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WO2018155913A1 - Procédé de différenciation en cellule de muscle squelettique à l'aide d'un composé de faible poids moléculaire - Google Patents

Procédé de différenciation en cellule de muscle squelettique à l'aide d'un composé de faible poids moléculaire Download PDF

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WO2018155913A1
WO2018155913A1 PCT/KR2018/002152 KR2018002152W WO2018155913A1 WO 2018155913 A1 WO2018155913 A1 WO 2018155913A1 KR 2018002152 W KR2018002152 W KR 2018002152W WO 2018155913 A1 WO2018155913 A1 WO 2018155913A1
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skeletal muscle
cells
differentiation
muscle cells
inhibitor
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김경규
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성균관대학교산학협력단
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Priority claimed from KR1020180020537A external-priority patent/KR102082185B1/ko
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts

Definitions

  • 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.
  • CiSMC 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.
  • muscle regeneration 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.
  • 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.
  • ESC embryonic stem cells
  • iPSC induced pluripotent stem cells
  • 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).
  • MSC mesenchymal Stem Cells
  • somatic cells differentiate somatic 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.
  • the time and efficiency is much superior.
  • 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.
  • iPSC 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cAMP cyclic AMP
  • 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.
  • 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.
  • BMP4 Bone morphogenetic protein 4
  • VEGF Vascular endothelial growth factor
  • GSK inhibitor Glycogen synthase kinase inhibitor
  • src tyrosine kinase inhibitor src tyrosine kinase inhibitor
  • 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.
  • cAMP signaling activator a signaling activator
  • 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,
  • a pan-histone deacetylase inhibitor Pan-histone deacetylase inhibitor
  • an ALK5 inhibitor activin A receptor type II-like kinase 5 inhibitor
  • cAMP signaling activator a composition for inducing differentiation of adult stem cells into skeletal muscle cells.
  • the somatic cells may be fibroblasts, and the fibroblasts may be mouse embryonic fibroblasts or mouse skin fibroblasts.
  • the adult stem cells may be mesenchymal stem cells (MSC), the mesenchymal stem cells may be adipocyte-derived stem cells (ADSC) have.
  • MSC mesenchymal stem cells
  • ADSC adipocyte-derived stem cells
  • 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.
  • the plate histone deacetylase inhibitor may be included in a concentration of 1 to 1000 ⁇ M, preferably 400 to 600 ⁇ M, more preferably 500 ⁇ M, but is not limited thereto. It is not.
  • concentration of pan-HDACi can be appropriately adjusted according to the type of pan-HDACi used and the cell to be differentiated.
  • 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
  • the ALK5 inhibitor may be included in a concentration of 1 to 100 ⁇ M, preferably 5 to 15 ⁇ M, more preferably 10 ⁇ M, but is not limited thereto.
  • concentration of the ALK5 inhibitor can be appropriately adjusted according to the type of ALK5 inhibitor used and the cells to be differentiated.
  • the cAMP signaling activator may include Forskolin, isoproterenol, NKH 477, isoprotereno (Chemical based), PACAP 1-27, or PACAP 1-38 (peptide based).
  • the cAMP signaling activator may be included in a concentration of 1 to 100 ⁇ M, preferably 40 to 60 ⁇ M, more preferably 50 ⁇ M, but is not limited thereto.
  • 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 ⁇ M ( ⁇ 10 ⁇ M) for Forskolin and 5 ⁇ M ( ⁇ 10 ⁇ M) for NKH477.
  • 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-
  • the GSK inhibitor may be included in a concentration of 0.001 to 100 ⁇ M, preferably 10 to 30 ⁇ M, more preferably 20 ⁇ M concentration, but is not limited thereto.
  • 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 ⁇ M ( ⁇ 10 ⁇ M) for Chir99021 and 10 nM ( ⁇ 10 nM) for AZD2858.
  • 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.
  • Activin A BMP4 (Bone morphogenetic protein 4)
  • VEGF Vascular endothelial growth factor
  • GSK inhibitor Glycogen synthase kinase inhibitor
  • src tyrosine kinase inhibitor src tyrosine kinase inhibitor
  • 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
  • Pan-histone deacetylase inhibitor Pan-histone deacetylase inhibitor
  • ALK5 inhibitor activin A receptor type II-like kinase 5 inhibitor
  • cAMP signaling activator 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.
  • 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).
  • GSK inhibitor Glycogen synthase kinase inhibitor
  • the somatic cells may be fibroblasts, and the fibroblasts may be mouse embryonic fibroblasts or mouse skin fibroblasts.
  • the adult stem cells may be mesenchymal stem cells (MSC), the mesenchymal stem cells may be adipocyte-derived stem cells (ADSC) have.
  • MSC mesenchymal stem cells
  • ADSC adipocyte-derived stem cells
  • 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.
  • the method may further comprise the step of maturing the cells differentiated into skeletal muscle cells by the method.
  • 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.
  • 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.
  • 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.
  • efficient skeletal muscle cells induce differentiation through inhibition of TGF-beta signaling and cAMP activation.
  • CiSMC 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.
  • 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.
  • FIG. 1 is a schematic diagram illustrating the differentiation process of cardiomyocytes and skeletal muscle cells.
  • FIG. 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.
  • 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.
  • (C) 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.
  • 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
  • (B) is a diagram quantifying MF20 positive cells
  • (C) is a flow cytometry diagram.
  • FIG. 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
  • (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.
  • FIG. 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. .
  • FIG. 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 ⁇ Actinin (low panel) in the cells after the cell maturation process by cytokines after induction of differentiation by VCRF.
  • FIG. 1 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).
  • FIG. 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.
  • FIG. 12 (A) is a diagram showing the expression of ⁇ SMA 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 ⁇ SMA 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.
  • skeletal muscle cell markers MyoD and alpha-actinin
  • 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,
  • Pan-histone deacetylase inhibitor Pan-histone deacetylase inhibitor
  • ALK5 inhibitor activin A receptor type II-like kinase 5 inhibitor
  • cAMP signaling activator cAMP signaling activator
  • composition for inducing differentiation may further include a GSK inhibitor (Glycogen synthase kinase inhibitor).
  • GSK inhibitor Glycogen synthase kinase inhibitor
  • Pan-histone deacetylase inhibitor pan-HDACi
  • pan-HDACi an inhibitor of an enzyme that removes an acetyl group from histones.
  • 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.
  • Glycogen synthase kinase inhibitor means an inhibitor that targets GSK1 / 2 involved in Wnt signaling.
  • 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-B
  • 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- ⁇ type I.
  • ALK5 activin A receptor type II-like kinase 5
  • the ALK5 is also referred to as a TGF- ⁇ type I receptor
  • the transforming growth factor- ⁇ 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 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- (pyri
  • 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.
  • 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.
  • BMP4 Bone morphogenetic protein 4
  • VEGF Vascular endothelial growth factor
  • GSK inhibitor Glycogen synthase kinase inhibitor
  • src tyrosine kinase inhibitor src tyrosine kinase inhibitor
  • 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)
  • composition for inducing maturation of skeletal muscle cells may further include one or more cytokines (cytokine) for inducing maturation of skeletal muscle cells.
  • cytokines cytokine
  • the type of starting 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.
  • 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.
  • somatic cells involved in the disease or somatic cells involved in the disease treatment can be used.
  • 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.
  • 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.
  • the medium in the case of inducing differentiation of adult stem cells into skeletal muscle cells, 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.
  • 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.
  • the differentiation induction method of the present invention 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.
  • 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.
  • 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, ⁇ -MEM ( ⁇ -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.
  • VCRF low molecular weight substances
  • 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
  • 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).
  • 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).
  • 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.
  • 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.
  • Valproic acid and sodium butyrate are 1 to 1000.
  • ⁇ M, chir99021 and AZD2858 can be added at concentrations of 0.001-100 nM, RepSox and SB431452 1-100 ⁇ M, Forskolin and NKH477 1-100 ⁇ M.
  • 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.
  • somatic cells were directly differentiated into skeletal muscle cells using only a combination of low molecular weight substances.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • markers specific for skeletal muscle cells can be detected by known biochemical or immunochemical methods, and such methods can be used without limitation.
  • 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.
  • 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.
  • physiological criteria may be additionally used.
  • 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.
  • 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.
  • 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.
  • a pharmaceutical carrier generally used for cell therapy.
  • 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).
  • 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.
  • ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mammal refers to a mammal that is the subject of treatment, observation or experiment, preferably human.
  • Mouse embryonic fibroblasts 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.
  • Mouse embryo fibroblasts 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 ⁇ -mercaptoethanol (Sigma) and 1 ⁇ penicillin / streptomycin).
  • Compound combinations of the VCRFPT or VCRF were 0.5 mM Valproic Acid (V), 10 ⁇ M CHIR99021 (C), 10 ⁇ M RepSox (R), 50 ⁇ M Forskolin (F), 5 ⁇ M Parnate (P), and 1 ⁇ M TTNPB ( T) (Medchemexpress). Fibroblasts induce differentiation for 6 days with changing medium every 3 days.
  • cytokine mixtures 25 ng / ml BMP4 (Preprotech), 10 ng / ml Activin A (R & D), 10 ng / ml VEGF (Preprotech), 10 ⁇ M CHIR99021 (Medchemexpress), 200 ⁇ g / ml Cultured in MRF containing Phospho Ascorbic Acid (Sigma), 0.15 mM Monothioglycerol (Sigma), and lx B27 supplement (Lifetechnologies).
  • cytokine mixtures 25 ng / ml BMP4 (Preprotech), 10 ng / ml Activin A (R & D), 10 ng / ml VEGF (Preprotech), 10 ⁇ M CHIR99021 (Medchemexpress), 200 ⁇ g / ml Cultured in MRF containing Phospho Ascorbic Acid (Sigma), 0.15 mM Monothioglycerol (Sigma), and lx B27 supplement (Lifetechnologies).
  • 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 ⁇ g / ml Phospho Ascorbic Acid, 10 ng / ml BMP4, 0.15 mM Monothioglycerol, 1 ⁇ B27 supplement, 10 ⁇ M CHIR99021, and 20 ng / ml FGF2).
  • 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.
  • mice 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).
  • 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.
  • 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.
  • the inducing chemical VCRFPT was treated with mouse embryonic fibroblasts for 9 days by the method of Example 1-2.
  • Fig. 1A 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).
  • muscle colonies formed by removing one or two components of the mixture at a time are quantitatively and qualitatively Evaluated.
  • Valproic acid HDAC inhibitor
  • Parnate epigenetic modulator
  • TTNPB retinoic acid receptor agonist
  • 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).
  • 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).
  • Example 3 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.
  • MyoD and Myogenin skeletal muscle cell specific markers
  • Mck and Myomaker mature muscle cell specific markers
  • 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
  • mRNA expression of the fibroblast direct differentiation step was analyzed for VCRF or VCRFPT treatment to confirm the effects of cytokines at each step.
  • 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).
  • Example 6 VCRF Induced by myocytes or of myotubes Identify the optimal compound or mixture needed for maturation
  • Example 3 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.
  • VEGF Vascular endothelial growth factor
  • PP1 src tyrosine kinase inhibitor
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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 (10 ⁇ M) was used in place of RepSox instead of RepSox, and NKH477 (5 ⁇ M) instead of Forskolin was used in place of ALK5 inhibitor.
  • NKH477 5 ⁇ M
  • Forskolin was used in place of ALK5 inhibitor.
  • sodium butyrate instead of valproic acid (500 ⁇ M) was used, and AZD2858 (10nM) was used instead of Chir99021 as a compound that performs the GSK inhibitor function.
  • 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.
  • 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.
  • 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.
  • human adipose-derived stem cells humanadipose-derived stem cells
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne une composition permettant d'induire la différenciation de cellules somatiques ou de cellules souches adultes en cellules de muscle squelettique. Par l'intermédiaire d'un procédé simple dans lequel des cellules sont cultivées dans un milieu de culture contenant la composition induisant la différenciation de la présente invention, des cellules somatiques peuvent être directement différenciées en cellules de muscle squelettique. La différenciation en cellules de muscle squelettique présente un taux de différenciation et une efficacité élevés et est économiquement avantageuse en termes de coût. De plus, les cellules de muscle squelettique obtenues ne présentent aucun risque d'oncogenèse après implantation du fait de la différenciation à partir de cellules somatiques, et présentent l'avantage d'être génétiquement stables en raison de l'utilisation d'un mélange de composés de faible poids moléculaire seuls, sans introduction de génétique. Selon la présente invention, la composition s'est également avérée induire rapidement et efficacement la différenciation de cellules souches mésenchymateuses en cellules de muscle squelettique. En particulier, il a été observé que l'absence d'inhibiteur de GSK dans la composition provoquait un effet positif sur l'efficacité de la différenciation. Apte à induire une différenciation en de nombreuses cellules de muscle squelettique en un court laps de temps, la présente invention est alors censée trouver des applications utiles dans le développement de produits de thérapie cellulaire économiques et efficaces pour le traitement de maladies du muscle squelettique.
PCT/KR2018/002152 2017-02-21 2018-02-21 Procédé de différenciation en cellule de muscle squelettique à l'aide d'un composé de faible poids moléculaire WO2018155913A1 (fr)

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KR10-2017-0022896 2017-02-21
KR20170022896 2017-02-21
KR1020180020537A KR102082185B1 (ko) 2017-02-21 2018-02-21 저분자 화합물을 이용한 골격근육세포 분화 방법
KR10-2018-0020537 2018-02-21

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CN114732809A (zh) * 2022-06-09 2022-07-12 广东省科学院微生物研究所(广东省微生物分析检测中心) 丁酸盐在制备缓解骨骼肌衰老的药物中的应用
WO2023012264A2 (fr) 2021-08-04 2023-02-09 Association Francaise Contre Les Myopathies Activateur pharmacologique et/ou genetique pour son utilisation pour preserver et regenerer la structure et la fonction musculaire en bloquant la senescence

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023012264A2 (fr) 2021-08-04 2023-02-09 Association Francaise Contre Les Myopathies Activateur pharmacologique et/ou genetique pour son utilisation pour preserver et regenerer la structure et la fonction musculaire en bloquant la senescence
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CN114732809A (zh) * 2022-06-09 2022-07-12 广东省科学院微生物研究所(广东省微生物分析检测中心) 丁酸盐在制备缓解骨骼肌衰老的药物中的应用

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