709, and miR-19b).
Figure 3a shows the results of transfection of 5′FAM-labeled control mimics to optimize transfection efficiency in fibroblasts. Bright-field microscopy images (left) and fluorescence microscopy images (right) are shown after transfection for 48 h in stage 1 medium. Scale bar = 100 μm.
Figure 3b shows the effect of various concentration combinations of miR-127, miR-709, and miR-19b on inducing Pdx1 expression in the stage 1 medium using qRT-PCR analysis. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance was determined by a two-tailed, paired Student's t-test. The data shown represent the mean ± SEM obtained from three independent experiments.
Figure 3c shows bright-field microscopic images of the morphological changes in MEFs treated with different combinations at stage 1 of differentiation (after 48 h). Scale bar = 100 μm.
Figure 4 shows the expression of pancreatic beta cell-related markers in acinar cells 266-6, a type of exocrine cell, following transfection with a combination of miRNA mimics and miRNAs of the present invention (miR-127 and miR-709).
Figure 4a shows the results of optimized transfection of 266-6 cells in Step 3 medium, showing bright-field microscopy images (left) and fluorescence microscopy images (right) after transfection for 48 hours in Step 3 medium.
Figure 4b shows the transcription levels of Pdx1, Ngn3, Insulin-1, Insulin-2, and Elastase in 266-6 cells transfected with miR-127 + miR-709 (Combination-3). Combination-3 decreased the expression of Elastase, a marker of acinar cells. * P < 0.05 and ** P < 0.01. Statistical significance was determined by a two-tailed, paired Student’s t-test. The data shown represent the means ± SEM obtained from three independent experiments.
Figure 5 shows the expression of pancreatic beta cell genes in human pancreatic ductal cell Capan-1 cells according to combinations of miR-127 and small molecules in the three steps. The small molecules used in the three steps, SB203580 (MAP kinase inhibitor), Nicotinamide (adjuvant), Exendin-4 (GLP receptor agonist), Bay K-8644 (calcium channel agonist), were treated with different combinations of miR-127 and the expression levels of beta cell markers Pax-6 and MafA were measured.

The present inventors have confirmed that when microRNA and small molecules are used in combination, direct differentiation into pancreatic beta cells is significantly better than when microRNA or small molecules are used alone or in a control group treated with microRNA alone or small molecules alone or not treated. Therefore, the present invention provides a composition for inducing direct differentiation of somatic cells into pancreatic beta cells, which comprises a specific microRNA as an effective ingredient, and more specifically, relates to a composition for inducing direct differentiation of somatic cells into pancreatic beta cells, which comprises one or more miRNAs selected from the group consisting of miR-127 and miR-709.

Hereinafter, the present invention will be described in detail.

In the present invention, “micro RNA (miRNA, microRNA) is a small noncoding RNA of ~22 nucleotides in length that acts as a negative regulator of gene expression by inhibiting mRNA translation or promoting mRNA degradation.

In the present invention, “miR-127” may include or consist of a base sequence represented by sequence number 1, but is not limited thereto.

In the present invention, “miR-709” may include or consist of a base sequence represented by sequence number 2, but is not limited thereto.

In the present invention, “miR-19b” may include or consist of a base sequence represented by SEQ ID NO: 5, but is not limited thereto.

In the present invention, miR-127 and miR-709 may be included in a molar concentration (M) ratio of 0.1 to 10:1, 1 to 3:1, 1:1 to 3, or 1:1, but is not limited thereto.

In the present invention, miR-127, miR-709, and miR-19b may be included in a molar concentration (M) ratio of 0.1 to 10:0.1 to 10:1, a molar concentration (M) ratio of 1 to 3:1 to 3:1 to 3, or a molar concentration (M) ratio of 1:1:1, but is not limited thereto.

In inducing the pancreatic beta cells of the present invention, the type of starting somatic cells (parent cells) is not particularly limited, and any somatic cell can be used. For example, in addition to somatic cells from the embryonic period, mature somatic cells can be used. When the induced pancreatic beta cells are used for the treatment of a disease, it is preferable to use somatic cells isolated from a patient, and for example, somatic cells involved in the disease or somatic cells involved in the treatment of the disease can be used. Meanwhile, the somatic cells of the present invention may be cells derived from a human pancreas, but are not limited thereto. The somatic cells may be fibroblasts, pancreatic duct cells, or exocrine cells, and the somatic cells in the present invention include all somatic cells derived from animals such as humans, mice, horses, sheep, pigs, goats, camels, antelopes, and dogs.

In the present invention, the term "pancreatic β cells" may be used interchangeably with "pancreatic β cell-like cells", which are one of the cells that make up the islets of Langerhans in the pancreas and are cells that produce and secrete insulin. If there is a problem with pancreatic β cells, there may be a problem with the production of insulin, which may cause diabetes. Therefore, such cells may be used to treat diabetes caused by a problem with the secretion of insulin by the pancreas.

The term “pancreatic progenitor cell” in the present invention refers to an endomesoderm cell that is typically capable of differentiating into pancreatic endocrine cells and pancreatic exocrine cells, and in the present invention, can be induced to differentiate into pancreatic beta cells.

In one embodiment of the present invention, it was confirmed that directly differentiated pancreatic beta cells highly expressed pancreatic-specific gene markers PDX1, Ngn3, Ins-1, and Ins-2 by treating somatic cells with microRNA and small molecule substances.

In the present invention, the composition may additionally include miR-19b, but is not limited thereto.

In the present invention, the composition may additionally contain one or more small molecule substances selected from the group consisting of a histone methyltransferase inhibitor, a retinoic acid agonist, an ALK-5 kinase inhibitor, a hedgehog inhibitor, a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, and a supplement, but is not limited thereto.

In the present invention, when the composition contains a histone methyltransferase inhibitor, it may be used for inducing differentiation of somatic cells into pancreatic endoderm cells, but is not limited thereto.

In the present invention, when the composition comprises a retinoic acid agonist, an ALK-5 kinase inhibitor, and a hedgehog inhibitor, it may be used for inducing differentiation of somatic cells or pancreatic endoderm cells into pancreatic progenitor cells, but is not limited thereto.

In the present invention, when the composition comprises a MAPK inhibitor, a calcium channel agonist, and a GLP receptor agonist, it may be used for inducing maturation of pancreatic beta cells, but is not limited thereto.

The term "maturation" in the present invention means that cells induced as pancreatic beta cells are converted into beta cells with more complete activity.

The term "retinoic acid agonist" in the present invention may preferably be a retinoic acid receptor agonist (RAR agonist), and may be at least one selected from the group consisting of TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid, arotinoid acid), phytanic acid, and retinoic acid (RA), but is not limited thereto. RAR receptors activate transcription by binding to a DNA sequence element known as a RAR response element (RARE) in the form of a heterodimer with a retinoid X receptor (known as RXR). The prior art includes a number of compounds which are RAR type receptor ligands. Among the prior art documents, examples that may be mentioned include patent US 6150413, which describes triaromatic compounds, patent US 6214878, which describes stilbene compounds, or patent US 6218128, which describes a group of bicyclic or tricyclic molecules. The medium of the present invention may comprise a retinoic acid agonist at, but is not limited to, 0.01 nM to 30 nM, 0.01 nM to 20 nM, 0.01 nM to 10 nM, 0.1 nM to 30 nM, 0.01 nM to 20 nM, 0.01 nM to 10 nM, 0.1 nM to 8 nM, 0.1 nM to 6 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, 0.1 nM to 1 nM, 0.1 nM to 0.8 nM, 0.1 nM to 0.7 nM, 0.3 nM to 1 nM, 0.3 nM to 0.7 nM, or about 0.5 nM.

In the present invention, "ALK-5 kinase inhibitor" means a substance that binds to the TGF-β type I receptor and interferes with the normal signaling process of TGF-β I. TGF-β type I (Transforming growth factor-β type I) is a multifunctional peptide that has various effects on cell proliferation, differentiation, and various types of cells, and this multifunctionality plays a pivotal role in the growth and differentiation of various tissues, such as adipogenesis, myogenesis, osteocytogenesis, and epithelial cell differentiation. The ALK-5 kinase inhibitor (TGF-β type I receptor inhibitor) is RepSox (1,5-Naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]); SB525334 (6-(2-tert-butyl-4-(6-methylpyridin-2-yl)-1H-imidazol-5-yl)quinoxaline); GW788388(4-(4-(3)-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide); SD-208 (2-(5-chloro-2-fluorophenyl)-N-(pyridin-4-yl)pteridin-4-amine); Galunisertib (LY2157299, 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide); EW-7197 (N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methanamine); LY2109761 (7-(2-morpholinoethoxy)-4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline); SB505124 (2-(4-(benzo[d][1,3]dioxol-5-yl)-2-tert-butyl-1H-imidazol-5-yl)-6-methylpyridine); LY364947 (Quinoline, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]); SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide); K02288 (3)-[(6-Amino-5-(3),4,5-trimethoxyphenyl)-3-pyridinyl]phenol]; or LDN-212854 (Quinoline, 5-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]), preferably Repsox, but is not limited thereto. The medium of the present invention may contain an ALK-5 kinase inhibitor at a concentration of 0.01 μM to 20 μM, preferably 0.1 to 10 μM, more preferably 0.5 μM to 5 μM, and most preferably 0.7 μM to 1.5 μM.

The term "hedgehog inhibitor" in the present invention may preferably be a sonic hedgehog inhibitor, and more preferably may be, but is not limited to, cyclopamine, mifepristone, GDC-0449 (Vismodegib), XL139 (BMS-833923), IPI926, IPI609 (IPI269609), LDE225, zervin, GANT61, furopamine, SAG, SANT-2, tomatidine, SANT74, SANT75, zerumbone or a derivative thereof. The medium of the present invention may contain a hedgehog inhibitor at a concentration of 0.05 μM to 20 μM, preferably 0.1 to 15 μM, more preferably 0.5 μM to 10 μM, even more preferably 0.5 μM to 5 μM, and most preferably 1 μM to 3 μM.

The term "MAPK inhibitor" as used herein refers to mitogen activated protein kinases (MAPK), a member of the proline-operated serine/threonine kinase family that activates their substrates by dual phosphorylation. Known MAPK inhibitors include, but are not limited to, known P38 kinase inhibitors, 1-Pyridinyl-2-phenylazole, SB 203580, SKF 86002, SKF 86096, SKF 104351, 1-Aryl-2-pyridinyl/ pyrimidinyl heterocycles, SB 242235, RO-32001195, SX-011, or BIRB-796, and the following literature [G. J. Hanson (Expert Opinions on Therapeutic Patents, 1997, 7, 729-733), J Hynes et al . (Current Topics in Medicinal Chemistry, 2005, 5, 967-985), C. Dominguez et al (Expert Opinions on Therapeutics Patents, 2005, 15, 801-816) and L. H. Pettus & R. P. Wurtz (Current Topics in Medicinal Chemistry, 2008, 8, 1452-1467). The MAPK inhibitor of the present invention may preferably be sb203580, but is not limited thereto. The medium of the present invention comprises a MAPK inhibitor at a concentration of 50 μM to 5000 μM, 0.001 to 2500 μM, 0.01 to 1000 μM, 0.01 to 900 μM, 0.01 to 800 μM, 0.01 to 700 μM, 0.01 to 600 μM, 0.01 to 500 μM, 0.01 to 400 μM, 0.01 to 300 μM, 0.01 to 200 μM, 0.01 to 100 μM, 0.01 to 90 μM, 0.01 to 80 μM, 0.01 to 70 μM, 0.01 to 60 μM, 0.01 to 50 μM, 0.01 to 40 μM, 0.01 to 30 μM, 0.01 to 20 μM, 0.01 to 10 μM, 0.01 to 5 μM, 0.01 to 3 μM, 0.01 to 2 μM, 0.01 to 1 μM, 0.5 to 10 μM, 0.5 to 7 μM, 0.5 to 5 μM, 0.5 to 3 μM, 0.5 to 1.5 μM, 0.7 to 1.3 μM, or about 1 μM.

The term “calcium channel agonist” in the present invention is also called “calcium channel opener” and is not limited to a substance that promotes ion transport through a calcium channel. The calcium channel agonist of the present invention may be at least one selected from the group consisting of Bay K-8644, FPL 64179, and CGP28392, but is not limited thereto. The medium of the present invention may contain the calcium channel agonist in an amount of 0.05 μM to 20 μM, preferably 0.1 to 15 μM, more preferably 0.5 μM to 10 μM, even more preferably 0.5 μM to 5 μM, and most preferably 1 μM to 3 μM.

The term “GLP receptor agonist” in the present invention may specifically be a GLP-1 receptor agonist, and encompasses all peptides having GLP agonistic activity, fragments, precursors, variants or derivatives thereof, and may include, without limitation, substances capable of activating a GLP receptor. The GLP receptor agonist of the present invention may be at least one selected from the group consisting of exendin-4, dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, and albiglutide, but is not limited thereto. The medium of the present invention may comprise a GLP receptor agonist at 1 to 500 ng/mL, 1 to 400 ng/mL, 1 to 300 ng/mL, 1 to 200 ng/mL, 1 to 100 ng/mL, 30 to 500 ng/mL, 30 to 400 ng/mL, 30 to 300 ng/mL, 30 to 200 ng/mL, 30 to 100 ng/mL, 30 to 90 ng/mL, 30 to 80 ng/mL, 30 to 70 ng/mL, 40 to 60 ng/mL, or about 50 ng/mL.

The term "supplement" in the present invention may be at least one selected from the group consisting of 2-phospho-L-ascorbic acid, B27, laminin, nicotinamide, and N2.

Furthermore, the present invention relates to a method for direct differentiation of somatic cells into pancreatic beta cells, comprising the step of culturing somatic cells in the presence of a composition comprising a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709.

In the present invention, the method may include or consist of, but is not limited to, (1) a step of inducing a somatic cell into a pancreatic endoderm cell; (2) a step of inducing a pancreatic endoderm cell into a pancreatic progenitor cell; and (3) a step of inducing a pancreatic progenitor cell into a pancreatic beta cell.

In the present invention, the method may include, or consist of, a step (3) of inducing pancreatic progenitor cells into pancreatic beta cells, but is not limited thereto.

In the present invention, if the somatic cells of the above method are fibroblasts, differentiation through three steps is required, but if the somatic cells are pancreatic ductal cells and exocrine cells having the properties of pancreatic progenitor cells, direct differentiation into pancreatic beta cells from the somatic cells is possible in the presence of the composition of (3).

In the present invention, the direct differentiation method may include, but is not limited to, the following steps:

(1) Step: Inducing a somatic cell into a pancreatic endoderm cell in the presence of a composition comprising a histone methyltransferase inhibitor; activin A, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709;

(2) Step: Inducing pancreatic endoderm cells into pancreatic progenitor cells in the presence of a composition comprising a retinoic acid agonist, an ALK-5 kinase inhibitor, a hedgehog inhibitor, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709; and

(3) Step: culturing somatic cells in the presence of a composition comprising a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709.

Step (1) of the present invention can be performed without limitation as long as it is possible to induce differentiation into pancreatic beta cells, but can be performed for 3 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, or 5 to 7 days, and more preferably can be performed for about 6 days. The supplement of the above step can preferably be 2-phospho-L-ascorbic acid.

The culture in step (2) above may be performed without limitation, but may be performed for 0.5 to 8 days, 0.5 to 7 days, 1 to 7 days, 2 to 6 days, 3 to 5 days, or about 4 days. However, the culture period is not limited thereto. The supplement in step (2) may preferably be 2-phospho-L-ascorbic acid.

Step (3) of the present invention can be performed without limitation as long as the cells in which the differentiation is induced can mature, but can be performed for 7 to 13 days, 8 to 12 days, 9 to 11 days, or about 10 days. However, the culture period is not limited thereto. The supplement of the step can be preferably at least one selected from the group consisting of 2-phospho-L-ascorbic acid, laminin, B27, and nicotinamide, and more preferably 2-phospho-L-ascorbic acid, laminin, B27, and nicotinamide.

When the direct differentiation method of the present invention is used, compared to conventionally known stem cell differentiation methods and chemical cell differentiation methods, there is an advantage in that there is no possibility of cancer development while causing less immune rejection.

The term "medium" of the present invention can use any basic medium known in the art without limitation. The basic medium can be manufactured by artificial synthesis, and a commercially manufactured medium can also be used. Examples of commercially manufactured media include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium. The medium may be a DMEM medium.

The culture medium for culturing the above somatic cells includes all culture mediums commonly used in the field for culturing fibroblasts. The culture medium used for culturing generally includes carbon sources, nitrogen sources, and trace element components.

Most methods for inducing somatic cells into pancreatic beta cells using the direct conversion method are carried out by introducing external genes. However, introducing genes using viruses can cause genomic instability due to random integration of external genes, which may cause cancer in patients in the future when clinically applied. For this reason, methods using small molecules without injecting external genes are gradually being proposed, but despite the recent active research on inducing direct differentiation using various small molecule compounds, at least one gene is used, and it is still not possible to convert human somatic cells into desired cells without gene introduction.

However, the present invention is a method that secures genetic stability by inducing pancreatic beta cells from somatic cells of a patient by treating micro RNA or a composition combining micro RNA and a low-molecular-weight substance without introducing external genes, and is designed to fundamentally solve the problem of genetic defects, which is a problem of existing cell transformation methods using genes, while lowering the possibility of cancer occurrence.

In the present invention, microRNA alone or a combination of a low molecular weight substance and microRNA was used to directly differentiate somatic cells into pancreatic beta cells. Accordingly, the microRNA of the present invention can be used as a treatment for diabetes or pancreatic cancer by itself, and has a very high potential for use as a cell therapy agent for patients.

Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating diabetes or pancreatic cancer, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-127 and miR-709.

In addition, the present invention provides a cell therapeutic agent for treating diabetes or pancreatic cancer, which comprises pancreatic beta cells directly differentiated by the direct differentiation method as an active ingredient.

In addition, the present invention provides a method for preventing or treating diabetes or pancreatic cancer, comprising a step of delivering into a living body a composition comprising at least one miRNA selected from the group consisting of miR-127 and miR-709, thereby inducing direct differentiation of somatic cells into beta cells in a living body.

In the present invention, the "diabetes", which is the subject of treatment or prevention, is a metabolic disorder syndrome characterized by hyperglycemia caused by an abnormality of insulin hormone produced in the beta cells of the pancreas or insulin resistance, and further by defects in both of these. This diabetes can be divided into insulin-dependent diabetes mellitus (IDDM, Type 1) and non-insulin-dependent diabetes mellitus (NIDDM, Type 2) caused by insulin resistance and impaired insulin secretion. In both type 1 and type 2 diabetes, various complications such as heart disease, intestinal disease, ophthalmic disease, neurological disease, and stroke occur, which is because chronic neurological disease and cardiovascular disease occur due to increased blood sugar and insulin levels for a long time, and acute complications are caused by short-term hypoglycemia and hyperglycemia reactions. Diabetes causes not only carbohydrate metabolism but also lipid and protein metabolism disorders as hyperglycemia persists chronically. The pathology is diverse and is directly caused by hyperglycemia, including diabetic peripheral neuropathy, diabetic retinopathy, diabetic nephropathy, diabetic cataracts, keratopathy, and diabetic arteriosclerosis in the retina, kidney, nerves, and cardiovascular system.

One of the important pathological phenomena that causes and aggravates diabetes is the death of pancreatic β cells due to hyperglycemia. The present inventors provide a method for producing beta cells that can be used for treating diabetes by converting somatic cells into pancreatic β cells. Accordingly, the diabetes in the present invention can be diabetes induced by glucose toxicity or aggravated or advanced diabetes, and more specifically, it can be selected from the group consisting of type 1 diabetes, type 2 diabetes, and gestational diabetes.

“Delivery” of the present invention may be via administration, but is not limited thereto.

The miRNA of the present invention may be included in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" as used herein includes a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base.

Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. They can also be prepared by heating an equimolar amount of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or by suction filtration of the precipitated salt.

Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium, potassium, etc., alkaline earth metals such as magnesium, and ammonium. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and then evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt, and also, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The content of the miRNA in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and for example, it can be 0.00001 to 99.9 wt% or 0.001 to 50 wt% based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, at least one selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipses, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigants, ointments, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

The additives of the tablets, powders, granules, capsules, pills and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethyl cellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinyl pyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

Additives that can be used in the liquid formulation according to the present invention include water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc.

The syrup according to the present invention may use a solution of white sugar, other sugars or sweeteners, and may also use a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity increasing agent, etc., as needed.

Purified water may be used in the emulsion according to the present invention, and an emulsifier, a preservative, a stabilizer, a fragrance, etc. may be used as needed.

The suspension according to the present invention may use suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, and the like. Surfactants, preservatives, stabilizers, colorants, and fragrances may also be used as needed.

The injection according to the present invention includes: solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, nonvolatile oils such as sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nitrile acid amide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO ₃ ), carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid disodium, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cisplatinum, sodium alginate, Tween 80, and aluminum monostearate.

The suppository according to the present invention comprises cocoa butter, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranette wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

In the present invention, the term "subject" means a subject requiring treatment of a disease, and is not limited to a vertebrate animal, but specifically can be applied to humans, mice, rats, guinea pigs, rabbits, monkeys, pigs, horses, cows, sheep, antelopes, dogs, cats, fish, and reptiles.

In the present invention, “administration” means providing a composition of the present invention to a subject by any appropriate method.

In the present invention, “prevention” means any act of suppressing or delaying the onset of a target disease, “treatment” means any act of improving or beneficially changing a target disease and its resulting metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any act of reducing a parameter related to a target disease, for example, the degree of symptoms, by administering a composition according to the present invention.

In addition, the present invention relates to a method for producing a cell therapy agent for treating diabetes or pancreatic cancer, comprising a step of mixing pancreatic beta cells differentiated by the above method with at least one selected from the group consisting of pharmaceutically acceptable carriers and excipients.

The term "cellular therapeutic agent" of the present invention refers to a medicine (US FDA regulations) used for the purposes of treatment, diagnosis, and prevention by cells and tissues isolated, cultured, and manufactured through special manipulation from humans, and means a medicine used for the purposes of treatment, diagnosis, and prevention of diseases by a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells by other methods to restore the function of cells or tissues.

The route of administration of the cell therapy composition of the present invention may be through any common route as long as it can reach the target tissue. It may be administered parenterally, for example, intraperitoneally, intravenously, intramuscularly, subcutaneously, or intradermally, but is not limited thereto.

The composition may be formulated into a suitable form together with a pharmaceutical carrier generally used in cell therapy. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not typically cause allergic reactions such as gastrointestinal upset, dizziness, or the like or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline solution, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The cell therapeutic agent according to the present invention can be manufactured in a unit dose form or manufactured by introducing into a multi-dose container by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. The pharmaceutically acceptable carrier included in the cell therapeutic agent of the present invention is one that is commonly used in formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition to the above components, the cell therapeutic agent of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, a preservative, etc.

Additionally, the composition may be administered by any device capable of transporting the cell therapy agent to the target cell.

The cell therapy composition of the present invention may contain a therapeutically effective amount of a cell therapy agent for treating a disease. The term "therapeutically effective amount" means an amount of an effective ingredient or pharmaceutical composition that is thought by a researcher, veterinarian, physician, or other clinician to induce a biological or medical response in a tissue system, animal, or human, including an amount that induces alleviation of symptoms of a disease or disorder being treated.

It is obvious to those skilled in the art that the cell therapy agent included in the composition of the present invention will vary depending on the desired effect. Therefore, the optimal cell therapy agent content can be easily determined by those skilled in the art, and can be adjusted according to various factors including the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. It is important to include an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors. For example, the daily dose of the stem cells of the present invention can be 1.0×10 ⁴ to 1.0×10 ¹¹ cells/kg body weight, preferably 1.0×10 ⁵ to 1.0×10 ⁹ cells/kg body weight, administered once or in several divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the dosage does not limit the scope of the present invention in any way.

In addition, in the treatment method of the present invention, a composition containing the cell therapy agent of the present invention as an active ingredient can be administered in a conventional manner via the rectal, intravenous therapy (i.v.), intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal route.

The present invention provides a treatment method comprising administering to a mammal a therapeutically effective amount of the cell therapy composition of the present invention. The term mammal as used herein refers to a mammal that is the subject of treatment, observation or experiment, and preferably refers to a human.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Experimental preparation and experimental method

1-1. Cell culture

Cells were cultured in DMEM (dulbecco's minimal essential medium, Welgene) supplemented with exosome-depleted 15% fetal bovine serum (FBS) (Gibco), 1% penicillin-streptomycin (Welgene), and 55 μM β-mercaptoethanol (β-ME) (Sigma) at 37°C in 5% _CO2 .

1-2. Isolation of exocrine cells from mice

Exocrine tissues were isolated from two 8-week-old male mice with reference to the literature [GC Chau, DU Im, TM Kang, JM Bae, W. Kim, S. Pyo, E.-Y. Moon, SHJJCB Um, mTOR controls ChREBP transcriptional activity and pancreatic β cell survival under diabetic stress, 216(7) (2017) 2091-2105.]. First, filtered 0.8 mg/ml collagenase P (Roche) dissolved in HBSS (Hank's Balanced Salt Solution, Wellgene) was injected into the mouse pancreas. The pancreatic tissues were incubated at 37 °C for 15 min with intermittent shaking. A 400 μm mesh (Sigma) was used to filter the digested suspension, and density gradient centrifugation on biocoll separation solution (Merck-Millipore) was used to separate all exocrine cells from the islets. Then, the cells were centrifuged at 2000 rpm for 20 min at 20 °C. The upper layer of the Biocol gradient contained acinar cells, whereas the pellet contained all other cells including ductal cells excluding islets and acinar cells. The acinar and ductal cells were carefully collected, mixed together, and filtered through a 70 μm mesh (Falcon). The cells were then washed three times with 1X HBSS, centrifuged at 1200 rpm for 3 min at 4 °C, and seeded onto 10 cm tissue culture dishes (TPP) containing RPMI (Roswell Park Memorial Institute) medium supplemented with 10% FBS and 1% P/S (penicillin/streptomycin) and cultured under 95% air and 5% CO ₂ .

1-3. Transfection

MEFs (5 × 10 ⁴ cells/well per 12-well plate) were transfected using jetMESSENGER (Polyplus-Transfection, Illkirch, France) reagent according to the manufacturer's instructions. The miRNA to be transfected (200 nM) (QIAGEN, Hilden, Germany) was diluted in mRNA buffer and 2 μL of jetMESSENGER reagent was added. The mixture was incubated at room temperature for 20 min and added to MEFs, followed by addition of stage-specific medium after 24 h.

266-6 cells (8 × 10 ⁴ cells/well) were transfected using RNAiMAX reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Transfections were performed in stage-specific media, and transfected cells were incubated for the desired time.

1-4. Immunostaining

After completing the three-stage differentiation, differentiated β-like cells were harvested and immunostained as described previously (Int. J. Mol. Sci. 2020 , 21, 665). Samples were visualized using a fluorescence microscope (IX71S1F3, Olympus, Tokyo, Japan). The antibodies used are shown in Table 1 below.

1-5. Direct differentiation of MEFs into pancreatic lineage using microRNA

As shown in Fig. 1, mouse embryonic fibroblasts (MEF) directed differentiation was performed by modifying a previous protocol. For cell directed differentiation, knockout DMEM (KO-DMEM) (Gibco) was used as a basal medium. Knockout DMEM contains 15% knockout serum replacement (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 5% FBS (exosome depleted), 1% GlutaMAX (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% nonessential amino acids (NEAA, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 0.5 mM β-ME (Sigma Aldrich, Inc., Saint Louis, MO, USA).

The three-step differentiation protocol of the present invention is as follows: Step 1, differentiation from MEFs into pancreatic endoderm cells; Step 2, differentiation from pancreatic endoderm cells into pancreatic progenitor-like cells; Step 3, differentiation from pancreatic progenitor-like cells into beta cell-like cells.

First, 5 × 10 ⁴ MEF cells/well were seeded into 12-well tissue culture plates in DMEM containing 10% FBS and 1 × P/S for 1 day. The following day, stage 1 differentiation medium was added. Stage 1 differentiation medium contains 1 μM Bix-01294 (MedchemExpress, Monmouth Junction, NJ, USA), 280 μM 2-phospho-L-ascorbic acid (pVc, Sigma Aldrich, Inc., Saint Louis, MO, USA), and 50 ng/mL activin A (R&D Systems, Minneapolis, MI, USA). Cells were maintained for 6 days after medium addition. The used medium was replaced every 3 days. After 6 days, stage 2 differentiation medium was added for 4 days. Step 2 differentiation medium contained small molecules including 0.5 nM TTNPB (MedchemExpress, Monmouth Junction, NJ, USA), 1 μM repsox (MedchemExpress, Monmouth Junction, NJ, USA), 2 μM cyclopamine (Tocris, Bristol, UK), and 280 μM pVc.

Stage 3 differentiation medium contained 1 μM SB203580 (MedchemExpress, Monmouth Junction, NJ, USA), 1× insulin-transferrin-selenium (ITS, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 10 mM nicotinamide (Sigma Aldrich, Inc., Saint Louis, MO, USA), 1 μg/mL laminin (Sigma Aldrich, Inc., Saint Louis, MO, USA), 50 ng/mL exendin-4 (MedchemExpress, Monmouth Junction, NJ, USA), 2 μM Bay K-8644 (Tocris, Bristol, UK), 1× B27 Plus supplement (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), and pVc. Cells were maintained in stage 3 medium for 10 days.

step Step Features Badge ingredients Step 1 Differentiation into endoderm cells BIX01294 (BIX), phospho-L-ascorbic acid (pVC), activin A, and micro RNA Step 2 Differentiation into pancreatic precursor cells TTNPB, repsox, cyclopamine, and pVC, micro RNA Step 3 Differentiation into beta cells SB203580, Nicotinamide, Extendin-A, Bay K-8644, Micro RNA

Micro RNA NAME SEQUENCE Sequence number miR-127-5p CUGAAGCUCAGAGGGCUCUGAU 1 miR-709 GGAGGCAGAGGCAGGAGGA 2 miR-127 MI0000154 precursor CCAGCCUGCUGAAGCUCAGAGGGCUCUGAUUCAGAAAGAUCAUCGGAUCCGUCUGAGCUUGGCUGGUCGG 3 miR-709 MI0004693 precursor UGUCCCGUUUCUCUGCUUCUACUCAGAAGUGCUCUGAGCAUAGAACUGUCCUGUUUGAGCAGCACUGGGGAGGCAGAGGCAGGAGGAU 4 miR-19b ugugcaaauccaugcaaaacuga 5

1-6. Gene expression study using qRT-PCR

Total cellular RNA was extracted using the manufacturer's Trizol (Invitrogen) method. 1 μg of total RNA was used for cDNA synthesis using PrimeScript 1st strand cDNA Synthesis Kit (Takara Clontech). Pancreatic lineage-specific marker analysis was performed using iQ SYBR Green Supermix (Biorad) using Real Time PCR (Biorad). qRT-PCR conditions were 40 cycles of 95 °C for 30 s, 60 °C for 15 s, and 72 °C for 15 s. The primers used in this study are shown in Table 4 below.

1-7. Statistical Analysis

Statistical significance was determined by a two-tailed, paired Student's t-test. The data presented were obtained from three independent experiments and are expressed as the mean ± SEM. p < 0.05 was considered statistically significant. Statistical analysis was performed using Prism v8.0 (GraphPad Software, Inc., San Diego, CA, USA).

Example 2. Conversion of MEFs into β-like cells using microRNA

Studies on the conversion of dox-inducible MEFs into endoderm-like cells using small molecules and the generation of functional pancreatic beta-cell-like cells have provided the basis for a directed differentiation approach. Accordingly, the inventors of the present invention hypothesized that if microRNAs act as transcriptional modifiers, they would induce pancreatic (endocrine)-specific transcriptional changes in MEFs in the 1-step medium depending on the presence or absence of small molecules such as epigenetic modifiers BIX01294 and pVC.

As shown in Figure 1a, the small molecule compound differentiation protocol includes three steps: Step 1, MEFs into pancreatic endoderm cells (PECs); Step 2, PECs into pancreatic progenitor-like cells (PPLCs); and Step 3, PPLCs into β-like cells (BLCs).

The present inventors established a final protocol for adding microRNA to the three-step protocol of Fig. 1a (see Fig. 1b).

Meanwhile, the increased expression of PDX1 indicates the onset of a pancreatic-specific program compared to microRNA-untreated cells. The formation of the pancreas begins with the differentiation of the definitive endoderm into the pancreatic endoderm. Pancreatic endoderm cells express the pancreatoduodenal homeobox gene, PDX1. In the absence of PDX1, the pancreas fails to develop beyond the formation of the ventral bud and dorsal bud. Thus, PDX1 expression characterizes an important step in pancreatic organogenesis. The mature pancreas contains exocrine and endocrine tissues, among other cell types. Exocrine and endocrine tissues are generated from the differentiation of the pancreatic endoderm. Therefore, the present inventors confirmed the expression of Pdx1 to confirm the differentiation into pancreatic beta cell-like cells.

As shown in Fig. 1c, when the small molecule compound differentiation protocol of Fig. 1a was used compared to the control medium (basal medium used), MEFs showed 2.9-fold and 2.6-fold induction of Pdx1 and insulin-2 expression.

Therefore, it was confirmed that the small molecule compound differentiation protocol of the present invention is suitable for use in direct differentiation of pancreatic beta cells.

Example 3. Confirmation of pdx1 differentiation efficacy of microRNA alone

In previous studies, we have confirmed that MIN6-derived exosomes are enriched with various miRNAs, including miR-486, miR-127, miR-19b, miR-494, and miR-709, which are related to the pancreatic lineage. Accordingly, we investigated whether these miRNAs could enhance the expression of pancreatic beta cell markers, either individually or in combination.

First, transfection experiments were optimized using 5' FAM (fluorescein amidite)-labeled control miRNA mimics before transfecting MEFs with miRNA mimics to simulate naturally occurring miRNAs. Control miRNA mimics were used that had no homology to mouse, rat, or human miRNAs. In addition, individual transfections were performed to select miRNAs that induced higher levels of Pdx1 during stage 1 differentiation.

As shown in Fig. 2a, among various miRNAs, miR-127 and miR-709 were confirmed to have the best effect in inducing the expression of Pdx1. Specifically, miR-127 was shown to induce 3.9 times that of the mimic, miR-709 was shown to induce 3.2 times that of the mimic, and miR-19b was shown to induce 1.6 times that of the mimic.

In the examples below, experiments were conducted using the three miRNAs above that had a significant effect on inducing Pdx1 expression.

Example 4. Confirmation of Pdx1 differentiation efficacy of microRNA combination

In Example 3, miR-127 and miR-709, which have a strong effect on inducing Pdx1 expression, were combined at different concentrations to confirm whether they increased the expression of pancreatic gene markers.

miR-127 miR-709 Combination-1 100nM 100nM Combination-2 133nM 66nM Combination-3 66nM 133nM

As shown in Fig. 2b, the highest transcription level of Pdx1 was observed at step 1 after transfection of miR-127+miR-709 combination-3.

In addition, as shown in Fig. 2c, it was confirmed that the expression level of Ngn3 was also significantly upregulated. Early activation of Ngn3 is known to exclusively induce glucagon-positive cells while depleting the pool of pancreatic progenitor cells.

In addition, as shown in Fig. 2d, high levels of Pdx1, Ngn3, Nkx6.1, and NeuroD1 were observed at step 2 after transfection of the miR-127+miR-709 combination-3. In particular, Pdx1 and Ngn3 were increased 4.8-fold and 4.1-fold, respectively, compared to the mimic-treated group.

In addition, as shown in Fig. 2e, the expression of pancreatic-specific gene markers including Pdx1 (5.4-fold), Ngn3 (2.8-fold), Nkx6.1 (6.2-fold), insulin-1 (2.7-fold), and insulin-2 (4.3-fold) were all significantly up-regulated in stage 3 cells after transfection of the miR-127+miR-709 combination-3 compared to the mimic-treated group.

That is, the results indicate that stepwise treatment of miR-127 and miR-709 can induce differentiation of MEFs in the presence of small molecule compounds and improve differentiation efficiency, thereby differentiating MEFs into beta-like cells.

In addition, we confirmed that combined treatment with miR-19b, in addition to miR-127 and miR-709, increased the expression of pancreatic gene markers.

miR-127 miR-709 miR-19b Combination-1 66nM 66nM 66nM Combination-2 160nM 20nM 20nM Combination-3 20nM 160nM 20nM Combination-4 20nM 20nM 160nM

First, as shown in Fig. 3a, the results of performing dose-dependent transfection confirmed that miRNA provided a transfection efficiency of approximately 80% with low cytotoxicity when treated at a dose of 200 nM.

Additionally, as shown in Fig. 3b, the highest transcription level of Pdx1 was observed at step 1 after transfection of the miR-127+miR-709+miR-19b combination-1.

In addition, as shown in Fig. 3c, different combinations of miR-127+miR-709+miR-19b increased cell proliferation, and in particular, cell proliferation was most increased in combination-3 compared to when transfected with miR-19b alone.

Example 5. Confirmation of the effect of microRNA combination on differentiation of exocrine cells into pancreatic beta cell-like cells

The present inventors confirmed through Examples 3 and 4 above that fibroblasts can be differentiated into pancreatic beta cell-like cells using the micro RNA combination of the present invention.

In addition, since exocrine and endocrine cells of the pancreas share a common developmental pathway, we investigated whether exocrine cells could be differentiated into pancreatic beta cell-like cells.

First, we tested the miR-127+miR-709 combination-3 on mouse acinar cell line 266-6 cells. First, the isolated exocrine cells were seeded in 6-well plates (coated with 1:10 dilution of Matrigel from BD Bioscience) in Step 3 medium. After 1 day in Step 3 medium, 50 μg/ml microRNA was added to one well and the other wells were not added microRNA. After the cells were cultured with microRNA in Step 3 medium for 2 days, fresh medium without microRNA was added to each well. After 7 days, the cells were harvested for RNA isolation using the Qiagen RNeasy kit, and gene profiling was performed using qRT-PCR.

As shown in Fig. 4a, 266-6 cells showed 70-80% transfection efficiency with the 5'FAM-tagged mimic control.

Additionally, the expression of pancreatic beta cell markers was confirmed when transfected 266-6 cells were cultured and grown in stage 3 medium.

As shown in Fig. 4b, it was confirmed that beta cell marker genes were increased even when the microRNA combination of the present invention was treated in exocrine cells. The level of Pdx1 was increased by more than 1.5 times compared to untreated cells. Ngn3 expression was also upregulated by 1.6 times in microRNA-treated cells. The levels of insulin-1 and insulin-2 were also found to increase by 1.8 times and 1.9 times, respectively. However, the expression of elastase (an acinar cell marker) was found to be rather decreased upon microRNA treatment.

That is, by using miR-127 and miR-709 together, somatic cells such as fibroblasts and exocrine cells can be directly differentiated into pancreatic beta cells.

Example 6. Confirmation of the effect of human pancreatic ductal cells’ beta cell-like cell differentiation according to the combination of micro RNA and small molecules

The present inventors confirmed that human pancreatic ductal cells, Capan-1, can be differentiated into pancreatic beta cell-like cells through a combination of miR-127 and a small molecule. In addition, they confirmed the expression of pancreatic beta cell markers depending on the presence or absence of the small molecule used in step 3.

As shown in Figure 5, it was confirmed that the expression of Pax-6 and MafA was significantly reduced in combination C deficient in nicotinamide.

This result indicates that Nicotinamide is essential in the three-step medium composition of the present invention.

In summary, the inventors of the present invention have clearly confirmed that direct differentiation into pancreatic beta cells is well achieved when microRNA alone or microRNA and a small molecule are used in combination. Therefore, the present inventors provide a composition for inducing direct differentiation of somatic cells into pancreatic beta cells, which comprises the microRNA of the present invention as an effective ingredient, and the produced pancreatic beta cells are expected to be useful for preventing, treating, and improving pancreatic-related diseases such as diabetes or pancreatic cancer. In addition, it is expected that the composition for inducing direct differentiation can be useful for preventing, treating, and improving pancreatic-related diseases such as diabetes or pancreatic cancer by directly injecting it into the body and inducing beta cells from somatic cells in the body.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> Research & Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Process and compositions for the direct differentiation of somatic cells into pancreatic beta cells using microRNA <130> MP20-181P1 <150> KR 10-2020-0123558 <151> 2020-09- 24 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-127-5p <400> 1 cugaagcuca gagggcucug au 22 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223>miR-709 <400> 2 ggaggcagag gcaggagga 19 <210> 3 <211> 70 <212> RNA <213> Artificial Sequence <220> <223>miR-127 MI0000154 precursor <400> 3 ccagccugcu gaagcucaga gggcucugau ucagaaagau caucggaucc gucugagcuu 60 ggcuggucgg 70 <210> 4 <211> 88 <212> RNA <213> Artificial Sequence <220> <223> miR-709 MI0004693 precursor <400> 4 ugucccguuu cucugcuucu acucagaagu gcucugagca uagaacuguc cuguuugagc 60 agcacugggg aggcagaggc aggaggau 88 <210> 5 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-19b <400> 5 gcaaauc caugcaaaac uga 23

Claims (21)

A composition for inducing direct differentiation of somatic cells into pancreatic beta cells, comprising one or more microRNAs selected from the group consisting of miR-127 and miR-709.
In the first paragraph,
A composition for inducing direct differentiation, characterized in that the somatic cells are at least one selected from the group consisting of fibroblasts, pancreatic duct cells, and exocrine cells.
In the first paragraph,
A composition for inducing direct differentiation, characterized in that the composition additionally contains miR-19b.
In the first paragraph,
A composition for inducing direct differentiation, characterized in that the composition further comprises at least one low-molecular-weight substance selected from the group consisting of a histone methyltransferase inhibitor, a retinoic acid agonist; an ALK-5 kinase inhibitor; a hedgehog inhibitor; a MAPK inhibitor; a calcium channel agonist; a GLP receptor agonist; and a supplement.
In paragraph 4,
The above histone methyltransferase inhibitors are BIX01294 (2-(Hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-6,7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinamine), 5-AZA-2'-deoxycytidine (5-aza-2'-deoxycytidine:DAC), Zebularine, 3'-Deazaneplanocin A hydrochloride, Lomeguatrib, Chaetocin, A composition for inducing direct differentiation, characterized in that at least one selected from the group consisting of 2,2',3S,3'S,5aR,5'aR,6,6'-octahydro-3,3'-bis(hydroxymethyl)-2,2'-dimethyl-[10bR,10'bR(11aS,11'aS)-bi-3,11a-epidithio-11aH-pyrazino[1',2':1,5]pyrrolo[2,3-b]indole]-1,1',4,4'-tetrone), and decitabine (Decitabine, 5-aza-2'-deoxycytidine).
In paragraph 4,
A composition for inducing direct differentiation, characterized in that the supplement is at least one selected from the group consisting of 2-phospho-L-ascorbic acid, B27, laminin, nicotinamide, and N2.
In paragraph 4,
A composition for inducing direct differentiation, characterized in that the retinoic acid agonist is at least one selected from the group consisting of TTNPB, phytanic acid, and retinoic acid.
In paragraph 4,
A composition for inducing direct differentiation, characterized in that the hedgehog inhibitor is at least one selected from the group consisting of cyclopamine, mifepristone, GDC-0449 (Vismodegib), XL139 (BMS-833923), IPI926, IPI609 (IPI269609), LDE225, zervin, GANT61, firmopamine, SAG, SANT-2, tomatidine, SANT74, SANT75, zerumbone and derivatives thereof.
In paragraph 4,
A composition for inducing direct differentiation, characterized in that the MAPK inhibitor is at least one selected from the group consisting of 1-Pyridinyl-2-phenylazole, SB 203580, SKF 86002, SKF 86096, SKF 104351, 1-Aryl-2-pyridinyl/pyrimidinyl heterocycles, SB 242235, RO-32001195, SX-011, and BIRB-796.
In paragraph 4,
The ALK-5 kinase inhibitor is RepSox (1,5-Naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]); SB525334 (6-(2-tert-butyl-4-(6-methylpyridin-2-yl)-1H-imidazol-5-yl)quinoxaline); GW788388 (4-(4-(3)-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide); SD-208 (2-(5-chloro-2-fluorophenyl)-N-(pyridin-4-yl)pteridin-4-amine); Galunisertib (LY2157299, 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide); EW-7197 (N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methanamine); LY2109761 (7-(2-morpholinoethoxy)-4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline); SB505124 (2-(4-(benzo[d][1,3]dioxol-5-yl)-2-tert-butyl-1H-imidazol-5-yl)-6-methylpyridine); LY364947 (Quinoline, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]); SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide); K02288 (3)-[(6-Amino-5-(3),4,5-trimethoxyphenyl)-3-pyridinyl]phenol]; A composition for inducing direct differentiation, characterized in that it comprises at least one selected from the group consisting of Quinoline, 5-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl] and LDN-212854 (Quinoline).
In paragraph 4,
A composition for inducing direct differentiation, characterized in that the calcium channel agonist is at least one selected from the group consisting of Bay K-8644, FPL 64179, and CGP28392.
In paragraph 4,
A composition for inducing direct differentiation, characterized in that the GLP receptor agonist is at least one selected from the group consisting of exendin-4, dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, and albiglutide.
A method for direct differentiation of somatic cells into pancreatic beta cells, comprising the step of culturing somatic cells in the presence of a composition comprising a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, a supplement; and one or more miRNAs selected from the group consisting of miR-127 and miR-709.
In Article 13,
A direct differentiation method, characterized in that the somatic cells are at least one selected from the group consisting of fibroblasts, pancreatic duct cells, and exocrine cells.
In Article 13,
The direct differentiation method is characterized by comprising the following steps:
(1) a step of inducing a somatic cell into a pancreatic endoderm cell in the presence of a composition comprising a histone methyltransferase inhibitor; activin A, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709;
(2) a step of inducing pancreatic endoderm cells into pancreatic progenitor cells in the presence of a composition comprising a retinoic acid agonist, an ALK-5 kinase inhibitor, a hedgehog inhibitor, a supplement; and one or more miRNAs selected from the group consisting of miR-127 and miR-709; and
(3) A step of culturing somatic cells in the presence of a composition comprising a MAPK inhibitor, a calcium channel agonist, a GLP receptor agonist, a supplement; and at least one miRNA selected from the group consisting of miR-127 and miR-709.
delete delete delete delete A method for producing a cell therapy agent for treating diabetes or pancreatic cancer, comprising a step of mixing pancreatic beta cells directly differentiated by the method of claim 13 with at least one selected from the group consisting of pharmaceutically acceptable carriers and excipients.
A cell therapeutic agent for treating diabetes or pancreatic cancer, comprising pancreatic beta cells directly differentiated by the method of Article 13 as an active ingredient.