Culture medium and method for efficiently inducing muscle stem cells

Abstract

A method for efficiently inducing muscle stem cells to obtain muscle fibers or skeletal muscle. The method comprises: firstly, using a suspension culture and three types of muscle stem cell induction culture mediums to carry out step-by-step induction; then using a culture medium containing bovine serum and a basic fibroblast growth factor BFGF for amplification; then using differentiation culture medium containing specific chemical small molecules for inducing differentiation. The method can be used for rapidly inducing pluripotent stem cells to obtain muscle stem cells and mature muscle fibers or skeletal muscle, and one process can obtain a significant amount of muscle stem cells for treatment and disease model simulation, and thus solving the problem that human muscle stem cells are difficult to obtain and amplify.

Description

A culture medium and method for efficiently inducing muscle stem cells Technical Field

[0001] The present invention relates to the technical field of pluripotent stem cell induction, and in particular to a culture medium and method for efficiently inducing muscle stem cells to obtain muscle fibers or skeletal muscles. Background Art

[0002] Muscles are the most abundant component of the human body and are crucial for maintaining movement and health. Under normal circumstances, when muscles are damaged, quiescent muscle stem cells located between the basement membrane and plasma membrane of muscle fibers are activated, proliferating and differentiating to produce new muscle fibers, thereby repairing the damaged muscles. Although adult muscle stem cells possess significant regenerative potential, progressive muscle necrosis caused by age-related muscle atrophy and diseases such as Duchenne muscular dystrophy often leads to a decline or complete loss of these stem cells' regenerative and repair abilities, ultimately leading to paralysis or even death.

[0003] In recent years, stem cell technology has brought great hope for the treatment of aging or muscle diseases such as DMD. However, a major challenge is still the difficulty in isolating, culturing in vitro, and expanding muscle stem cells in large quantities, resulting in an insufficient supply of muscle stem cells and severely limiting the clinical application of stem cell therapy.

[0004] Summary of the Invention

[0005] In response to the technical problems existing in the prior art, one of the objectives of the present invention is to provide a method for efficiently inducing muscle stem cells. This method can obtain a considerable amount of muscle stem cells at one time for treatment and simulation of disease models, which effectively solves the problem of difficulty in obtaining and amplifying human muscle stem cells.

[0006] The present invention adopts the following technical solution to achieve the above technical objectives: The present invention provides a method for efficiently inducing muscle stem cells, comprising the following steps:

[0007] After expanding the human pluripotent stem cells to the point where they can be induced to differentiate, the cells are digested with a cell digestion buffer until the cell confluency reaches 60-80%, and then placed on a low-adhesion plate and suspended in a first muscle stem cell induction medium for one day; the first muscle stem cell induction medium comprises stem cell basal medium and 5-10 μM ROCK inhibitor Y-27632;

[0008] On the third day, the culture medium is replaced with a second muscle stem cell induction medium and cultured for 5 to 7 days to obtain muscle stem cell spheres; wherein the second muscle stem cell induction medium includes stem cell basal medium and 10 to 15 μM glycogen synthase kinase-3 inhibitor CHIR99021;

[0009] The muscle stem cell spheres are digested with cell digestion solution and then centrifuged and transferred to adherent culture. A third muscle stem cell induction medium is added to a well plate coated with a collagen solution and cultured for one day. The culture medium is then changed to a muscle stem cell proliferation medium and cultured for 2 to 5 generations. The third muscle stem cell induction medium comprises a stem cell basal medium, 10 to 15 μM glycogen synthase kinase-3 inhibitor CHIR99021, and 5 to 10 μM ROCK inhibitor Y-27632. The muscle stem cell proliferation medium comprises a cell growth basal medium, bovine serum, and basic fibroblast growth factor (BFGF), wherein the volume concentration of the bovine serum is 10 to 20%, and the concentration of the basic fibroblast growth factor (BFGF) is 2 to 5 ng / mL.

[0010] After the expansion of the muscle stem cells, the original muscle stem cell proliferation medium is discarded, and a muscle stem cell differentiation medium is added to induce differentiation for 6 to 8 days to obtain mature muscle fibers; wherein, the muscle stem cell differentiation medium includes mammalian cell growth basal medium, horse serum, tyrosine kinase receptor A agonist and / or prostaglandin synthase inhibitor, the tyrosine kinase receptor A agonist is amitriptyline, the concentration is 1 to 5 μM, the prostaglandin synthase inhibitor is benzyldamine, the concentration is 1 to 5 μM, and the volume proportion of horse serum is 2 to 5%.

[0011] The method provided by the present invention obtains muscle stem cell spheres with higher purity through suspension culture when obtaining mature muscle fibers. After digesting the muscle stem cell spheres into single cells, they are rapidly expanded on a 2D level. During the differentiation of muscle stem cells into mature muscle fibers, they are cultured in a differentiation medium containing specific small molecule inhibitors. This can quickly induce pluripotent stem cells to obtain muscle stem cells and mature muscle fibers or skeletal muscles, and a considerable amount of muscle stem cells can be obtained at one time for treatment and disease model simulation, which can effectively solve the problem of difficulty in obtaining and expanding human muscle stem cells.

[0012] As a preferred embodiment, the basal culture medium in the muscle stem cell differentiation medium is DMEM medium; the basal culture medium in the first muscle stem cell induction medium is mTeSR1 medium, and the basal culture medium in the second and third muscle stem cell induction medium is E6 medium; the basal culture medium in the muscle stem cell proliferation medium is F10 medium.

[0013] As a preferred embodiment, the collagen solution is prepared by adding acetic acid solution to collagen I rat tail solution.

[0014] As a preferred embodiment, the method for expanding human pluripotent stem cells to the point where they can be induced to differentiate is as follows: growing and maintaining the human pluripotent stem cells on a Matrigel-coated plate in mTeSR1 culture medium.

[0015] As a preferred embodiment, human pluripotent stem cells are obtained by reprogramming human blood monocytes using Episome reprogramming plasmids.

[0016] A second object of the present invention is to provide a culture medium for efficiently inducing muscle stem cells, including the above-mentioned muscle stem cell differentiation culture medium.

[0017] As a preferred embodiment, it also includes the above-mentioned first muscle stem cell induction medium, second muscle stem cell induction medium, third muscle stem cell induction medium and muscle stem cell proliferation medium.

[0018] A third object of the present invention is to provide a kit for efficiently inducing muscle stem cells, comprising the above-mentioned culture medium.

[0019] The method for efficiently inducing muscle stem cells provided by the present invention can obtain a considerable amount of muscle stem cells at one time for treatment and simulation of disease models, solving the problems of difficulty in obtaining and amplifying human muscle stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 shows the expression level of the MF20 marker in the muscle fibers obtained after 7 days of induction differentiation in the Examples and Comparative Examples;

[0021] FIG2 shows the expression level of Myogenin marker in muscle fibers obtained after 7 days of induction of differentiation in Examples and Comparative Examples;

[0022] FIG3 shows the expression level of the Dystrophin marker in the muscle fibers obtained after 7 days of induction and differentiation in the Examples and Comparative Examples;

[0023] FIG4 shows the expression levels of PAX7 and PAX3 markers in muscle stem cells obtained after induced differentiation in Example 1 and Comparative Example 7;

[0024] Figure 5 shows the microscopic observation results of cells in Example 1 of the present invention at different stages, wherein a is the microscopic observation result of human peripheral blood mononuclear cells before reprogramming, b is the microscopic observation result of induced pluripotent stem cells cultured under a wall after 10 passages after reprogramming, c is the microscopic observation result when induced pluripotent stem cells were switched to suspension culture, d is the observation result of induced muscle stem cell suspension spheres, e is the microscopic observation result of induced muscle stem cell suspension spheres after digestion and adherence culture for 1 day, and f is the microscopic observation result of muscle fibers obtained after differentiation. DETAILED DESCRIPTION

[0025] Below in conjunction with specific embodiment, the present invention is described in further detail so that those skilled in the art can understand the present invention more clearly.The following embodiments are only used to illustrate the present invention, but are not used to limit the scope of the present invention.Based on the specific embodiments in the present invention, those of ordinary skill in the art, without making creative work, all other embodiments obtained belong to protection scope of the present invention.

[0026] Unless otherwise specified, all reagents used in the examples of the present invention are commercially available. Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0027] Example 1

[0028] This embodiment provides a method for obtaining muscle stem cells, comprising the following steps:

[0029] (1) Acquisition of pluripotent stem cells: Induced pluripotent stem cells (iPS) were obtained by reprogramming human blood monocytes from a healthy donor using Episome reprogramming plasmids (purchased from Thermo Fisher Scientific) on a Celetrix EX+ electroporation instrument. The induced pluripotent stem cells were grown and maintained on six-well plates coated with Matrigel (purchased from Corning) in mTeSR1 medium.

[0030] (2) Induced differentiation: Induced differentiation was initiated after amplification of induced pluripotent stem cells to the 20th generation. When the confluence reached about 70%, the cells were digested with cell digestion solution (Accutase, purchased from Stemcell Technologies) for 5 minutes. The cells were then re-placed on a low-adsorption plate for suspension culture and placed on a shaker in an incubator. The cells were then cultured for 24 hours using the first muscle stem cell induction medium (prepared by adding 5 μM ROCK inhibitor Y-27632 to mTeSR1 medium) and cultured for 24 hours. On the third day, the cells were replaced with the second muscle stem cell induction medium (prepared by adding 10 μM glycogen synthase kinase-3 inhibitor CHIR99021 to E6 medium) and cultured for 6 days to obtain muscle stem cell spheres.

[0031] Next, the muscle stem cell spheres were digested with cell digestion solution (Accutase) for 5 minutes, and then centrifuged and transferred to adherent culture. Six-well plates coated with collagen solution were added with the third muscle stem cell induction medium (E6 medium supplemented with 10 μM glycogen synthase kinase-3 inhibitor CHIR99021 and 5 μM ROCK inhibitor Y-27632) and cultured for 1 day;

[0032] The collagen solution was prepared as follows: 6.6 mL of Collagen I Rat Tail solution (purchased from Thermo Fisher Scientific) was added with 0.02 mM acetic acid solution and the volume was adjusted to 50 mL to obtain the collagen solution.

[0033] (3) Muscle stem cell proliferation: On the second day, the medium was replaced with muscle stem cell proliferation medium and expanded for 2 to 5 generations. The muscle stem cell proliferation medium contains 20% fetal bovine serum and 2.5 ng / mL basic fibroblast growth factor (BFGF) at a volume ratio of 20%.

[0034] (4) After the expansion is completed, the original proliferation medium of the muscle stem cells is discarded, and a newly prepared muscle stem cell differentiation medium is added to induce differentiation for seven days; wherein, the muscle stem cell differentiation medium is: a high-glucose DMEM medium containing 2% horse serum by volume, 3 μM tyrosine kinase receptor A agonist amitriptyline (Amitriptyline), 2.5 μM prostaglandin synthase inhibitor benzyldamine (Benzydamine), and 4% serum substitute (Ultroser Serum Substitute, abbreviated as UG) by volume.

[0035] Example 2

[0036] This embodiment provides a method for obtaining muscle stem cells, comprising the following steps:

[0037] (1) Acquisition of pluripotent stem cells: Induced pluripotent stem cells (iPS) were obtained by reprogramming human blood monocytes from a healthy donor using Episome reprogramming plasmids (purchased from Thermo Fisher Scientific) on a Celetrix EX+ electroporation instrument. The induced pluripotent stem cells were grown and maintained on six-well plates coated with Matrigel (purchased from Corning) in mTeSR1 medium.

[0038] (2) Induced differentiation: Induced differentiation was initiated after amplification of induced pluripotent stem cells to the 20th generation. When the confluence reached about 70%, the cells were digested with cell digestion solution (Accutase, purchased from Stemcell Technologies) for 5 minutes. The cells were then re-placed on a low-adsorption plate for suspension culture and placed on a shaker in an incubator. The cells were then cultured for 24 hours using the first muscle stem cell induction medium (prepared by adding 5 μM ROCK inhibitor Y-27632 to mTeSR1 medium) and cultured for 24 hours. On the third day, the cells were replaced with the second muscle stem cell induction medium (prepared by adding 10 μM glycogen synthase kinase-3 inhibitor CHIR99021 to E6 medium) and cultured for 6 days to obtain muscle stem cell spheres.

[0039] Next, the muscle stem cell spheres were digested with cell digestion solution (Accutase) for 5 minutes, and then centrifuged and transferred to adherent culture. Six-well plates coated with collagen solution were added with the third muscle stem cell induction medium (E6 medium supplemented with 10 μM glycogen synthase kinase-3 inhibitor CHIR99021 and 5 μM ROCK inhibitor Y-27632) and cultured for 1 day;

[0040] The collagen solution was prepared as follows: 6.6 mL of Collagen I Rat Tail solution (purchased from Thermo Fisher Scientific) was added with 0.02 mM acetic acid solution and the volume was adjusted to 50 mL to obtain the collagen solution.

[0041] (3) Muscle stem cell proliferation: On the second day, the medium was replaced with muscle stem cell proliferation medium containing 20% ​​by volume of North American fetal bovine serum and 2.5 ng / mL basic fibroblast growth factor (BFGF) for 2 to 5 generations.

[0042] (4) After the expansion is completed, the original proliferation medium of the muscle stem cells is discarded, and a newly prepared muscle stem cell differentiation medium is added to induce differentiation for seven days. The muscle stem cell differentiation medium is a high-glucose DMEM medium containing 2% horse serum by volume, 3 μM tyrosine kinase receptor A agonist amitriptyline, and 4% serum substitute (Ultroser Serum Substitute, abbreviated as UG) by volume.

[0043] Example 3

[0044] This embodiment provides a method for obtaining muscle stem cells, comprising the following steps:

[0045] (1) Acquisition of pluripotent stem cells: Induced pluripotent stem cells (iPS) were obtained by reprogramming human blood monocytes from a healthy donor using Episome reprogramming plasmids (purchased from Thermo Fisher Scientific) on a Celetrix EX+ electroporation instrument. The induced pluripotent stem cells were grown and maintained on six-well plates coated with Matrigel (purchased from Corning) in mTeSR1 medium.

[0046] (2) Induced differentiation: Induced differentiation was initiated after amplification of induced pluripotent stem cells to the 20th generation. When the confluence reached about 70%, the cells were digested with cell digestion solution (Accutase, purchased from Stemcell Technologies) for 5 minutes. The cells were then re-placed on a low-adsorption plate for suspension culture and placed on a shaker in an incubator. The cells were then cultured for 24 hours using the first muscle stem cell induction medium (prepared by adding 5 μM ROCK inhibitor Y-27632 to mTeSR1 medium) and cultured for 24 hours. On the third day, the cells were replaced with the second muscle stem cell induction medium (prepared by adding 10 μM glycogen synthase kinase-3 inhibitor CHIR99021 to E6 medium) and cultured for 6 days to obtain muscle stem cell spheres.

[0047] Next, the muscle stem cell spheres were digested with cell digestion solution (Accutase) for 5 minutes, and then centrifuged and transferred to adherent culture. Six-well plates coated with collagen solution were added with the third muscle stem cell induction medium (E6 medium supplemented with 10 μM glycogen synthase kinase-3 inhibitor CHIR99021 and 5 μM ROCK inhibitor Y-27632) and cultured for 1 day;

[0048] The collagen solution was prepared as follows: 6.6 mL of Collagen I Rat Tail solution (purchased from Thermo Fisher Scientific) was added with 0.02 mM acetic acid solution and the volume was adjusted to 50 mL to obtain the collagen solution.

[0049] (3) Muscle stem cell proliferation: On the second day, the medium was replaced with muscle stem cell proliferation medium containing 20% ​​by volume of North American fetal bovine serum and 2.5 ng / mL basic fibroblast growth factor (BFGF) for 2 to 5 generations.

[0050] (4) After the expansion is completed, the original proliferation medium of the muscle stem cells is discarded, and a newly prepared muscle stem cell differentiation medium is added to induce differentiation for seven days; wherein, the muscle stem cell differentiation medium is: a high-glucose DMEM medium containing 2% horse serum by volume, 2.5 μM prostaglandin synthase inhibitor benzyldamine (Benzydamine), and 4% serum substitute (Ultroser Serum Substitute, abbreviated as UG) by volume.

[0051] Comparative Examples 1 to 6

[0052] Comparative Examples 1 to 6 all provide a method for obtaining muscle stem cells, which differ from Examples 1 to 3 in that the muscle stem cell differentiation culture medium is different, wherein:

[0053] The muscle stem cell differentiation medium in Comparative Example 1 is a high-glucose DMEM medium containing 2% horse serum by volume and 4% serum substitute (Ultroser Serum Substitute, abbreviated as UG) by volume;

[0054] The muscle stem cell differentiation medium in Comparative Example 2 is a high-glucose DMEM medium containing 2% horse serum by volume, 3 μM β1 adrenergic receptor blocker Atenolol, and 4% serum substitute (UG) by volume;

[0055] The muscle stem cell differentiation medium in Comparative Example 3 was a high-glucose DMEM medium containing 2% horse serum, 5 μM escin, and 4% serum substitute (UG) by volume;

[0056] The muscle stem cell differentiation medium in Comparative Example 4 was a high-glucose DMEM medium containing 2% horse serum, 3 μM Fipexide, and 4% serum substitute (UG) by volume;

[0057] The muscle stem cell differentiation medium in Comparative Example 5 was a high-glucose DMEM medium containing 2% horse serum, 1 μM urethane, and 4% serum substitute (UG) by volume;

[0058] The muscle stem cell differentiation medium in Comparative Example 6 is a high-glucose DMEM medium containing 2% horse serum, 3 μM Miltefosine, and 4% Ultroser Serum Substitute (UG) by volume.

[0059] Comparative Example 7 Traditional induction method

[0060] This comparative example provides a method for obtaining muscle stem cells, which is a traditional method commonly used in the art for obtaining muscle stem cells, as follows:

[0061] (1) Acquisition of pluripotent stem cells: Induced pluripotent stem cells (iPS) were obtained by reprogramming human blood monocytes from a healthy donor using Episome reprogramming plasmids (purchased from Thermo Fisher Scientific) on a Celetrix EX+ electroporation instrument. The induced pluripotent stem cells were grown and maintained on six-well plates coated with Matrigel (purchased from Corning) in mTeSR1 medium.

[0062] (2) Induction of differentiation: When the density of pluripotent stem cells reached about 30%, the DMEM medium containing 10 μM CHIR99021, 0.5 μM LDN193189, 10 ng / mL bFGF, 1% non-essential amino acid NEAA by volume, and 1% N-2 by volume was replaced for three days of induction; the DMEM medium containing 10 μM DAPT, 20 ng / mL bFGF, 1% non-essential amino acid NEAA by volume, and 1% N-2 by volume was replaced for two days of induction; the DMEM medium containing 10 μM DAPT, 10 ng / mL HGF, 20 ng / mL bFGF, 1% non-essential amino acid NEAA by volume, and 1% N-2 by volume was replaced for two days of induction; the DMEM medium containing 10 μM DAPT, 10 ng / mL HGF, 20 ng / mL bFGF, 1% non-essential amino acid NEAA by volume, and 1% N-2 by volume was replaced for two days of induction; the DMEM medium containing 10 μM DAPT, 10 ng / mL Muscle stem cells were obtained by inducing for four days with a DMEM culture medium containing HGF, 10% KSR by volume, 0.1 mM β-ME, 1% non-essential amino acid NEAA by volume, and 1% N-2 by volume.

[0063] Result analysis:

[0064] (1) On the last day of induction, the muscle fibers obtained in Examples 1 to 3 and Comparative Examples 1 to 6 were subjected to immunofluorescence staining experiments to detect the expression of three mature muscle fiber markers, MF20, Dystrophin, and Myogenin. The fluorescence levels were then quantified and statistically analyzed using Image J software. The results are shown in Table 1 and Figures 1 to 3:

[0065] Table 1 Expression of mature muscle fiber markers in muscle fibers obtained in Examples and Comparative Examples

[0066] From the above results, it can be seen that 7 days after adding differentiation medium, no mature muscle fiber markers were detected in the experimental group (Comparative Example 2) added with the small molecule compound Atenolol, which indicates that the small molecule compound Atenolol has a lethal effect on stem cells;

[0067] Compared with the comparative example 1 without adding small molecule compounds, the expression levels of each marker (i.e., the proportion of positive cells) in the treatment groups with the addition of aescin, fepivacet, urethane, and miltefosine all decreased to a certain extent, indicating that the small molecule compounds aescin, fepivacet, urethane, and miltefosine have a mild inhibitory effect on the differentiation of stem cells;

[0068] Compared with Comparative Example 1 in which no small molecule compounds were added, the expression levels of various markers in the treatment groups to which the small molecule compounds amitriptyline and benzydamine were added separately and to which the small molecule compounds amitriptyline and benzydamine were added together were significantly increased, indicating that the small molecule compounds amitriptyline and benzydamine, whether used alone or together, have a promoting effect on the differentiation of muscle stem cells, and in the treatment group to which amitriptyline and benzydamine were added simultaneously, the induction efficiency was 1.2 times higher than that of the treatment group to which amitriptyline was added alone, and the efficiency of the treatment group to which benzydamine was added alone was 1.4 times higher.

[0069] (2) To ensure that the initial induction density was the same, the muscle stem cells obtained in step (2) of Example 1 and the muscle stem cells obtained by the traditional method (Comparative Example 7) were subjected to immunofluorescence experiments to detect the expression of two muscle stem cell markers, PAX7 and PAX3. The results are shown in Figure 4 and Table 2:

[0070] Table 2 Comparison of muscle stem cell induction efficiency between the method described in Example 1 and the traditional method

[0071] From the above results, it can be seen that the induction efficiency of muscle stem cells by the method described in the present invention is significantly higher than that of traditional induction methods.

[0072] Figure 5 shows the microscopic observation results of cells at different stages in Example 1 of the present invention, wherein a is the microscopic observation result of human peripheral blood mononuclear cells before reprogramming, b is the microscopic observation result of induced pluripotent stem cells cultured under a wall after 10 passages after reprogramming, c is the microscopic observation result when induced pluripotent stem cells were switched to suspension culture, d is the observation result of induced muscle stem cell suspension spheres, e is the microscopic observation result of induced muscle stem cell suspension spheres cultured under a wall for 1 day after digestion, and f is the microscopic observation result of muscle fibers obtained after differentiation, indicating that a large number of mature muscle fibers can be successfully obtained using the method of the present invention.

[0073] Through further experiments, the inventors found that when the content of ROCK inhibitor Y-27632 in the first muscle stem cell induction medium is 5-10 μM, the content of glycogen synthase kinase-3 inhibitor CHIR99021 in the second muscle stem cell induction medium is 10-15 μM, the content of glycogen synthase kinase-3 inhibitor CHIR99021 in the third muscle stem cell induction medium is 10-15 μM, the content of ROCK inhibitor Y-27632 is 5-10 μM, the volume concentration of bovine serum in the muscle stem cell proliferation medium is 10-20%, the concentration of basic fibroblast growth factor BFGF is 2-5 ng / mL, the concentration of tyrosine kinase receptor A agonist amitriptyline in the muscle stem cell differentiation medium is 1-5 μM, the concentration of prostaglandin synthase inhibitor benzydamine is 1-5 μM, and the volume proportion of horse serum is 2-5%, a considerable amount of muscle fibers can be obtained.

[0074] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for efficiently inducing muscle stem cells, characterized in that: The following steps are involved: After the human pluripotent stem cells are expanded to the point where they can be induced to differentiate, the cells are digested with a cell digestion solution when the cell confluence reaches 60-80%, and the cells are placed on a low-adsorption plate and suspended and cultured in a first muscle stem cell induction medium for 1 day; wherein the first muscle stem cell induction medium includes a stem cell basal medium and 5-10 μM ROCK inhibitor Y-27632; On the third day, the culture medium is replaced with a second muscle stem cell induction medium for 5 to 7 days to obtain muscle stem cell spheres; wherein the second muscle stem cell induction medium includes a stem cell basal medium and 10 to 15 μM glycogen synthase kinase-3 inhibitor CHIR99021; The muscle stem cell balls are digested with cell digestion solution and then centrifuged and transferred to adherent culture, and the well plates coated with collagen solution are added with a third muscle stem cell induction medium and cultured for 1 day, and then replaced with a muscle stem cell proliferation medium for culture, and expanded for 2 to 5 generations; wherein the third muscle stem cell induction medium includes stem cell basal medium, 10 to 15 μM glycogen synthase kinase-3 inhibitor CHIR99021 and 5 to 10 μM ROCK inhibitor Y-27632; the muscle stem cell proliferation medium includes cell growth basal medium, bovine serum and basic fibroblast growth factor BFGF, wherein the volume concentration of the bovine serum is 10 to 20%, and the concentration of the basic fibroblast growth factor BFGF is 2 to 5 ng / mL; After the expansion is completed, the original muscle stem cell proliferation medium is discarded, and a muscle stem cell differentiation medium is added to induce differentiation for 6 to 8 days to obtain mature muscle fibers; wherein the muscle stem cell differentiation medium includes mammalian cell growth basal medium, horse serum, tyrosine kinase receptor A agonist and / or prostaglandin synthetase inhibitor, the tyrosine kinase receptor A agonist is amitriptyline with a concentration of 1 to 5 μM, the prostaglandin synthetase inhibitor is benzydamine with a concentration of 1 to 5 μM, and the volume proportion of horse serum is 2 to 5%.

2. The method for efficiently inducing muscle stem cells according to claim 1, characterized in that: The basal culture medium in the muscle stem cell differentiation culture medium is DMEM culture medium; and / or the basal culture medium in the first muscle stem cell induction culture medium is mTeSR1 culture medium, and the basal culture medium in the second and third muscle stem cell induction culture mediums is E6 culture medium; and / or the basal culture medium in the muscle stem cell proliferation culture medium is F10 culture medium.

3. The method for efficiently inducing muscle stem cells according to claim 1, characterized in that: The collagen solution is prepared by adding acetic acid solution to collagen I rat tail solution.

4. The method for efficiently inducing muscle stem cells according to claim 1, characterized in that: The method for expanding human pluripotent stem cells until they can be induced to differentiate is as follows: growing and maintaining the human pluripotent stem cells on a Matrigel-coated plate in mTeSR1 medium.

5. The method for efficiently inducing muscle stem cells according to claim 1, characterized in that: Human pluripotent stem cells were derived from human blood monocytes reprogrammed using Episome reprogramming plasmids.

6. A culture medium for efficiently inducing muscle stem cells, characterized in that: The invention comprises the muscle stem cell differentiation medium as claimed in claim 1.

7. The culture medium for efficiently inducing muscle stem cells according to claim 6, characterized in that: It also includes the first muscle stem cell induction medium, the second muscle stem cell induction medium, the third muscle stem cell induction medium and the muscle stem cell proliferation medium described in claim 1.

8. A kit for efficiently inducing muscle stem cells, characterized in that: The method comprises the culture medium according to any one of claims 6 to 7.

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