KR101636533B1 - Composition for inducing dedifferentiation of somatic cell into muscle stem cell and method for preparing muscle stem cell using the same - Google Patents

Abstract

The present invention relates to a composition for inducing de-differentiation from somatic cells to muscle stem cells, and a method for producing muscle stem cells using the same, and the present invention can produce muscle stem cell-like cells by a simple method.

Description

TECHNICAL FIELD The present invention relates to a composition for inducing the differentiation of somatic cells into muscle stem cells and a method for preparing muscle stem cells using the same.

The present invention relates to a composition for inducing de-differentiation from somatic cells to muscle stem cells, and a method for producing muscle stem cells using the same.

Stem cells are the cells that can be the basis for all cells and tissues. Thus, the technique of proliferating stem cells through cultivation and then differentiating them into specific cells has the potential to treat various diseases in terms of cell therapy. Stem cells are divided into pluripotency, multipotency or unipotency according to their differentiation ability. The types of stem cells are embryonic stem cells (inner cells of pre-implantation embryo), adult stem cells And pluripotent stem cells (degenerated stem cells: cells in which somatic cells are re-induced by inserting genes and proteins). Embryonic stem cells have unlimited potential to differentiate into tissues that constitute 210 organs in the body through proper stimulation, but it is difficult to control the differentiation with the bioethical problem of cell extraction, cancer cellization such as teratoma, And it is known that there are many difficulties in application to clinical trials. Because of this difficulty, inducible pluripotent stem cells (iPSCs), which have been induced to artificially deplete the differentiated cells to have pluripotent, have attracted much attention. However, induction of pluripotent stem cells is not easy to regulate, and it has been reported that cancer cell metaplasia may occur. Adult stem cells can be extracted from the patients themselves, and the problem of immune rejection is excluded, making them the most commonly used treatment for diseases. However, it is theoretically infinitely proliferating adult stem cells, but it is actually difficult to proliferate adult stem cells on in vitro, and it is not easy to separate large amounts of cells from patients.

On the other hand, the separation of muscle stem cells from muscle to obtain muscle stem cells also has various obstacles. First, the amount of tissue that can be secured is unreasonably low, and thus the efficiency of stem cell isolation deteriorates. Moreover, since the development of a culture solution capable of maintaining the proliferation state on Invitro is at a novice level, the differentiation can proceed in a certain amount of time in the culture solution have. In order to overcome these drawbacks, muscle stem cells which can differentiate into muscle in differentiation medium are produced by expressing the central transcription factors of muscle stem cells in degenerated stem cells. These muscle stem cells are first made by forming degenerative stem cells, then by expressing the muscle-related genes, and then differentiating stem cells into muscle. The muscle-related gene expression is induced by the injection of tetracycline (Tet-On System) in which the stem cells are transformed into muscle stem cells. However, the method of manufacturing the above-mentioned muscle stem cells has a disadvantage in that if the tetracycline is not continuously injected, the muscle stem cells may have a risk of returning to the degenerated stem cells again. After producing the degenerated stem cells, Lt; RTI ID = 0.0 > and / or < / RTI > differentiating it into separate steps.

Therefore, the present inventors have made intensive efforts to solve these drawbacks, and as a result, it has been found that when YAMANAKA factor and a gene that plays a pivotal role in muscle differentiation are overexpressed in somatic cells, direct mutagenesis It is possible to form muscle stem cells, thus completing the present invention.

It is an object of the present invention to provide a composition for inducing muscle stem cell de-differentiation from somatic cells.

Another object of the present invention is to provide a method for producing muscle stem cells.

It is still another object of the present invention to provide muscle stem cell-like cells produced by the above method.

One aspect of the present invention relates to a method of expressing a protein selected from the group consisting of Paired box 3 (Pax3), Esrrb (estrogen related receptor beta) and reprogramming-inducing genes or polynucleotides encoding the same, A composition for inducing the differentiation is provided.

The term " de-differentiation "in the present invention means a process by which differentiated cells can be restored to a state having a new type of differentiation potential. In addition, the de-differentiation can be used in the same sense as the cell reprogramming.

The term " dedifferentiation inducing factor "is a substance that can induce finally differentiated cells to be differentiated into induced pluripotent stem cells having a potential of a new type of differentiation, and can be named as reprogramming inducers. The dedifferentiation inducing factor may include any substance that induces the differentiation of differentiated cells.

Specifically, the dedifferentiation inducing factor may be any one selected from the group consisting of Oct4, Sox2, KlF4, c-Myc, Nanog and Lin-28. And may be two or more, preferably three or more, selected from the group consisting of degenerated Oct4, Sox2, Klf4, c-Myc, Nanog and Lin-28. More preferably, the de-differentiation inducing factor may be Sox2, Klf4, and c-Myc. In one embodiment of the present invention, when Sox2, Klf4, and c-Myc were introduced into somatic cells as de-differentiation factors, it was confirmed that somatic cells were differentiated into muscle stem cells.

The term "somatic cell" in the present invention means a cell constituting an adult with limited ability to differentiate and regenerate. Specifically, the somatic cells may be somatic cells constituting skin, hair, fat, blood, etc. of an individual, and may be fibroblasts, but are not limited thereto. The somatic cell may be a fibroblast of the embryonic period.

The somatic cells may be naturally occurring somatic cells or genetically modified somatic cells. The somatic cell may be derived from an animal, including a human, and preferably also from a human or a mouse.

The term "muscle stem cell (MSC) " in the present invention refers to a cell having the characteristics, including transformation-free proliferation, infinite proliferation, or self-renewal ability, and ability to differentiate into muscle , Self-renewal ability or infinite proliferative ability, and muscle differentiation ability. The self-reproducing ability and muscle differentiation ability can be confirmed as markers. In addition, the muscle stem cells may express Pax7 or Myf5 while exhibiting specific self-renewal ability. In one embodiment of the present invention, it has been confirmed that Myf5, which is a specific expression marker of muscle stem cells, is expressed in the cells in which the differentiation is induced by the composition.

The composition of the present invention comprises a Pax3 protein or a polynucleotide encoding it, an Esrrb protein or a polynucleotide encoding the same, and a polynucleotide encoding a de-differentiation-inducing factor protein. The Pax3, Esrrb and dedifferentiation inducing factors may be human or mouse-derived. In addition, each protein of Pax3, Esrrb and dedifferentiation factor may be a protein having a wild type amino acid sequence thereof. Each of the polynucleotides encoding each protein of Pax3, Esrrb and dedifferentiation inducing factor may be a sequence encoding a wild-type protein, and one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof . The polynucleotides may be isolated from nature or may be prepared by chemical synthesis. The polynucleotide having the nucleotide sequence coding for Pax3, Esrrb, and the differentiation inducing factor protein may be a single-stranded or double-stranded DNA molecule (genome, cDNA) or an RNA molecule.

Methods for delivering or introducing Pax3, Esrrb and a polynucleotide encoding a dedifferentiation inducer protein or a protein thereof into a somatic cell can be used without limitation as a method for providing a nucleic acid molecule or protein to a cell commonly used in the art, Preferably, the active ingredients contained in the composition of the present invention may be administered to a culture medium of somatic cells or injected directly into somatic cells.

The Pax3, Esrrb, and de-differentiation inducing factors may be directly injected into the differentiated cells by any method known in the art. For example, microinjection, electroporation electroporation, particle bombardment, direct muscle injection, insulator, and transposon.

Polynucleotides encoding the Pax3, Esrrb and dedifferentiation inducing factor proteins can be introduced into cells with naked DNA in the form of a vector (Wolff et al. Science 1990: Wolff et al. J Cell Sci. 103: 1249-59 ), Liposome (liposome), cationic polymer, and the like. Liposomes are phospholipid membranes prepared by mixing cationic phospholipids such as DOTMA and DOTAP for gene introduction. When a cationic liposome and an anionic nucleic acid are mixed at a certain ratio, a nucleic acid-liposome complex is formed. Preferably, the polynucleotide may be provided in a vector form. The polynucleotides may be obtained from viruses obtained from packaging cells transformed with viral vectors inserted with respective polynucleotides, messenger RNAs produced by in vitro transcription, or proteins produced in various cell lines. Can be used. The packaging cell may be selected from a variety of cells known in the art depending on the viral vector used.

As used herein, the term "vector" refers to an expression vector capable of expressing a desired protein in a suitable host cell, including a necessary regulatory element operably linked to the expression of the gene insert. The vector of the present invention may include an expression regulatory element such as a promoter, an initiation codon, a stop codon, a polyadenylation signal, and the like. In addition, the expression vector may include a selectable marker for selecting a host cell containing the vector, and may contain a replication origin if the expression vector is a replicable expression vector. The vector may be self-replicating or integrated into the host DNA.

The vector includes a plasmid vector, a viral vector, and the like. Preferably, it is a viral vector. The viral vectors may be selected from the group consisting of lentiviruses, retroviruses, for example, HIV (human immunodeficiency virus), Murineleukemia virus (MLV) ASLV (Avian sarcoma / leukosis), Spleen necrosis virus (SNV), Rous sarcoma virus but are not limited to, vectors derived from adenoviruses, adeno-associated viruses, herpes simplex viruses, Sendai viruses and the like. Preferably, a lentiviral vector can be used. In one embodiment, pJM and FUW vectors containing selectable markers for furomycin as lentiviral vectors for the Pax3, Sox2, Klf4, c-Myc, and Esrrb genes were used.

The Pax3, Esrrb and dedifferentiation inducing factors contained in the composition of the present invention can be introduced into somatic cells by different vectors, some of them can be introduced by the same vector, and Pax3, Both Esrrb and dedifferentiation inducers can be introduced into somatic cells.

In one embodiment, a pJM-Pax3 vector was prepared by inserting the nucleotide sequence - (NCBI accession No. NT_039173) encoding the Pax3 protein into the pJM vector and the three nucleotide sequences encoding the Sox2, Klf4, and c-Myc proteins (FUW-SKM) was purchased from Addgene (Plasmid # 20325) and Esrrb was inserted into the FUW vector (FUW-Esrrb; Addgene Plasmid # 40798) A 293FT cell, which is a packaging cell that produces a high-frequency virus capable of infecting cells, is finally transformed into a virus expressing Pax3 protein, a virus expressing Sox2, Klf4, c-Myc protein and a virus expressing Esrrb protein And infected fibroblasts with these viruses.

Another aspect of the present invention is a method for producing a recombinant vector comprising the steps of: introducing Pax3, Esrrb and a dedifferentiation inducer protein or a polynucleotide encoding the same into somatic cells; And culturing the somatic cells into which the protein or polynucleotide has been introduced in a growth medium of muscle stem cells.

In the method for producing muscle stem cells from somatic cells of the present invention, the de-differentiation-inducing factor may be at least one selected from the group consisting of Oct4, Sox2, Klf4, c-Myc, Nanog and Lin-28 have. And may be two or more, preferably three or more, selected from the group consisting of degenerated Oct4, Sox2, Klf4, c-Myc, Nanog and Lin-28. More preferably, the de-differentiation inducing factor may be Sox2, Klf4 and c-Myc.

The somatic cells are as described above. The somatic cells may also be preferably fibroblasts.

And culturing the somatic cells in a medium before the step of introducing Pax3, Esrrb and a dediffering inducer protein or a polynucleotide encoding the same into the somatic cells. The culture medium for culturing the somatic cells includes all media conventionally used in somatic cell culture in the art. The medium used for the culture generally includes a carbon source, a nitrogen source and a trace element component. In a specific embodiment of the present invention, fibroblasts are cultured in DMEM (high glucose, w / o sodium pyruvate) + 10% FBS (fetal bovine serum) + 0.1 mM non-essential amino acids + 1% penicillin / streptomycin + 0.1 mM Lt; / RTI > ethanol.

The introduction of the protein and the polynucleotide into somatic cells is as described above. Specifically, a virus comprising the polynucleotide may be used. The virus may preferably be lentivirus.

The method for producing a muscle stem cell according to the present invention is characterized in that somatic cells into which the protein or polynucleotide has been introduced are introduced into the growth medium of muscle stem cells by introducing the proteins of Pax3, Esrrb and a differentiation inducing factor or a polynucleotide encoding these proteins into somatic cells, Lt; / RTI >

The muscle stem cell growth medium includes, but is not limited to, any medium conventionally used for muscle stem cell culture or proliferation. Specifically, the growth medium of the muscle stem cells may include FBS and 6 to 15% HS (horse serum). In one embodiment, DMEM (high glucose) + 1% penicillin / streptomycin + 5 ng / ml bFGF + 10% FBS + 10% HS (horse serum) was used. When cultured under the above culture conditions, cells such as muscle stem cells can be formed.

The step of culturing the somatic cells in the growth medium of muscle stem cells may be a change in cell shape and culturing until the changed cells are up to 70% in the culture container. The cultured cells can show favorable expression of the major gene (Myf5) expressed in muscle stem cells.

The method for producing muscle stem cells from the somatic cells of the present invention may further include the step of culturing the cultured muscle stem cells into single cells and culturing the cells in a muscle stem cell growth medium. The step of isolating the cultured muscle stem cells usually involves separating into single cells using a fluorescence activated cell sorter (FACS). In one embodiment, the cells having a change in cell morphology are treated with trypsin to separate the cells from each other into single cells. Then, single cells are separated from each other in a 96-well culture container so that one cell can exist per well. Respectively. By separating and culturing the cells into single cells, the muscle stem cells can have a certain stage and shape.

The method for producing muscle stem cells from the somatic cells of the present invention is a direct cross-differentiation method in which the stem cells are directly differentiated from the somatic cells into the stem cells without passing through the inducible pluripotent stem cells. vitro), the ability to differentiate into muscle is very similar to that of muscle stem cells known to those skilled in the art.

Another aspect of the present invention provides muscle stem cells prepared by the method for producing muscle stem cells from somatic cells according to the present invention.

The method for producing muscle stem cells from the somatic cells is as described above.

The muscle stem cells prepared in the present invention show self-proliferation in the culture solution and show differentiation ability to differentiate into muscle in the differentiation solution.

The muscle stem cells may express Myf5 or MyoD, for example, muscle stem cell growth markers. In one embodiment, when the cultured muscle stem cells continue to be cultured in the growth medium, the expression level of Myf5 is high, but when cultured in the differentiation medium, the expression level of Myf5 tends to decrease. In the case of MyoD, the expression level was similar to that of muscle cells in the differentiation medium. In one embodiment, when the muscle stem cells were completely differentiated, it was observed that myogenin and MHC were expressed.

According to one embodiment of the present invention, it is possible to reverse-differentiate into stem cells directly from somatic cells without going through the process of induced pluripotent stem cells, thereby shortening the culturing time, cost reduction, and work effort. In addition, the muscle stem cells prepared according to one embodiment are extremely useful because they can avoid ethical problems using human embryos or rejection after transplantation. Therefore, the present invention can be efficiently used for the production of muscle stem cells, and the produced muscle stem cells can be utilized as a system for evaluating the drug efficacy or toxicity of a cell therapeutic agent, a compound, a drug, and a toxin.

FIG. 1A shows results obtained by introducing Pax3, Sox2, Klf4, c-Myc, and Esrrb genes into mouse somatic cells and culturing them in the culture conditions of muscle stem cells. The cells thus formed were named iMLC (induced Myoblast like cell) (MEF: mouse embryonic fibroblast).
FIG. 1B shows the results of PCR to confirm the expression of muscle marker genes (Pax3, Pax7, MyoD and Myf5) in the transformed cells (MEF: mouse embryonic fibroblast, C2C12: myoblast cell line)
FIG. 1C shows a method of isolating single cells using FACS from iMLC cells labeled with SSEA-3, subculturing them in 96-well, 24-well, and 6-well culture dishes (96W, 24W, 6W, respectively) (M: MEF; C: C2C12; 1A12: Single cell in column A, row 12 of the 96 well culture vessel, column A, row 9, column A of the 96 well culture vessel, Single cell).
Fig. 2 shows the results of induction of differentiation into single muscle cells expressing Myf5. FIG. 2A is a result of observation of root canal formation at 40x and 200x, and FIG. 2b is a photograph showing fluorescence staining of myogenin and myotube, a muscle differentiation marker, with troponin.
Figure 3a shows the results of RT-PCR confirmed that muscle stem cell growth and differentiation markers (Myf5, MyoD, myogenin, and MHC (myosin heavy chain)) are expressed at the RNA level in the cells in which muscle differentiation is observed .
FIG. 3B shows the result of fluorescence staining that the muscle stem cell growth and differentiation markers (MyoD, myogenin, and MHC (myosin heavy chain)) are expressed at the protein level in the cells in which muscle differentiation is observed.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example  1. In somatic cells Pax3 , Sox2 , Klf4 , c- Myc , And Esrrb  Gene introduction

1.1. Preparation of mouse fibroblasts for gene transfer

Mouse fibroblasts (MEF) were used as somatic cells for the introduction of Pax3, Sox2, Klf4, c-Myc, and Esrrb genes. Mouse fibroblasts were obtained from an embryo at C56 / B6 strain mice (C56 / B6 strain mice) at 13.5 days of gestation. Specifically, the embryo tissue from which bones and organs were removed was finely cut with scissors and suspended in PBS. Then, the cells were filtered through a suspended cell network to remove cell debris and cells were collected by centrifugation. To the collected cells were added DMEM (high glucose, w / o sodium pyruvate) + 10% FBS (Fetal bovine serum) + 0.1 mM non-essential amino acid + 1% penicillin / streptomycin + 0.1 mM- Ethanol) medium was added, and the cells were suspended and cultured in a tissue culture flask. At the 2nd passage, 2 x 10 ⁵ cells were inoculated into 6 well culture dishes for gene transfer.

1.2. Introduction of Pax3, Sox2, Klf4, c-Myc, and Esrrb genes in mouse fibroblasts

Lentivirus was used to introduce the gene. For this, virus particles were prepared using a 293FT cell line. Specifically, pJM puro Pax3 prepared by inserting the mouse Pax3 (NCBI accession No. NT_039173) gene into the pJM vector and FUW-SKM (Sox2 + Klf4) prepared by removing Oct4 from the FUW-Sox2 + Oct4 + Klf4 + c- + c-Myc) and FUW-Esrrb vector plasmids were separated and mixed in a 1: 3 ratio with a packaging mix (Invitrogen). The NCBI accession numbers of the respective Sox2, Klf4, c-Myc, and Esrrb gene sequences contained in the vector are as follows: Esrrb: NM011934; Sox2: NM 011443; [0228] Klf4: NM 010637; And c-Myc: NM 010849. The cells were then transfected into 293FT cell lines (Clontech) using lipofectamine 2000 (Invitrogen). After 24 hours, the medium was changed and high-frequency virus was generated at 48 hours and 96 hours. The supernatant was filtered with a 0.45 filter (Millipore) to remove cell debris, and the titer was confirmed using a kit (lenti-x go stix, Clontech) that confirmed the virus titer. A polybrene (6 / ml) (Sigma) was added to the virus supernatant with high titer to sequentially infect mouse fibroblasts inoculated at 6 wells with a virus containing Pax3, Sox3 + Klf4 + c-Myc Virus and Esrrb. ≪ / RTI >

Example 2. Derivation of somatic cells into which Pax3, Sox2, Klf4, c-Myc, and Esrrb genes were introduced into muscle stem cells

2.1. Culture of transgenic fibroblasts

10% FBS + 10% HS) in the culture medium (DMEM + 1% penicillin / streptomycin + 5 ng / ml bFGF + 10% FBS) supplemented with fibroblasts of mice in which Pax3, Sox2, Klf4, c- Lt; / RTI > As a result, it was confirmed that cells with large and small nuclei appeared to grow together (FIG. 1A). On the other hand, it was confirmed that the surrounding somatic cells die due to the increase of cytoplasm. When smaller cells were cultured, mouse somatic cells disappeared and only small nuclei remained in the culture vessel. The cells were subcultured while being transferred to 6 wells of 60 mm and 100 mm containers to obtain a sufficient number of cells.

2.2 Expression of muscle-related genes

Fig. 1B shows the results of RNA isolation of the transformed cells with Invitrogen and 0.5-mg RNA synthesis of the cDNA at 37 for 1 hour with MMLV reverse transcriptase (Invitrogen). PCR primers (1 ml), cDNA (1 ml) and H ₂ O (18 ml) were mixed with each other using PCR premix (Bioneer) to confirm the expression of muscle-related genes. PCR was performed for 30-35 cycles under the conditions of 95 30 seconds, 55-60 30 seconds, and 72 30 seconds. The PCR result was confirmed by electrophoresis on 0.8-1% agarose gel. Mouse fibroblast cDNA was used as a negative control and C2C12 (muscle cell line; ATCC) cDNA was used as a positive control. As a result, it was confirmed that Myf5, which is a muscle cell marker, was expressed, but the expression of other markers was observed to be insignificant.

2.3. Single cell separation and culture

Since these cells are not cells with a certain stage and shape, the muscle stem cell-like cell line was isolated from these cells (Fig. 1C). Specifically, the cells were first treated with trypsin and cultured at 37 for 2 minutes to allow the cells to fall into single cells. The growth medium was added thereto, and the cells were floated so that the single cells were observed under a microscope. Then, the growth medium was removed by centrifugation, and the cells were washed once with PBS. Thereafter, 1 ml of SSEA-3 antibody to which FITC was added per million cells was added, reacted for 30 minutes, centrifuged at 4, and cells were washed 3 times with PBS. Cells were supplemented with 2% FBS solution and then applied to FACS. Cells labeled with SSEA-3 antibody were shifted to the right as a whole compared to the unlabeled control (Cont), and a total of 20% of the cells were labeled with SSEA-3 and separated into FACS. The FACS menu was used for single cell separation, and the separated cells were set so that one cell was dropped in one well of a 96-well culture container. Cells transferred to a 96 well culture vessel were cultured in muscle stem cell growth medium. After 10 days, each well of a 96-well culture vessel was observed under a microscope to observe the cells. Each cell becomes a cell line derived from one cell. The cells were transferred to 24 wells, 12 wells, and 6 wells, and partially stored for storage in liquid nitrogen. For the analysis, the expression of the marker gene Myf5 was confirmed by RT-PCR. At this time, mouse fibroblast (MEF; M) was used as a negative control and muscle cells (C2C12; C) were used as a positive control. As a result, it was confirmed that Myf5 was expressed in almost all single cells (Fig. 1C).

Example  3. Myf5 Induction of differentiation into single muscle cells

Single cell-derived cells expressing Myf5 were transferred to a 6-well culture container and cultured in a growth medium to a cell density of 70%. Cells were then cultured in muscle differentiation medium (DMEM + 2% HS + 1% penicillin / streptomycin) to induce differentiation into muscle. On the eighth day of muscle differentiation culture, the formation of myotube, which is observed when myofiber is formed in muscle stem cells, is observed (Fig. 2a). When the differentiated cells were stained with myogenin and troponin, multinuclei and root canal formation were observed due to fusion of myocytes when the root canal was formed. In the case of polynuclear cells, myogenin, a transcription factor of muscle differentiation, And the expression of troponin expressed in the cytoplasm of the root canal was also confirmed, indicating that the isolated single cell was a muscle cell (Fig. 2B). In addition, these single cells continued to proliferate without changes in morphology until passage 30, and proliferation was strong enough to subculture once a day, indicating the nature of stem cells.

Example  4. Muscle Stem Cells in Muscle Differentiated Cell Lines Markers  Check for expression

We examined the expression of muscle stem cell markers (Myf5, MyoD, Myogenin, and MHC) related to proliferation and differentiation in single cells with observed muscle differentiation at RNA level and protein level. First, with regard to identification of growth markers, isolated single cells were grown in growth medium (muscle stem cell culture medium) for 2 days and then used in experiments. For experiments related to identification of differentiation markers, isolated single cells were grown in growth medium 70%, then replaced with the muscle differentiation medium, and the differentiation was induced by changing the medium every day until the root canal was observed (10 days).

FIG. 3A shows the results of confirming the expression at the RNA level of the markers involved in growth and differentiation after culturing cells in which muscle differentiation was observed to be transferred to 6 wells. Specifically, RNA was isolated from cells and cDNA was synthesized and analyzed using PCR. As a result, Myf5 expression was high in the growth medium and slightly decreased in the differentiation medium. MyoD, which plays a central role in differentiation, showed an increase in expression of C2C12 in the differentiation medium. MHC expression was observed only in the differentiation medium when myogenin and root canal were formed when the muscle stem cells completely entered into differentiation.

FIG. 3B shows the results of confirming the expression of the markers involved in growth and differentiation at the protein level by transferring the cells in which muscle differentiation was observed to a chamber slide. After 10 days of culture in the differentiation medium, the medium was removed and washed briefly with PBS. Cells were fixed with 4% PFA, reacted with 0.1% Triton X-100, and reacted with blocking solution for 1 hour. After that, MyoD, myogenin and MHC antibody were added and reacted for 4 to 12 hours. After washing with PBS, secondary antibody with FITC and TRITC was added and allowed to react at room temperature for 2 hours. The cells were stained with DAPI and stained with a confocal microscope. As a result, it was confirmed that the muscle stem cell growth and differentiation markers (MyoD, Myogenin, MHC: myosin heavy chain) were expressed at the protein level in the cell line of the present invention.

Claims (13)

A composition for direct crossover differentiation from somatic cells to muscle stem cells, comprising Pax3, Esrrb, and a reprogramming-inducing genes protein or a polynucleotide encoding the same, wherein the dedifferentiation inducing factor is Sox2, Klf4, c-Myc. < / RTI > delete delete The composition according to claim 1, wherein said somatic cells are derived from human or mouse. The composition according to claim 1, wherein the somatic cell is a fibroblast. Introducing Pax3, Esrrb and a dedifferentiation inducer protein or a polynucleotide encoding the same into somatic cells; And
And culturing the somatic cells into which the protein or polynucleotide has been introduced in a growth medium of muscle stem cells, wherein the de-differentiation inducing factor is Sox2, Klf4 and c-Myc A method for producing muscle stem cells from somatic cells. delete 7. The method according to claim 6, wherein the introduction of the polynucleotide utilizes a virus comprising a vector comprising the polynucleotide. 9. The method of claim 8, wherein the virus is a retrovirus. The method according to claim 6, wherein the culture medium of muscle stem cells is a medium containing bFGF and HS (horse serum). [Claim 7] The method according to claim 6, further comprising separating the cultured muscle stem cells into single cells and culturing the cells in a muscle stem cell growth medium. delete delete

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