WO2018155913A1 - Method for differentiation into skeletal muscle cell using low-molecular weight compound - Google Patents

Abstract

The present invention relates to a composition for inducing the differentiation of somatic cells or adult stem cells into skeletal muscle cells. Though a simple method in which cells are cultured in a culture medium containing the differentiation-inducing composition of the present invention, somatic cells can be directly differentiated into skeletal muscle cells. The differentiation into skeletal muscle cells shows an elevated differentiation rate and efficiency and is economically advantageous in terms of cost. In addition, the skeletal muscle cells obtained have no risks of oncogenesis after implantation because of differentiation from somatic cells, and has the advantage of being genetically stable because of using a mixture of low-molecular weight compounds alone, without genetic introduction. In the present invention, the composition was also found to fast and effectively induce the differentiation of mesenchymal stem cells into skeletal muscle cells. Particularly, it was observed that the absence of a GSK inhibitor in the composition elicited a positive effect on differentiation efficiency. Capable of inducing differentiation into a great deal of skeletal muscle cells within a short time period, therefore, the present invention is expected to find useful applications in the development of economical and effective cell therapy products for treatment of skeletal muscle diseases.

Description

Skeletal muscle cell differentiation using low molecular weight compounds

The present invention relates to a method of differentiating somatic or adult stem cells into skeletal muscle cells, and more specifically, chemically induced skeletal muscle cells (CiSMC :) using a low molecular weight material without genetic introduction to safe and high efficiency somatic cells or adult stem cells. Chemically induced skeletal muscle cells).

Hereditary muscle diseases such as muscular dysfunction caused by genetic mutations, muscle underdevelopment, and muscle degeneration, muscle loss caused by muscle or nerve damage or aging, and muscle damage accompanied by excessive muscle wasting. Treatment requires muscle regeneration. New born babies have relatively good muscle regenerative ability, but adults have reduced muscle regenerative capacity due to aging of muscle stem and progenitor cells. In addition, even if muscle regeneration is possible to some extent, if the muscle damage caused by trauma or disease is large, the possibility of new muscle differentiation is reduced, traditional medical practice is difficult to expect permanent recovery of muscle damage. Therefore, the transplantation of muscle cells or muscle precursor cells is required for the treatment of such muscle diseases. However, the treatment by this muscle transplantation has a problem that side effects due to the immune rejection reaction occurs when the transplantation of muscle or muscle progenitor cells derived from an immune-type matched donor.

In order to fabricate the cells necessary for cell therapy, embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) have been studied in many ways. In this case, undifferentiated stem cells may be contained, and after the undifferentiated stem cells are transplanted into a living body, teratoma is formed to cause cancer. Therefore, cell therapy using stem cells differentiated from stem cells or stem cell therapy using stem cells is currently clinically applied only in very limited fields such as retinal diseases.

As a method for solving the above problem, mesenchymal cells with very differentiation capacity are not used without using highly differentiated pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells (iPSCs). (Mesenchymal Stem Cells: MSC) or somatic cells (differentiated somatic cells) have been actively studied to directly differentiate into desired cells. The method of directly producing cells for cell therapy using direct differentiation does not include pluripotent stem cells, so there is no possibility of cancer, and it is free from ethical problems caused by using embryonic stem cells. In addition, compared to the process of de-differentiating somatic cells to make iPSCs and re-differentiating them, the time and efficiency is much superior. Above all, the advantage of this direct differentiation is that it is possible to self-treat the cells directly using the patient's own cells without the immune rejection reaction occurring when using the cells of others.

In the case of cell differentiation using iPSC, the cell differentiation process occurs in all stages of biological development, while direct differentiation using somatic cells or adult stem cells of the patient is performed to express genes expressed in the final differentiated cells. Cells are differentiated through forced or induced expression, so they do not go through the intermediate cells produced during stem cell differentiation. Due to the characteristics of direct differentiation that the differentiation method is simple, the time required for cell differentiation can be reduced as well as the cost required for cell differentiation, compared to the method of making a cell to be used as a therapeutic agent using stem cells. For example, to differentiate skeletal muscles using iPSCs, iPSCs should be differentiated into mesoderm, and myoblasts should be made through mesenchymal mesoderm, myocytes, and myoblasts. Can differentiate.

Direct differentiation using somatic cells has to play an important role in the cells to be differentiated and to selectively express marker genes that characterize the cells. Since the expression of these marker genes is regulated by the master transcription factor of the cells, The most popular method for differentiation is to force expression of the master transcription factor. For example, direct differentiation into skeletal muscle can be achieved by forced expression of the transcription factor MyoD, which is a master transcription factor of myotube cells and a marker of myotubes. However, for the selective expression of this specific gene, it is necessary to put an external gene into the cell. In this process, the stability of the virus used as a gene transfer medium has not been confirmed, and it also inhibits the stability of the genome in the process of inserting the external gene into the genome. There is a possibility of causing unwanted side effects.

As an alternative to solve the problems caused by the direct differentiation by gene introduction, a chemically induced direct differentiation method that expresses a specific gene in a specific cell by regulating a specific signal or epigenetic signal through a low molecular compound, not a gene insertion, Is being studied.

In addition to the advantages presented above, the direct differentiation method using low molecular materials has the advantages of ease of cell transfer, reproducibility of results, efficiency of differentiation and expansion of research scope. Most importantly, it is more stable than other cell therapies in the absence of genetic engineering, and thus can be easily used for clinical purposes.

Human cells are known to have about 24,000 genes, of which only about 10,000 genes are known to be expressed in each cell. To date, it is known that there are about 200 cells of different shapes and functions in the human body. These different cells are commonly expressed in essential genes necessary to perform the common functions of the cells, but the difference between these cells is determined by the expression of different genes. Therefore, for direct differentiation of cells, it is necessary to express specific genes that influence the characteristics of the cells in order to prevent differentiation of specific genes of the original cell. However, since the expression of thousands of genes changes when two cells are very different in character, precisely adjusting the expression of these genes is currently impossible. For this reason, in order to discover the conditions for direct differentiation of cells using the small molecule compounds of the cells, a lot of experiments have to find the conditions for inducing such a large number of genetic changes. That is, compounds that control various functions of cells such as cell signal regulation, epigenetics, metabolic regulation, etc. should be treated in various concentrations to find the conditions closest to the desired gene expression.

Direct differentiation of various cells using low molecular weight compounds has been reported. For example, a method of making heart muscle from fibroblasts using small molecule compounds has already been reported (Korean Patent No. 10-1539132 and Science, 2016, 352, 1216-1220.). However, a method of chemically differentiating skeletal muscle that can be used for muscle-related diseases has not been reported yet.

On the other hand, skeletal muscle and heart muscle undergoes a completely differentiation process as shown in Figure 1, the marker gene of each cell is also different, both cells have different characteristics. Specifically, skeletal muscle is differentiated into muscle tubes or muscle fibers through mesoderm, mesenchymal mesoderm, myoelectric progenitor cells, and myoblasts, whereas cardiomyocytes are made through mesodermal mesoderm and mesodermal mesoderm. It will go through a differentiation process. In other words, since cardiomyocytes and skeletal muscle cells express completely different genes, it is necessary to regulate different cell signaling or epigenetics in order to selectively control the expression of these genes to make each cell. In general, in order to confirm the expression of a specific cell, a method of confirming the expression of a representative gene (marker) that occurs only in that cell is used. As shown in FIG. 1, the expression markers of heart muscle cells and skeletal muscle cells are completely different. You can check it.

This technique is a method for chemically replacing adult stem cells, which are completely undifferentiated, such as fibroblasts or adipose-derived mesenchymal cells, using skeletal muscle cells, using chemical methods. It is simpler and more efficient than using skeletal muscle cells. In addition, the cells produced by such a method is unlikely to develop cancer, so it is highly likely to be used as a cell therapy in terms of stability. On the other hand, since it is less likely to modify the genome compared to the direct differentiation method by genetic engineering, it is also an advantageous method in terms of stability. First of all, it is possible to use a cell of a patient in need of transplantation, thereby developing a cell therapy product without an immune rejection reaction. In addition, by inducing direct differentiation of cells using only low molecular weight compounds, it is more cost effective and time efficient than other known cell differentiation methods.

The present invention invented a technique for directly differentiating adult cells, such as somatic cells or mesenchymal cells, into osteomyocytes by treating various compounds known to modulate cell function in various combinations. It proved that the skeletal muscle has the cellular activity. Therefore, a substance derived from differentiated osteomyocytes or differentiated osteomyocytes may be used as a therapeutic agent for bone muscle related diseases.

The present invention has been made to solve the above-mentioned problems in the prior art, the present inventors have made a diligent effort to find a method for directly converting adult cells such as somatic cells or mesenchymal cells into muscle cells, (1) histone deacetylase Open chromatin formation through inhibition of (2) activating Wnt / beta-catenin signaling through glycogen synthase kinase inhibitors, (3) inhibiting TFG-beta signaling through ALK-5 kinase inhibitors, and (4) via cAMP signaling activator The present invention was completed by confirming that cyclic AMP (cAMP), a cell signaling material, is required for the direct differentiation of adult cells into skeletal muscle cells.

Accordingly, an object of the present invention is a pan-histone deacetylase inhibitor (Pan-histone deacetylase inhibitor), GSK inhibitor (Glycogen synthease kinase inhibitor), ALK5 inhibitor (activin A receptor type II-like kinase 5 inhibitor), and cAMP signaling activator ( It provides a composition for inducing differentiation of somatic cells or adult stem cells into skeletal muscle cells, including cAMP signaling activator) as an active ingredient.

In addition, an object of the present invention is a method of inducing differentiation of somatic cells or adult stem cells into skeletal muscle cells comprising the step of culturing somatic cells or adult stem cells in a culture medium comprising the composition for inducing differentiation and Activin A after the step , Bone morphogenetic protein 4 (BMP4), Vascular endothelial growth factor (VEGF), Glycogen synthase kinase inhibitor (GSK inhibitor), and src tyrosine kinase inhibitor. It is to provide a method of inducing differentiation into skeletal muscle cells further comprising the step of maturing the differentiation-induced cells.

It is also an object of the present invention to provide a cell therapy agent for treating skeletal muscle disease, including cells differentiated into skeletal muscle cells by the above method.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pan-histone deacetylase inhibitor (Pan-histone deacetylase inhibitor), GSK inhibitor (Glycogen synthase kinase inhibitor), ALK5 inhibitor (activin A receptor type II-like kinase 5 inhibitor), and cAMP It provides a composition for inducing differentiation of somatic cells or adult stem cells into skeletal muscle cells comprising a signaling activator (cAMP signaling activator) as an active ingredient.

In addition, the present invention includes a pan-histone deacetylase inhibitor (Pan-histone deacetylase inhibitor), an ALK5 inhibitor (activin A receptor type II-like kinase 5 inhibitor), and a cAMP signaling activator (cAMP signaling activator) as an active ingredient, Provided is a composition for inducing differentiation of adult stem cells into skeletal muscle cells.

In another embodiment of the present invention, the somatic cells may be fibroblasts, and the fibroblasts may be mouse embryonic fibroblasts or mouse skin fibroblasts.

In another embodiment of the present invention, the adult stem cells may be mesenchymal stem cells (MSC), the mesenchymal stem cells may be adipocyte-derived stem cells (ADSC) have.

In another embodiment of the present invention, the panhistone deacetylase inhibitor is Valproic acid (Valproic acid), Sodium butyrate, Suberoylanilide hydroxamic acid (Suberoylanilide hydroxamic acid), Hydroxamic acid (hydroxamic acid), between Click Tetrapeptide (cyclic tetrapeptide), depsipeptides, Trichostatin A, Borinostat, Belinostat, Panobinostat, Benzamide, Benzamide Notinostat, or butyrate.

In another embodiment of the present invention, the plate histone deacetylase inhibitor may be included in a concentration of 1 to 1000 μM, preferably 400 to 600 μM, more preferably 500 μM, but is not limited thereto. It is not. The concentration of pan-HDACi can be appropriately adjusted according to the type of pan-HDACi used and the cell to be differentiated.

In another embodiment, the ALK5 inhibitor is RepSox (1,5-Naphthyridine, 2- [3- (6-methyl-2-pyridinyl) -1H-pyrazol-4-yl]); SB431452 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] benzamide; 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]).

In another embodiment of the present invention, the ALK5 inhibitor may be included in a concentration of 1 to 100 μM, preferably 5 to 15 μM, more preferably 10 μM, but is not limited thereto. The concentration of the ALK5 inhibitor can be appropriately adjusted according to the type of ALK5 inhibitor used and the cells to be differentiated.

In another embodiment of the present invention, the cAMP signaling activator may include Forskolin, isoproterenol, NKH 477, isoprotereno (Chemical based), PACAP 1-27, or PACAP 1-38 (peptide based). .

In another embodiment of the present invention, the cAMP signaling activator may be included in a concentration of 1 to 100 μM, preferably 40 to 60 μM, more preferably 50 μM, but is not limited thereto. The concentration of the cAMP signaling activator can be appropriately adjusted according to the type of cAMP signaling activator used and the cells to be differentiated. For example, appropriate differentiation can be identified when included at concentrations of 50 μM (± 10 μM) for Forskolin and 5 μM (± 10 μM) for NKH477.

In another embodiment of the invention, the GSK inhibitor is Chir99021 (6- (2- (4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino ) ethylamino) nicotinonitrile); 1-azakenpaullone (9-Bromo-7,12-dihydro-pyrido [3 ', 2': 2,3] azepino [4,5-b] indol-6 (5H) -one); AZD2858; 3-amino-6- (4-((4-methylpiperazin-1-yl) sulfonyl) phenyl) -N- (pyridin-3-yl) pyrazine-2-carboxamide; BIO ((2'Z, 3'E) -6-Bromoindirubin-3'-oxime); ARA014418 (N- (4-Methoxybenzyl) -N '-(5-nitro-l, 3-thiazol-2-yl) urea); Indirubin-3'-monoxime; 5-Iodo-indirubin-3'-monoxime; kenpaullone (9-Bromo-7,12-dihydroindolo- [3,2-d] [1] benzazepin-6 (5H) -one); SB-415286 (3-[(3-Chloro-4-hydroxyphenyl) amino] -4- (2-nitro-phenyl) -1H-pyrrole-2,5-dione); SB-216763 (3- (2,4-Dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione); Maybridge SEW00923SC (2-anilino-5-phenyl-1,3,4-oxadiazole); (Z) -5- (2,3-Methylenedioxyphenyl) -imidazolidine-2,4-dione; TWS 119 (3- (6- (3-aminophenyl) -7H-pyrrolo [2,3-d] pyrimidin-4-yloxy) phenol); Chir98014 (N2- (2- (4- (2,4-dichlorophenyl) -5- (1 H-imidazol-1-yl) pyrimidin-2-ylamino) ethyl) -5-nitropyridine-2,6-diamine); SB415286 (3- (3-chloro-4-hydroxyphenylamino) -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione); Tideglusib (2- (1-naphthalenyl) -4- (phenylmethyl)); Or LY2090314 (3-imidazo [1,2-a] pyridin-3-yl-4- [1,2,3,4-tetrahydro-2- (1-piperidinylcarbonyl) -pyrrolo [3,2, jk] [1 , 4] benzodiazepin-7-yl]).

In another embodiment of the present invention, the GSK inhibitor may be included in a concentration of 0.001 to 100 μM, preferably 10 to 30 μM, more preferably 20 μM concentration, but is not limited thereto. The concentration of the GSK inhibitor can be appropriately adjusted according to the type of GSK inhibitor used and the cells to be differentiated. For example, appropriate differentiation can be identified when included at concentrations of 20 μM (± 10 μM) for Chir99021 and 10 nM (± 10 nM) for AZD2858.

In addition, the present invention includes as an active ingredient at least one compound selected from the group consisting of Activin A, BMP4 (Bone morphogenetic protein 4), VEGF (Vascular endothelial growth factor), GSK inhibitor (Glycogen synthase kinase inhibitor) and src tyrosine kinase inhibitor To provide a composition for inducing maturation of skeletal muscle cells.

In addition, the present invention is a medium containing a pan-histone deacetylase inhibitor (Pan-histone deacetylase inhibitor), an ALK5 inhibitor (activin A receptor type II-like kinase 5 inhibitor), and a cAMP signaling activator (cAMP signaling activator) as an active ingredient It provides a method of inducing differentiation of somatic cells or adult stem cells into skeletal muscle cells, comprising the step of culturing somatic cells or adult stem cells.

In one embodiment of the invention, the medium may be a method of inducing differentiation of somatic cells into skeletal muscle cells further comprising a GSK inhibitor (Glycogen synthase kinase inhibitor).

In another embodiment of the present invention, the somatic cells may be fibroblasts, and the fibroblasts may be mouse embryonic fibroblasts or mouse skin fibroblasts.

In another embodiment of the present invention, the adult stem cells may be mesenchymal stem cells (MSC), the mesenchymal stem cells may be adipocyte-derived stem cells (ADSC) have.

In another embodiment of the present invention, the culture may be performed for 5 to 25 days, in the case of inducing differentiation of somatic cells into skeletal muscle cells, preferably for 4 to 12 days, more preferably for 6 to 8 days It may be performed, and may be performed for 20 to 25 days in the case of induction of differentiation of adult stem cells into skeletal muscle cells.

In another embodiment of the present invention, the method may further comprise the step of maturing the cells differentiated into skeletal muscle cells by the method.

In another embodiment of the present invention, the step of maturing the differentiation-induced cells into skeletal muscle cells is Activin A, BMP4 (Bone morphogenetic protein 4), VEGF (Vascular endothelial growth factor), GSK inhibitor (Glycogen synthase kinase inhibitor), And src kinase inhibitor may be to culture the cells in a medium containing at least one compound selected from the group consisting of.

In another embodiment of the present invention, the culturing of the cells in the maturation step may be performed for 1 to 5 days, preferably for 3 days.

The present invention also provides a cell therapy agent for treating skeletal muscle disease, which comprises cells differentiated into skeletal muscle cells by the above method.

In one embodiment, the skeletal muscle disease includes skeletal muscle disease caused by genetic and acquired factors, specifically Becker muscular dystrophy, Congenital muscular dystrophy, Duchenne muscular dystrophy, Duchenne muscular dystrophy Distal muscular dystrophy, Emery-Dreifuss musculardystrophy, Facioscapulohumeral musculardystrophy, Limb-girdle musculardystrophy, Muscular dystrophy, Myotonic dystrophy (Myotonic dystrophy) Muscular dystrophy, which is one or more congenital skeletal muscle diseases selected from the group consisting of oculopharyngeal musculardystrophys, may be muscle dysfunction caused by acquired factors, for example, physical cell death, muscle cell death due to inflammation, metabolic abnormalities, aging, etc. Decrease, Dystrophy, muscular sclerosis, and the like muscle sprains, muscular or inflammatory diseases.

The present invention also provides a method for preventing or treating skeletal muscle disease, comprising administering the cell therapy agent to a subject.

The present invention also provides the use of cells induced to differentiate into skeletal muscle cells by the above method for producing a cell therapy for the prevention or treatment of skeletal muscle disease.

The present invention relates to a method for differentiating into skeletal muscle cells, and more specifically, to directly differentiate somatic cells or adult stem cells into chemically induced skeletal muscle cells (CiSMC) using only low molecular weight material without gene introduction. It is about. The present inventors have found that the conditions of differentiation of skeletal muscle cells by treating fibroblasts by combining a combination of epigenex of cells and various low molecular substances that regulate signal transmission, and as a result, activate histone acetylase and inhibit Wnt signals using low molecular substances. We found that efficient skeletal muscle cells induce differentiation through inhibition of TGF-beta signaling and cAMP activation.

The direct differentiation of CiSMC was confirmed that the major marker factor expressed in mammalian osteomyocytes was well expressed, and that CiSMC exhibited spontaneous contraction similar to that of mammalian osteomyocytes. It was confirmed that they are functionally similar to osteomyocytes. Therefore, technology that directly differentiates adult cells such as fibroblasts and mesenchymal cells into skeletal muscle cells through a simple method of growing cells by treating only low-molecular substances in a culture medium without genetic manipulation is to develop safe cell therapy using autologous cells derived from patients. It is expected to be useful.

 In addition, the skeletal muscle cells produced by the present invention prevents, treats, and prevents and treats musculoskeletal disorders, including muscle damage caused by accidents, diseases, or diseases, and Becker musculardystrophy and Congenital musculardystrophy. It is expected that the present invention may be usefully used as a cell therapy composition for improving and improvement.

1 is a schematic diagram illustrating the differentiation process of cardiomyocytes and skeletal muscle cells.

Figure 2 (A) is a schematic diagram illustrating the process of inducing differentiation into skeletal muscle cells by treating a mixture containing six compounds (VCRFPT) by separating the MEF, (B) is an immunostaining of MF20 and MyoD Co-expression (upper panel), sarcomeric a Actinin expression (middle panel), and myogenin expression (lower panel). (C) shows the expression of skeletal muscle specific transcripts quantified by qRT-PCR analysis. The figure shown.

3A and 3B are diagrams confirming that the mixture composed of VCRF is an optimal mixture for inducing differentiation of fibroblasts into skeletal muscle cells.

Specifically, (A) of FIG. 3A is a diagram schematically illustrating a process of inducing differentiation into skeletal muscle cells by treating a mixture composed of a combination of different low molecular weight compounds to MEF, and (B) treating the mixture having different configurations. Quantitatively showing the incidence of MF20 positive colonies, (C) is immunofluorescent staining of the MF20 positive colonies, (D) is the co-expression of MF20 and MyoD (top panel), sarcomeric a Actinin Expression (middle panel), and myogenin expression (lower panel) is confirmed, (E) is a diagram quantifying the expression level of MF20 and MyoD by qRT-PCR analysis, (F) is optimal for musculoskeletal muscle differentiation Figure VCRF showing a mixture configuration of.

In addition, (A) of FIG. 3B is a diagram analyzing cells differentiated by Facs analysis after inducing differentiation of MEF using a mixture composed of different low molecular weight compounds, and (B) is a diagram quantifying MF20 positive cells. , (C) is a flow cytometry diagram.

4 is a view confirming the optimum culture period for inducing differentiation into skeletal muscle cells, (A) is a diagram schematically showing the differentiation into skeletal muscle cells according to the period of incubating MEF in a medium containing VCRF, (B ) Is a diagram showing the number of Sarcomeric actinin positive cells by flow cytometry, (C) is a diagram showing the expression level of MyoD1, Myogenin, Myomaker, and Mck.

Figure 5 shows the expression level of skeletal muscle specific marker according to the presence or absence of cytokines, (A) is a marker specific to premyogenic mesoderm, (B) is specific to myogenic precursor (myogenic precursor) Red marker, (C) is a diagram showing the quantification of the expression level specific markers in mature muscle cells (mature muscle).

FIG. 6 shows a mixture consisting of BMP4, Activin A, Chir99021, and VEFG as an optimal mixture for maturation of cells induced to differentiate into skeletal muscle cells, and (A) is a method of maturing MEF into skeletal muscle after an ACRF induction step. (B) is a diagram showing the degree of maturation of skeletal muscle cells with or without cytokines after induction of differentiation with VCRF or VCRFPT, (C) is an immunofluorescent staining of MF20 positive colonies, (D) is a diagram confirming the co-expression of MF20 and MyoD (upper panel), the expression of sarcomeric a Actinin (middle panel), and the expression of myogenin (lower panel), (E) is the qRT expression level of MF20 and MyoD -Quantified by PCR analysis.

7 is a view showing the optimal compound required for the maturation of the cells induced differentiation into skeletal muscle cells, (A) is a schematic diagram of the process of maturation into skeletal muscle further comprising a src tyrosine kinase inhibitor (PP1) after induction of differentiation (B) is the chemical structure of the src tyrosine kinase inhibitor, (C) is a diagram confirming the co-expression of MF20 and MyoD in mature cells containing additional PP1, (D) is sarcomeric a Actinin and Co-expression of MyoD, (E) is a diagram confirming the co-expression of MF20 and MyoD in cells matured with cytokines alone without the addition of PP1, (F) is a diagram confirming the co-expression of sarcomeric a Actinin and MyoD. .

8 is a diagram schematically illustrating a process of producing FSP1-dTomato progeny through Fsp1-cre: R26RtdTomato mouse crossing.

9A shows co-expression of dTomato and MF20 (1st panel), co-expression of dTomato and sarcomeric a Actinin (2nd panel), and co-expression of dTomato and MyoD in cells differentiated by VCRF without cytokine maturation (1st panel). Panel), co-expression of dTomato and Myogenin (4th panel).

Figure 9B (A) is a diagram confirming the co-expression of dTomato and MF20 (top panel), co-expression of dTomato and sarcomeric a Actinin (low panel) in cells differentiated by VCRFPT without cytokine-induced cell maturation, (B) is a diagram confirming the co-expression of dTomato and MF20 (top panel), the co-expression of dTomato and sarcomeric α Actinin (low panel) in the cells after the cell maturation process by cytokines after induction of differentiation by VCRF.

(A) is a diagram schematically illustrating the function performed by the VCRF compound, (B) co-expression of MF20 and MyoD in cells differentiated using a mixture containing Valproic acid, CHIR99021, SB431542, and NKH477. Top panel), confirming the expression of sarcomeric a Actinin and MyoD co-expression (bottom panel), (C) is MF20 and MyoD in cells differentiated using a mixture comprising sodium butyrate, AZD2858, SB431542, and NKH477 It is the figure which confirmed the co-expression of (high panel), the co-expression of sarcomeric a Actinin, and MyoD (lower panel).

11 is a diagram confirming the expression of co-expression of MF20 and MyoD (upper panel), co-expression of sarcomeric a Actinin and MyoD (lower panel) by treating dermal fibroblasts with VCRF.

12 (A) is a diagram showing the expression of αSMA in cells induced by differentiation into skeletal muscle cells by treating a mixture consisting of Valproic Acid, Repsox, and Forskolin in adipose derived stem cells, (B) is Valproic Acid, and SB431542 , NKH422 treated with the mixture consisting of the expression of αSMA in the cells induced differentiation into skeletal muscle cells.

FIG. 13 shows that skeletal muscle cell markers (MyoD and alpha-actinin) are expressed when the composition for inducing differentiation with skeletal muscle cells of the present invention is expressed, but cardiomyocyte markers (cTNT and Nkx2.5) are not expressed.

The present invention is a somatic cell or a pan-histone deacetylase inhibitor (Pan-histone deacetylase inhibitor), ALK5 inhibitor (activin A receptor type II-like kinase 5 inhibitor), and cAMP signaling activator (cAMP signaling activator) as an active ingredient, Provided is a composition for inducing differentiation of adult stem cells into skeletal muscle cells.

In the present invention, the composition for inducing differentiation may further include a GSK inhibitor (Glycogen synthase kinase inhibitor).

In the present invention, "Pan-histone deacetylase inhibitor (pan-HDACi)" means an inhibitor of an enzyme that removes an acetyl group from histones. Non-limiting examples of the plate histone deacetylase inhibitors include Valproic acid, Sodium butyrate, Suberoylanilide hydroxamic acid, Hydroxamic acid, Cyclic tetrapeptide ( Cyclic tetrapeptide, depsipeptides, Trichostatin A, Verinostat, Velinostat, Belinostat, Panobinostat, Benzamide, Entinostat ), And butyrate, but the pan-HDACi is not limited as long as it performs a function of inhibiting an enzyme that removes an acetyl group from a histone, preferably Valproic Acid or butyrate, more preferably. Valproic Acid or sodium butyrate.

In the present invention, "Glycogen synthase kinase inhibitor" means an inhibitor that targets GSK1 / 2 involved in Wnt signaling. Non-limiting examples of the GSK inhibitor include Chir99021 (6- (2- (4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino) ethylamino) nicotinonitrile); 1-azakenpaullone (9-Bromo-7,12-dihydro-pyrido [3 ', 2': 2,3] azepino [4,5-b] indol-6 (5H) -one); AZD2858; 3-amino-6- (4-((4-methylpiperazin-1-yl) sulfonyl) phenyl) -N- (pyridin-3-yl) pyrazine-2-carboxamide; BIO ((2'Z, 3'E) -6-Bromoindirubin-3'-oxime); ARA014418 (N- (4-Methoxybenzyl) -N '-(5-nitro-l, 3-thiazol-2-yl) urea); Indirubin-3'-monoxime; 5-Iodo-indirubin-3'-monoxime; kenpaullone (9-Bromo-7,12-dihydroindolo- [3,2-d] [1] benzazepin-6 (5H) -one); SB-415286 (3-[(3-Chloro-4-hydroxyphenyl) amino] -4- (2-nitro-phenyl) -1H-pyrrole-2,5-dione); SB-216763 (3- (2,4-Dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione); Maybridge SEW00923SC (2-anilino-5-phenyl-1,3,4-oxadiazole); (Z) -5- (2,3-Methylenedioxyphenyl) -imidazolidine-2,4-dione; TWS 119 (3- (6- (3-aminophenyl) -7H-pyrrolo [2,3-d] pyrimidin-4-yloxy) phenol); Chir98014 (N2- (2- (4- (2,4-dichlorophenyl) -5- (1 H-imidazol-1-yl) pyrimidin-2-ylamino) ethyl) -5-nitropyridine-2,6-diamine); SB415286 (3- (3-chloro-4-hydroxyphenylamino) -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione); Tideglusib (2- (1-naphthalenyl) -4- (phenylmethyl)); And LY2090314 (3-imidazo [1,2-a] pyridin-3-yl-4- [1,2,3,4-tetrahydro-2- (1-piperidinylcarbonyl) -pyrrolo [3,2, jk] [1 , 4] benzodiazepin-7-yl]), and the GSK inhibitor is not limited as long as it targets GSK1 / 2. Preferably, the GSK inhibitor may be Chir 99021 or AZD2858.

In the present invention, "ALK-5 kinase inhibitor" refers to a substance that binds to ALK5 (activin A receptor type II-like kinase 5) and interferes with the normal signaling process of TGF-β type I. The ALK5 is also referred to as a TGF-β type I receptor, and the transforming growth factor-β type I is a multifunctional peptide which has various functions on cell proliferation, differentiation and various kinds of cells. Plays a pivotal role in the growth and differentiation of various tissues such as adipocyte formation, myocyte formation, bone cell formation, epithelial cell differentiation. Non-limiting examples of the ALK5 inhibitor (TGF-β type I receptor inhibitor) include RepSox (1,5-Naphthyridine, 2- [3- (6-methyl-2-pyridinyl) -1H- pyrazol-4-yl]); SB431452 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] benzamide; 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]), and the ALK5 inhibitor is not limited as long as it binds to the TGF-β type I receptor and interferes with the normal signaling process of TGF-β I. However, preferably Repsox or SB431452.

In the present invention, "cAMP signaling activator" means a substance that activates a cAMP signal. Non-limiting examples of the cAMP signaling activator include Forskolin, isoproterenol, NKH 477, isoprotereno (Chemical based), PACAP 1-27, and PACAP 1-38 (peptide based), the cAMP signaling activator The activating cAMP signal is not limited, but may be preferably Forskolin or NKH477.

In another aspect of the invention, the present invention is one or more compounds selected from the group consisting of Activin A, Bone morphogenetic protein 4 (BMP4), Vascular endothelial growth factor (VEGF), Glycogen synthase kinase inhibitor (GSK inhibitor) and src tyrosine kinase inhibitor It provides a composition for inducing maturation of skeletal muscle cells, comprising as an active ingredient.

In the present invention, src tyrosine kinase inhibitor means a substance that interferes with phosphorylation of src. Non-limiting examples of src tyrosine kinase inhibitors include PP1 (1- (1,1-dimethylethyl) -3- (4-methylphenyl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine), PP2 (3 -(4-chlorophenyl) -1- (1,1-dimethylethyl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine), SU6656 (2,3-dihydro-N, N-dimethyl-2- oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl) methylene] -1H-indole-5-sulfonamide), and Dasatinib (N- (2-chloro-6-methylphenyl) -2-[[6- [4- (2-hydroxyethyl) -1-piperazinyl] -2-methyl-4-pyrimidinyl] amino] -5-thiazolecarboxamide), and the src tyrosine kinase inhibitor inhibits phosphorylation of src. The material that is interfering is not limited, but may preferably be PP1.

The composition for inducing maturation of skeletal muscle cells may further include one or more cytokines (cytokine) for inducing maturation of skeletal muscle cells.

In inducing the skeletal muscle cells of the present invention, the type of starting cells (parent cells) is not particularly limited, and somatic cells or adult stem cells can be used. The somatic cell is not limited to the type thereof. For example, the somatic cell may be a mature somatic cell in addition to the somatic cell of the embryonic period, but may preferably be a fibroblast. Adult stem cells are not limited to their kind, but may be mesenchymal stem cells, and more preferably fat-derived mesenchymal stem cells. When the induced skeletal muscle cells are used for the treatment of a disease, it is preferable to use cells separated from the patient. For example, somatic cells involved in the disease or somatic cells involved in the disease treatment can be used. In the present invention, fibroblasts and adult stem cells include all fibroblasts and adult stem cells derived from animals such as humans, mice, horses, sheep, pigs, goats, camels, antelopes, and dogs.

As another aspect of the present invention, the present invention comprises the step of culturing somatic cells or adult stem cells in a medium containing a plate histone deacetylase inhibitor, GSK inhibitor, ALK5 inhibitor, and cAMP signaling active agent as an active ingredient, Provided is a method for inducing differentiation of adult stem cells into skeletal muscle cells.

In the method of inducing differentiation, in the case of inducing differentiation of adult stem cells into skeletal muscle cells, the medium may not include a GSK inhibitor, and the culture may be performed without limitation as long as the culture can induce differentiation into skeletal muscle cells. Although, preferably, it may be performed for 5 to 25 days, more specifically, for 4 to 12 days for somatic cells, 20 to 25 days for adult stem cells.

In addition, in the differentiation-inducing method, after the step, Activin A, BMP4 (Bone morphogenetic protein 4), VEGF (Vascular endothelial growth factor), GSK inhibitor (Glycogen synthase kinase inhibitor), and src tyrosine kinase inhibitor are selected. The method may further comprise the step of maturing the differentiation-induced cells into skeletal muscle cells in a medium containing at least one compound as an active ingredient, the maturation step is limited if the differentiation-induced cells are mature period It can be done without, but preferably may be performed for 1 to 5 days, more preferably for 2 to 4 days. In addition, when the differentiation induction method of the present invention is used, there is an advantage in that differentiation to a desired cell can be efficiently induced with only a shorter time of treatment compared with the known chemically induced cell differentiation method.

The medium for culturing the somatic or adult stem cells includes all of the medium conventionally used for culturing fibroblasts or mesenchymal stem cells in the art. The culture medium used for the culture generally contains a carbon source, a nitrogen source and a trace element component. Although not limited thereto, the medium preferably includes DMEM / F12, N2, B27, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).

The medium for culturing of the present invention can be used without limitation a basal medium known in the art. The basal medium may be prepared by artificially synthesizing, or a commercially prepared medium may be used. Examples of commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal). essential medium), G-MEM (Glasgow's Minimal Essential Medium) and Isocove's Modified Dulbecco's Medium, and the like, but are not limited thereto.

In the embodiment of the present invention, four types of low molecular weight substances (VCRF) which are expected to induce skeletal muscle cells more efficiently among various low molecular weight substances such as various differentiation inducing substances were selected and directly differentiated through the selected low molecular weight substances. As a result, spontaneous contraction phenomenon similar to muscle cells was observed through the treatment of four kinds of low molecular weight substances, and it was confirmed that the direct differentiation with a very high yield when inducing similar skeletal muscle cells in this manner (Example 2 to 4).

In addition, in the embodiment of the present invention, the same effect was confirmed even in the case of treating other compounds performing the same function as each compound constituting the VCRF. Specifically, SB431452 was used instead of RepSox for compounds that performed ALK5 inhibitor function, NKH477 was used for Forskolin for compounds that performed cAMP signaling activator, and sodium butyrate instead of valproic acid for compounds that performed HDAC inhibitor function. Compounds that perform the GSK inhibitor function were confirmed to have the same effect using AZD2858 instead of Chir99021 (see Example 8).

In addition, in the embodiment of the present invention, it was confirmed that the differentiation of not only embryonic fibroblasts but also skin fibroblasts into musculoskeletal cells using the composition for inducing differentiation of the present invention (see Example 9), and also fibroblasts In addition, it was confirmed that the composition for inducing differentiation of the present invention is effective in inducing differentiation of human adipose derived stem cells into musculoskeletal cells (see Example 10).

In addition, in the embodiment of the present invention, by confirming that the cardiomyocytes are not produced at all under the conditions of direct differentiation of skeletal muscle cells, the cardiomyocyte differentiation does not occur but only skeletal muscle cell differentiation with the composition and differentiation method proposed in the present invention. (See Example 11).

In addition, in the embodiment of the present invention, in inducing the differentiation of adipose derived stem cells into musculoskeletal cells, a greater number of muscle cells were observed when VRF was treated than when VCRF was treated. It was confirmed that the lack of GSK inhibitor is more effective in inducing differentiation into musculoskeletal cells (see Example 10). From the above it is apparent that the composition for inducing differentiation of adult stem cells into musculoskeletal cells does not include a GSK inhibitor, thereby increasing the differentiation efficiency.

Skeletal muscle cells can be efficiently induced by the composition for inducing differentiation comprising the four low molecular weight compounds. The differentiation inducing composition may be one containing no GSK inhibitor. In addition, the concentration of each of the low molecular weight compounds constituting the composition for inducing differentiation is not particularly limited as long as it can induce differentiation of somatic cells or adult stem cells into skeletal muscle cells. For example, Valproic acid and sodium butyrate are 1 to 1000. μM, chir99021 and AZD2858 can be added at concentrations of 0.001-100 nM, RepSox and SB431452 1-100 μM, Forskolin and NKH477 1-100 μM.

Most of the methods of inducing somatic cells into skeletal muscle cells by using a direct conversion method are performed by introducing an external gene. However, introducing a gene using a virus causes genetic instability due to random integration of an external gene, which may cause cancer in future clinical applications. For this reason, methods for using small molecules without gradually injecting external genes have been proposed. However, despite the recent progress in research on inducing direct differentiation using various low molecular weight compounds, at least one gene has been used, and without introducing a gene, it is still unable to switch from human somatic cells to desired skeletal muscle cells.

However, the present invention is a method of ensuring the genetic stability of inducing skeletal muscle cells from somatic cells without introducing foreign genes, and is designed to solve the method of inducing genetic defects using existing genes.

In the present invention, somatic cells were directly differentiated into skeletal muscle cells using only a combination of low molecular weight substances. Thus, by overcoming many of the problems with the existing technology, it is very likely to be used as a cell therapy for patients.

In another aspect of the present invention, the present invention provides a cell therapy agent for treating skeletal muscle disease, including cells induced to differentiate into skeletal muscle cells by the above method and / or skeletal muscle cells differentiated by the above method. As cells included in the cell therapy herein, cells differentiated into skeletal muscle cells, differentiated skeletal muscle cells, and differentiated skeletal muscle cells may be mixed with each other.

In the present invention, the term "skeletal muscle cell" refers to a cell that performs the function of skeletal muscle, and there is no limitation as long as the cell performs the above function, but may be fetal skeletal muscle cells or adult skeletal muscle cells. "Induced differentiation into skeletal muscle cells" includes, without limitation, cells of all differentiation stages, such as myeloid progenitors, muscle progenitors, myoblasts, etc., having the ability to become functional skeletal muscle cells in the future, as described below. By at least one, preferably a plurality of methods means at least one, preferably a cell which can be identified by a plurality of markers or criteria. Expression of various markers specific for skeletal muscle cells can be detected by known biochemical or immunochemical methods, and such methods can be used without limitation. In this method, marker specific polyclonal antibodies or monoclonal antibodies that bind to skeletal muscle progenitor cells or skeletal muscle cells can be used. Antibodies that target individual specific markers can be used commercially or without limitation, those prepared by known methods. Examples of markers specific for skeletal muscle progenitor cells or skeletal muscle cells include MF20, sarcomeric Actinin, MyoD and Myogenin. Alternatively, expression of skeletal muscle progenitor or skeletal muscle cell specific markers is not limited to specific methods, but amplifies mRNA encoding any marker protein, which is reverse transcriptase mediated polymerase chain reaction (RT-PCR) or hybridization assay, It can be confirmed by molecular biological methods commonly used for detection and interpretation. Nucleic acid sequences encoding marker proteins specific for skeletal muscle progenitors or skeletal muscle cells are already known and can be obtained from public databases such as GenBank, which facilitates marker specific sequences necessary for use as primers or probes. You can decide. In addition, to confirm the differentiation of somatic cells into skeletal muscle cells, physiological criteria may be additionally used.

In the present invention, the skeletal muscle disease refers to a disease caused by damage to skeletal muscle caused by genetic or acquired factors, a decrease in the number of skeletal muscle cells, a weakening of the function of skeletal muscle cells, and the non-limiting example of skeletal muscle disease is Becker. Becker musculardystrophy, Congenital musculardystrophy, Duchenne musculardystrophy, Distal musculardystrophy, Emery-Dreifuss musculardystrophy, Muscular dystrophy (Limb-girdle musculardystrophy), Myotonic musculardystrophy, and Oculopharyngeal musculardystrophy. Other factors include acquired reduction, muscular dystrophy, muscular sclerosis, gold sprain, and inflammatory muscle disease. have.

As used herein, the term "cellular therapeutic agent" refers to a medicinal product (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special manipulation from humans. Or through a series of actions such as proliferating and screening living autologous, allogeneic, or heterologous cells in vitro or otherwise altering the biological properties of a cell to restore tissue function. Means the drug used for the purpose.

In the present invention, the term "treatment" means any action that improves or benefits the condition of the disease by administration of the cell therapy agent.

The route of administration of the cell therapy of the invention can be administered via any general route as long as it can reach the desired tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration may be, but is not limited thereto.

The cell therapy agent may be formulated in a suitable form with a pharmaceutical carrier generally used for cell therapy. 'Pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not cause an allergic or similar reaction, such as gastrointestinal disorders, dizziness or the like, when administered to a human. Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, carriers for parenteral administration such as aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, 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 references (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

In addition, the cell therapeutic agent may be administered by any device capable of differentiation-induced cells or differentiation-induced skeletal muscle cells to the target site.

The cell therapy agent of the present invention may include a therapeutically effective amount of the cell therapy composition for the treatment of the disease. By `` therapeutically effective amount '' is meant the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician. This includes the amount that induces alleviation of the symptoms of the disease or disorder being treated.

It is apparent to those skilled in the art that the composition included in the cell therapy of the present invention will vary depending on the desired effect. Therefore, the optimum content can be easily determined by one skilled in the art, and the type of disease, the severity of the disease, the amount of other components contained in the composition, the type of formulation, and the patient's age, weight, general state of health, sex and diet, administration It can be adjusted according to various factors including time, route of administration and rate of release of the composition, duration of treatment, and drugs used simultaneously. In consideration of all the above factors, it is important to include an amount that can achieve the maximum effect in a minimum amount without side effects. For example, the daily dose of the differentiation-induced cells or the differentiation-induced skeletal muscle cells of the present invention is 1.0 × 10 ⁴ to 1.0 × 10 ¹⁰ cells / kg body weight, preferably 1.0 × 10 ⁵ to 1.0 × 10 ⁹ The cell / kg body weight can be administered once or in divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore, the dosage may be It does not limit the scope of the present invention in terms of aspects.

In addition, in the method of treatment of the present invention, the cell therapy of the present invention is conventionally administered through rectal, intravenous (intravenous therapy, iv), intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. May be administered in such a manner.

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

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Experiment Preparation and Experimental Method

1-1. Animal experiment (cell isolation and culture)

Mouse embryonic fibroblasts (MEFs) were isolated from C57BL.6 mouse embryos at day 13.5 of embryonic stage. The head, spinal cord and internal organs were carefully removed to eliminate the potential for contamination of neural crest cells. The remaining portion of tissue was cut with 0.25% trypsin-EDTA (GIBCO) and trypsinized to prepare a single cell suspension. It was then incubated in high-glucose DMEM (Welgene) with 10% FBS, 1% penicillin and streptomycin (Welgene). For lineage tracing experiments, FSP-Cre mice were crossed with R26RtdTomato mice (Jackson laboratories) to produce mice with fibroblasts that specifically express tdTomato in MEF. MEF was identified by a procedure similar to the above.

1-2. Generation of skeletal muscle

Mouse embryo fibroblasts (MEF) were precoated with 1: 100 Matrigel (BD Biosciences) at room temperature for 2 hours, then seeded in 35 mm plates at a density of 60,000 cells per plate, and compounds of VCRFPT or VCRF MuscleReprogramming medium containing a combination of knockout DMEM (Gibco), 15% knockout serum replacement, 5% FBS (Gibco), 1% Glutamax (Gibco), 1% non-essential amino acid (Gibco), 0.1 mM β-mercaptoethanol (Sigma) and 1 × penicillin / streptomycin). Compound combinations of the VCRFPT or VCRF were 0.5 mM Valproic Acid (V), 10 μM CHIR99021 (C), 10 μM RepSox (R), 50 μM Forskolin (F), 5 μM Parnate (P), and 1 μM TTNPB ( T) (Medchemexpress). Fibroblasts induce differentiation for 6 days with changing medium every 3 days.

6 days after induction of differentiation, various cytokine mixtures (25 ng / ml BMP4 (Preprotech), 10 ng / ml Activin A (R & D), 10 ng / ml VEGF (Preprotech), 10 μM CHIR99021 (Medchemexpress), 200 μg / ml Cultured in MRF containing Phospho Ascorbic Acid (Sigma), 0.15 mM Monothioglycerol (Sigma), and lx B27 supplement (Lifetechnologies). For Pax3, after the induction phase, the positive myodermal expansion MEF is EM (DMEM / F12 (Welgene), 1% penicillin and streptomycin, 0.5% BSA (Gibco), 1% Glutamax, 1% non-essential amino acid (Gibco), 200 Μg / ml Phospho Ascorbic Acid, 10 ng / ml BMP4, 0.15 mM Monothioglycerol, 1 × B27 supplement, 10 μM CHIR99021, and 20 ng / ml FGF2).

1-3. Immunostaining

Skeletal muscle differentiated from mouse embryonic fibroblasts after the induction or maturation stages were harvested on day 6 or 9, respectively, washed twice with 1x PBS (Welgene) and then with 10% 4% paraformaldehyde (Sigma-Aldrich). It was fixed for minutes and PBS containing 0.25% Triton X-100 (USB Corporation) was treated at 22 ° C. for 10 minutes, followed by washing with PBS twice for 5 minutes each. Block for 60 minutes with blocking solution containing 1% BSA (Amresco), 22.52 mg / ml glycine (Affymetrix), and 0.1% Tween 20 (Affymetrix) in PBS and overnight at 4 ° C. with appropriate primary antibody diluted with blocking solution. Stained. The antibodies used were mouse monoclonal MF20 (DSHB, dilution 1:20), mouse monoclonal anti-sarcomeric actinin (A7732, Sigma-Aldrich, dilution 1: 100), rabbit polyclonal anti MyoD (C-20) (sc -304, Santacruz dilution 1:20), mouse monoclonal anti myogenin (ab1835, Abcam, dilution 1:50), and mouse anti Pax3 (MAB2457-SP, R & D, dilution 1: 100). After incubation with primary antibody, cells were washed three times in PBST and diluted 1: 100 to Alexa-488-conjugated goat secondary anti-mouse antibody (A11001, Invitrogen) or Alexa-563-conjugated goat secondary anti-rabbit antibody ( A21428, Invitrogen) was stirred at room temperature for 2 hours. Cells were incubated with 1 μg / ml DAPI (D9542, Sigma-Aldrich) for 5 minutes at room temperature to stain nuclei and visualized using fluorescence microscopy (IX71S1F3, Olympus).

1-4. QuantitativeRT-PCR

Mouse skeletal muscles derived from embryonic fibroblasts were harvested at 6 or 9 days after induction or maturation, respectively, and total RNA was extracted with RNeasy Mini Kit (QIAGEN), and 5 μg of total RNA was collected from RNA to cDNA EcoDry Premix (Oligo). dT, Clontech) to cDNA. qRT-PCR was performed using a SYBR Green PCR Master Mix (Bio-Rad) on a Bio-Rad Prime PCR instrument, and total RNA was used to assess the mRNA levels of cardiac marker genes from cardiomyocytes derived from P19 cells. . The qRT-PCR conditions were 40 cycles of 30 seconds at 95 ° C, 15 seconds at 60 ° C, and 15 seconds at 72 ° C. Primers used in this study are shown in Table 1 below.

Prime name Forward Primer (5’-3 ’) SEQ ID NO: Reverse Primer (5’-3 ’) SEQ ID NO:  Tcf15 TTCTGTCTCAGCAACCAGCG One GGCTACACCCCTCACTTTCAA 2  Mesp1 GCTCGGTCCCCGTTTAAGC 3 ACGATGGGTCCCACGATTCT 4  Tbx6 ATGTACCATCCACGAGAGTTGT 5 CCAAATCAGGGTAGCGGTAAC 6  Six1 ATGCTGCCGTCGTTTGGTT 7 GGTGATTGTGAGGCGAGAACT 8  Six4 CCACGGTTTTTCCCTGACCC 9 GGTTGCATAGTTAGTGTTGCTGA 10  Myf5 CACCACCAACCCTAACCAGAG 11 AGGCTGTAATAGTTCTCCACCTG 12  Nppa GTGCGGTGTCCAACACAGAT 13 TCCAATCCTGTCAATCCTACCC 14  Myl2 ATCGACAAGAATGACCTAAGGGA 15 ATTTTTCACGTTCACTCGTCCT 16  Lbx1 CGTCCGTGCGGAGAAGTTAC 17 CCTCCAGCCCCTTAAAGGTCT 18  Pax7 CTCAGTGAGTTCGATTAGCCG 19 AGACGGTTCCCTTTGTCGC 20  Pax3 TTTCACCTCAGGTAATGGGACT 21 GAACGTCCAAGGCTTACTTTGT 22  Myog GCAATGCACTGGAGTTCG 23 ACGATGGACGTAAGGGAGTG 24  Myod1 CCCCGGCGGCAGAATGGCTACG 25 GGTCTGGGTTCCCTGTTCTGTGT 26  Myh6 CAACAACCCATACGACTACGC 27 ACATCAAAGGGCCACTATCAGTG 28  Myh3 GCATAGCTGCACCTTTCCTC 29 GGCCATGTCCTCAATCTTGT 30  Msgn1 CTTCTGACACCGCTGGTCTG 31 GTGACTGCCGTAGCCATCG 32  Meox1 TGGCCTATGCAGAATCCATTCC 33 TGGATCTGAGCTGCGCATGTG 34  T CTGGACTTCGTGACGGCTG 35 TGACTTTGCTGAAAGACACAGG 36  Actb AAATCGTGCGTGACATCAAA 37 AAGGAAGGCTGGAAAAGAGC 38

1-5. Flow cytometry quantitation

Differentiated CiSMCs were harvested and cell fixation and permeability increased using FIX & PERM (Thermofisher). Subsequently, the cells were treated in antibody dilution buffer (1 × PBS, 5% BSA, 0.1% Tween20), and mouse monoclonal antibodies bound to the markers were added to the cells and bound overnight. Primary conjugated cells were washed with 1 × PBS and treated with Alexa-488 bound secondary antibodies for 3 hours. Subsequently, the cells were washed with 1x PBS, and then put into a cell culture medium, and flow cytometry was attempted.

Example  2. Chemical Induction Skeletal muscle cell  ( Cismc Identification of chemicals that induce differentiation

In order to induce skeletal muscle production without genetic manipulation, the inducing chemical VCRFPT was treated with mouse embryonic fibroblasts for 9 days by the method of Example 1-2.

As a result, as shown in Fig. 1A, it was confirmed that some myocytes contract naturally, and on day 6, myocytes or myotube-like structures were confirmed (hereinafter, in all subsequent experiments). Use 6 days processing).

Furthermore, in order to confirm and verify that the myocyte / myotube-like structure has properties of skeletal muscle, expression of skeletal muscle markers was confirmed according to the methods of Examples 1-3 and 1-4.

As a result, as shown in FIG. 2, all myocyte / myotube-like structures were immunopositive against MF20 (pan MHC marker), sarcomeric Actinin, and mature skeletal muscle markers such as MyoD or Myogenin, and cytoplasmic positions of MF20 and sarcomeric actinin (Cytosolic). localization), MyoD nuclear localization, and myogenesis and co-expression of these markers were confirmed (B). In addition, it was confirmed that the expression of mature skeletal muscle markers MyoD and Myogenein increased several times as compared to the control group treated with nothing (C). These results indicate that the myocyte / myotube-like structure has the properties of skeletal muscle, and thus chemical-induced skeletal muscle synthesis is possible.

Example  3. Fibroblasts Into skeletal muscle cells  Identify the optimal mixture needed to induce differentiation

Based on the results of Example 2, in order to identify the minimum mixture critical for skeletal muscle induction and optimize the induction process, muscle colonies formed by removing one or two components of the mixture at a time are quantitatively and qualitatively Evaluated.

As a result, as can be seen in Figures 3a and 3b, it was confirmed that the chemicals that exhibit the effect of significantly weakening the differentiation induction into skeletal muscle cells when removed includes Valproic acid, Chir99021, Repsox and Forskolin. More specifically, the removal of Chir99021 (Wnt signaling activator) showed the weakest effect on muscle formation so that MF20-positive cells could not be observed, followed by Repsox (TGF β inhibitor) and Forskolin (cAMP agonist). appear. In addition, the removal of Valproic acid (HDAC inhibitor) also showed a significant decrease in the number of colonies, whereas the removal of Parnate (epigenetic modulator) or TTNPB (retinoic acid receptor agonist) showed a moderate effect when compared to the mixture (VCRFPT). It was confirmed that appeared (B). In other words, if you remove two components of Valproic acid, Chir 99021, Repsox and Forskoin at one time, the number of colonies is reduced to a similar level, whereas if you remove Parnate and TTNPB, colony formation is more than doubled. , Removal of TTNPB was confirmed to improve colony formation.

In addition, MF20 immunopositive colony qualitative results confirmed that VCRF forms the maximum number of colonies, then VCRFP, and then VCRF and VCRFPT form similar numbers of colonies (C). In addition, enhanced MF20 positive colony was confirmed to increase sarcomeric actinin, MyoD and Myogenin expression (D), and further confirmed that MyoD and Myogenin expression is expressed at a significant level when VCRF treatment (E).

As a result of the synthesis, it was confirmed that the combination of fibroblasts (Chir99021, Repsox, Forskolin, and Valproic acid) optimized for induction of differentiation of fibroblasts into skeletal muscle cells (F).

Example  4. Fibroblasts Into skeletal muscle cells  Identify the optimal incubation period for inducing differentiation

Based on the results of Example 3, the differentiation efficiency into skeletal muscle cells according to the period of culturing the fibroblasts treated with VCRF was confirmed. More specifically, the fibroblasts are cultured in a medium containing a mixture of VCRF, Sarcomeric actinin positive cells using flow cytometry according to Example 1-5 at 6, 8, 10, 12, 15 days after the culture It was measured. In addition, the expression levels of skeletal muscle cell specific markers (MyoD and Myogenin) and mature muscle cell specific markers (Mck and Myomaker) at 6, 8, 10, 12, and 15 days were examined.

As a result, as shown in FIG. 4, as the incubation period became longer, the number of Sarcomeric actinin-positive cells increased, and the largest number could be measured after 10 days of culture. However, it was confirmed that the number of Sarcomeric actinin-positive cells increased and decreased after 10 days of culture (B). As a result of measurement of marker expression level, it was confirmed that the expression bands of skeletal muscle cell-specific markers and mature muscle cell-specific markers appeared the thickest after 10 days of culture as in the flow cytometry (C).

The results indicate that induction of differentiation into skeletal muscle cells, skeletal muscle cells can be obtained with high efficiency by culturing fibroblasts for 4 to 12 days in a medium containing VCRF.

Example  5. Into skeletal muscle cells  Identify the effects of cytokines on induction or maturation

Based on the results of Example, mRNA expression of the fibroblast direct differentiation step was analyzed for VCRF or VCRFPT treatment to confirm the effects of cytokines at each step.

As a result, as shown in FIG. 5, cytokine treatment was found to enhance the expression of premyogenic mesoderm specific markers (Msgn1 and T), as compared to both VCRF and VCRFPT, and myogenic precursors. It has been shown to enhance the expression of myogenic precursor specific markers (pax3 and pax7) and mature muscle cell specific markers (MyoD and Myogenin).

These results indicate that the efficiency of direct differentiation of fibroblasts to CiSMC when treated with cytokines with the chemicals of the present invention (VCRF), and VCRF, compared with VCRFPT, with or without cytokines It can be seen that it has a high efficiency.

Example  6. VCRF  Induced by myocytes  or of myotubes  Identify the optimal compound or mixture needed for maturation

Based on the results of Example 3 above, in order to further mature myocytes or myotubes induced by VCRF, BMP4, Activin A, along with several cytokines known to be involved in muscle differentiation after an initial induction phase for 6 days The maturation of skeletal muscle cells was confirmed by culturing the cells in a medium containing Chir99021, and a mixture containing Vascular endothelial growth factor (VEGF) or src tyrosine kinase inhibitor (PP1). A schematic diagram of the experiment is shown in Figs. 6A and 7A.

After culturing in a medium further comprising a mixture comprising BMP4, Activin A, Chir99021, and vascular endothelial cell growth factor (VEGF), as shown in FIGS. 6 (B) and (C), after the initial induction step, Three days of treatment with BMP4, Chir99021, Activin A and VEGF resulted in an improvement in muscle maturation. Spindle-like cell structures could be observed (B). Furthermore, the cytokine treatment improved the range of MF20 expression as depicted in thicker and more prominent tubular structures, and after three days did not improve maturation further (C). After stage 3, mature myocytes obtained by further maturation for 3 days showed a pronounced multinuclear structure expressing MyoD and Myogenin with simultaneous expression of MF20 and sarcomeric actinin (4D), and cytokines at the transcription level of MyoD and Myogenin The effect was similar (E).

On the other hand, as a result of culturing in a medium additionally containing src tyrosine kinase inhibitor (PP1), as shown in Figure 7, when the maturation in the medium containing PP1 for 3 days after the initial induction step shows the mature state of the muscle It was confirmed that the muscle cells matured by treatment with PP1 had a thicker spindle-like multinucleated cell structure than the cytokines alone.

Example  7. Chemical Induction Skeletal muscle cells  ( Cismc Identification of Differentiated Skeletal Muscle Cells from Fibroblasts by Lineage Tracking

Mouse embryonic fibroblasts (MEFs) isolated from embryonic (day 13.5) mouse embryos may contain myogenic progenitor cells as impurities. The contained muscle precursor cells can differentiate into skeletal muscle cells during the culture period. Thus, lineage tracing was performed using Fsp1-cre: R26RtdTomato mice to confirm that the obtained skeletal muscle cells were not differentiated from muscle precursor cells contained as impurities and to verify that they were differentiated from fibroblasts. More specifically, FSP1-dTomato progeny were obtained, MEFs were isolated from the FSP1-dTomato mouse embryos, and treated with VCRF or VCRFPT for 6 days to confirm the expression level of myocyte-specific markers and dTomato, and additional cytokine. The expression level of the marker with or without treatment was also confirmed. A schematic diagram of the experiment is shown in FIG. 8.

As a result, as shown in Figures 9a and 9b, regardless of the presence or absence of cytokine treatment, it was confirmed that the skeletal muscle cells obtained by treatment with VCRF or VCRFPT simultaneously express FSP1-dTomato and MF20 markers.

Since dTomato fluorescence is controlled by the FSP1 protein, when considering fluorescence only in fibroblast-derived cells, the results indicate that skeletal muscle cells obtained following VCRF or VCRFPT treatment have differentiated from fibroblasts.

Example  8. Fibroblasts Using a Mixture of Different Compounds of the Same Function Into skeletal muscle cells  Confirmation of differentiation induction effect

According to the results of Example 3, the mixture (VCRF) containing Valproic acid (V), CHIR99021 (C), RepSox (R), and Forskolin (F) was most effective for inducing differentiation of fibroblasts into skeletal muscle cells. Valproic acid acts as an HDAC inhibitor, CHIR99021 acts as a GSK inhibitor, RepSox acts as an ALK5 inhibitor, and Forskolin acts as a cAMP signaling activator, performing the same function as each compound used above. It was also confirmed that effective fibroblast differentiation induction into skeletal muscle cells was achieved even when other compounds were used. Specifically, in the same manner as in Example 3, only the compound contained in the differentiation inducing mixture was tested. SB431452 (10μM) was used in place of RepSox instead of RepSox, and NKH477 (5μM) instead of Forskolin was used in place of ALK5 inhibitor.Sodium butyrate instead of valproic acid (500μM) was used, and AZD2858 (10nM) was used instead of Chir99021 as a compound that performs the GSK inhibitor function.

As a result, as can be seen in FIG. 10, even when inducing differentiation using a mixture containing Valproic acid, CHIR99021, SB431542, and NKH477, differentiation of fibroblasts into skeletal muscle cells was effectively induced (B), Similar results were obtained when inducing differentiation using a mixture comprising sodium butyrate, AZD2858, SB431542, and NKH477 (C).

From the above results, it can be seen that differentiation from fibroblasts to skeletal muscle cells can be effectively induced not only by the mixture of the compounds of the above combination but also by mixing with other compounds having the same function as the above compounds.

Example 9 Confirmation of Differentiation Induction Effect of Skin Fibroblasts into Skeletal Muscle Cells

Through the results of the above examples, mouse embryo fibroblasts in a medium containing a histone deacetylase inhibitor, a GSK inhibitor, a ALK5 inhibitor, and a cAMP signaling activator (MEF) was cultured it was confirmed that can effectively induce differentiation into skeletal muscle cells. In this embodiment, in order to determine whether the differentiation from normal somatic cells to skeletal muscle cells using the mixture of the compound is possible, the skin fibroblasts (not the embryonic fibroblasts) by culturing in the medium containing the mixture skeletal muscle cells Expression of specific differentiation markers was confirmed. Skin tissues of young mice were treated with collagenase at 37 ° C. overnight, and then fibroblasts were grown on cell culture dishes. The experimental method is the same as in the case of MEF.

As a result, as shown in FIG. By confirming the expression of representative markers of skeletal muscle, it was confirmed that skin-derived fibroblasts also differentiate into skeletal muscle under differentiation conditions of the present invention. These results indicate that fibroblasts can be differentiated into skeletal muscle under differentiation conditions of the present invention regardless of which tissue they are isolated from.

Example 10 Confirmation of Induction Effect of Mesenchymal Stem Cells into Skeletal Muscle Cells

Through the results of Example 9, it was confirmed that the differentiation into skeletal muscle cells can be effectively induced by culturing somatic cells in a medium containing a histone deacetylase inhibitor, a GSK inhibitor, an ALK5 inhibitor, and a cAMP signaling activator. In this embodiment, in order to determine whether induction of differentiation of adult stem cells into skeletal muscle cells rather than somatic cells using a mixture of the compounds, human adipose-derived stem cells (humanadipose-derived stem cells) medium containing the mixture Cultured at, the expression of skeletal muscle cell-specific markers were confirmed whether the differentiation of human adipose derived adult stem cells skeletal muscle. Human adipose derived stem cells used for skeletal muscle differentiation were purchased from Lonza and cultured in high glucose DMEM (Gibco) and 15% fetal bovine serum (Gibco). The experimental method is the same as in the case of MEF.

Chemical cocktail Abundance of Muscles Valproic Acid, Chir99021, Repsox, Forskolin, + Valproic Acid, Repsox - Valproic Acid, Repsox, Forskolin ++ Repsox, Forskolin, Chir99021 - Repsox, Forskolin - Valproic Acid, Forskolin -

As can be seen in Table 2 above, when VCRF was treated to adipose derived stem cells, it was confirmed that differentiation into skeletal muscle cells was induced like somatic cells. Differentiation was attempted with various combinations of the four VCRF compounds used for differentiation to identify more detailed conditions related to adipose derived adult stem cell differentiation (Table 2). These results confirm that no differentiation occurs at all in the absence of Valporic acid, Repsox, or Forskolin, indicating that HDAC inhibitor, ALK-5 inhibitor, and cAMP activator are essential for differentiation. However, it was confirmed that the differentiation efficiency was excellent when the GSK inhibitor was not included (FIG. 12 (A)). Also, in order to confirm the importance of signal regulation related to differentiation, as in the case of fibroblasts, other ALK- It was confirmed that differentiation occurs with the same efficiency when the cAMP signaling activator NKH422, instead of 5 inhibitor SB431542 and Forskolin, was treated (Fig. 12 (B)). These results indicate that adult stem cells can be differentiated into skeletal muscle cells by using not only the specific compound used in this embodiment but also the same kind of compound having the function of each chemical.

Example 11. Confirmation of Induction of Skeletal Muscle Cell Specific Differentiation of VCFR

In order to confirm that the differentiation by the differentiation inducing composition of the present invention is skeletal muscle cell specific differentiation and does not induce differentiation into cardiomyocytes, experiments were carried out by the methods of Examples 1-2 to 1-4.

As a result, as shown in FIG. 13, it was confirmed that skeletal muscle cell markers (MyoD and alpha-actinin) were expressed in the cells treated with VCFR, but not cardiomyocyte markers (cTNT and Nkx2.5). From the above results, it can be seen from the present invention that no cardiomyocytes are produced at all in the conditions of direct differentiation into skeletal muscle cells, but only differentiation into skeletal muscle cells occurs.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The present invention relates to a composition for inducing differentiation of somatic cells or adult stem cells into skeletal muscle cells, and the like, obtaining directly differentiated skeletal muscle cells from somatic cells through a simple method of culturing cells in a culture medium comprising the composition for inducing differentiation of the present invention. can do. The obtained skeletal muscle cells are differentiated from somatic cells, there is no risk of cancer after transplantation, and there is an advantage of genetically stable using only a mixture of low molecular weight compounds without gene introduction. In addition, the present invention was confirmed that the composition also induced differentiation of mesenchymal stem cells into skeletal muscle cells quickly and efficiently, and in particular, the lack of GSK inhibitor in the composition was confirmed to have a positive effect on the differentiation efficiency. Therefore, the present invention induces differentiation into a large amount of skeletal muscle cells in a shorter time, thereby developing an economical and efficient cell therapy for treating skeletal muscle diseases caused by genetic factors, congenital factors, acquired factors, and endogenous and exogenous factors. It is expected to be useful for.

Claims (20)

Somatic or adult stem cells comprising a pan-histone deacetylase inhibitor, an ALK5 inhibitor (activin A receptor type II-like kinase 5 inhibitor), and a cAMP signaling activator as active ingredients Composition for inducing differentiation into skeletal muscle cells. The method of claim 1, The composition further comprises a GSK inhibitor (Glycogen synthase kinase inhibitor), composition for inducing differentiation of somatic cells into skeletal muscle cells. The method of claim 1, The somatic cells are fibroblasts (fibroblast), composition for inducing differentiation into skeletal muscle cells. The method of claim 1, The adult stem cells, characterized in that the mesenchymal stem cells, composition for inducing differentiation into skeletal muscle cells. The method of claim 1, The plate histone deacetylase inhibitors include Valproic acid, Sodium butyrate, Suberoylanilide hydroxamic acid, Hydroxamic acid, Cyclic tetrapeptide, Depsi Peptides (depsipeptides), Trichostatin A, Verinostat, Velinostat, Belinostat, Panobinostat, Benzamide, Entinostat, and Butylate Butyrate, characterized in that any one or more selected from the group consisting of, composition for inducing differentiation into skeletal muscle cells. The method of claim 1, The ALK5 inhibitor is RepSox (1,5-Naphthyridine, 2- [3- (6-methyl-2-pyridinyl) -1H-pyrazol-4-yl]); SB431452 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1 H -imidazol-2-yl] benzamide; 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]), at least one selected from the group consisting of, composition for inducing differentiation into skeletal muscle cells. The method of claim 1, The cAMP signaling activator is at least one selected from the group consisting of Forskolin, isoproterenol, NKH 477, isoprotereno (Chemical based), PACAP 1-27, and PACAP 1-38 (peptide based), Composition for inducing differentiation into skeletal muscle cells. The method of claim 2, The GSK inhibitors include: Chir99021 (6- (2- (4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino) ethylamino) nicotinonitrile); 1-azakenpaullone (9-Bromo-7,12-dihydro-pyrido [3 ', 2': 2,3] azepino [4,5-b] indol-6 (5H) -one); AZD2858; 3-amino-6- (4-((4-methylpiperazin-1-yl) sulfonyl) phenyl) -N- (pyridin-3-yl) pyrazine-2-carboxamide; BIO ((2'Z, 3'E) -6-Bromoindirubin-3'-oxime); ARA014418 (N- (4-Methoxybenzyl) -N '-(5-nitro-l, 3-thiazol-2-yl) urea); Indirubin-3'-monoxime; 5-Iodo-indirubin-3'-monoxime; kenpaullone (9-Bromo-7,12-dihydroindolo- [3,2-d] [1] benzazepin-6 (5H) -one); SB-415286 (3-[(3-Chloro-4-hydroxyphenyl) amino] -4- (2-nitro-phenyl) -1H-pyrrole-2,5-dione); SB-216763 (3- (2,4-Dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione); Maybridge SEW00923SC (2-anilino-5-phenyl-1,3,4-oxadiazole); (Z) -5- (2,3-Methylenedioxyphenyl) -imidazolidine-2,4-dione; TWS 119 (3- (6- (3-aminophenyl) -7H-pyrrolo [2,3-d] pyrimidin-4-yloxy) phenol); Chir98014 (N2- (2- (4- (2,4-dichlorophenyl) -5- (1 H-imidazol-1-yl) pyrimidin-2-ylamino) ethyl) -5-nitropyridine-2,6-diamine); SB415286 (3- (3-chloro-4-hydroxyphenylamino) -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione); Tideglusib (2- (1-naphthalenyl) -4- (phenylmethyl)); And LY2090314 (3-imidazo [1,2-a] pyridin-3-yl-4- [1,2,3,4-tetrahydro-2- (1-piperidinylcarbonyl) -pyrrolo [3,2, jk] [1 , 4] benzodiazepin-7-yl]), the composition for inducing differentiation into skeletal muscle cells, characterized in that at least one selected from the group consisting of. Skeletal muscle cells comprising at least one compound selected from the group consisting of Activin A, BMP4 (Bone morphogenetic protein 4), Vascular endothelial growth factor (VEGF), GSK inhibitor (Glycogen synthase kinase inhibitor) and src tyrosine kinase inhibitor A composition for inducing maturation. Somatic or adult stems in a medium containing a pan-histone deacetylase inhibitor, an ALK5 inhibitor (activin A receptor type II-like kinase 5 inhibitor), and a cAMP signaling activator as an active ingredient A method of inducing differentiation of somatic cells or adult stem cells into skeletal muscle cells, comprising culturing the cells. The method of claim 10, The medium further comprises a GSK inhibitor (Glycogen synthase kinase inhibitor), differentiation induction into skeletal muscle cells. The method of claim 10, The somatic cells are fibroblasts, characterized in that differentiation into skeletal muscle cells. The method of claim 10, The adult stem cells, characterized in that the mesenchymal stem cells, the method of inducing differentiation into skeletal muscle cells. The method of claim 10, The culture is performed for 5 to 25 days, characterized in that the differentiation into skeletal muscle cells. The method of claim 10, The method further comprises at least one compound selected from the group consisting of Activin A, Bone morphogenetic protein 4 (BMP4), Vascular endothelial growth factor (VEGF), Glycogen synthase kinase inhibitor (GSK) inhibitor, and src tyrosine kinase inhibitor. A method for inducing differentiation into skeletal muscle cells, further comprising the step of maturing the cells induced to differentiate into skeletal muscle cells in a medium containing the active ingredient. The method of claim 15, The maturation step is characterized in that performed for 1 to 5 days, differentiation into skeletal muscle cells. Cell therapy for the treatment of skeletal muscle disease, comprising skeletal muscle cells differentiated by the method of claim 10. The method of claim 17, The skeletal muscle diseases include Becker muscular dystrophy, Congenital musculardystrophy, Duchenne muscular dystrophy, Distal musculardystrophy, Emery-Dreifuss muscular dystrophy musculardystrophy), limb-girdle musculardystrophy, myotonic musculardystrophy, ocular pharyngeal atrophy, myopathy, muscular dystrophy, muscular sclerosis, muscular sprains, and inflammatory muscle disease Cell therapy. A method for preventing or treating skeletal muscle disease, comprising administering the cell therapy agent of claim 17 to a subject. Use of skeletal muscle cells differentiated by the method of claim 10 for the manufacture of a medicament for the treatment of skeletal muscle disease.

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