CN116536255B - Culture medium and method for efficiently inducing muscle stem cells - Google Patents
Culture medium and method for efficiently inducing muscle stem cells Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0659—Satellite cells
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2510/00—Genetically modified cells
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of pluripotent stem cell induction, in particular to a method for efficiently inducing muscle stem cells to obtain muscle fibers or skeletal muscles. The method comprises the steps of carrying out step-by-step induction by adopting suspension culture and three muscle stem cell induction culture mediums, then adopting a culture medium containing bovine serum and alkaline fibroblast growth factors for amplification, and then adopting a differentiation culture medium containing specific chemical small molecules for induction differentiation.
Description
Technical Field
The invention relates to the technical field of pluripotent stem cell induction, in particular to a culture medium and a method for efficiently inducing muscle stem cells to obtain muscle fibers or skeletal muscles.
Background
Muscles are the most abundant components of the human body and are vital to maintaining body movement and life health. Under normal conditions, if the muscle is damaged, the resting muscle stem cells located between the basal and plasma membranes of the muscle fiber are activated and proliferate and differentiate to produce new muscle fiber, thereby repairing the damaged muscle. Although adult muscle stem cells have strong regeneration potential, progressive muscle necrosis caused by diseases such as aging muscular atrophy and duchenne muscular dystrophy often results in the reduction or complete loss of the regeneration and repair ability of the muscle stem cells, and finally leads to paralysis and even death of patients.
In recent years, stem cell technology brings great hope for treating muscle diseases such as aging or DMD, but still faces a great challenge that separation, in vitro culture and mass expansion of muscle stem cells are difficult to achieve, so that insufficient supply of the muscle stem cells is caused, and clinical application of stem cell therapy is severely limited.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, one of the purposes of the invention is to provide a method for efficiently inducing muscle stem cells, by adopting the method, a considerable amount of muscle stem cells can be obtained at one time for treatment and simulation of disease models, and the problem of difficult acquisition and expansion of human muscle stem cells is well solved.
The invention adopts the following technical scheme to realize the technical purposes: the invention provides a method for efficiently inducing muscle stem cells, which comprises the following steps:
after expanding the human pluripotent stem cells to be induced and differentiated, digesting the cells with a cell digestive juice when the cell fusion degree reaches 60-80%, and placing the cells into a low-adsorptivity plate and adding a first muscle stem cell induction culture medium for suspension culture for 1 day; the first muscle stem cell induction culture medium comprises a stem cell basal culture medium and 5-10 mu M of ROCK inhibitor Y-27632;
on the 3 rd day, replacing the culture medium with a second muscle stem cell induction culture medium for culturing for 5-7 days to obtain muscle stem cell spheres; wherein the second muscle stem cell induction culture medium comprises a stem cell basal culture medium and 10-15 mu M glycogen synthase kinase-3 inhibitor CHIR99021;
digesting muscle stem cell balls by using a cell digestive juice, performing centrifugal transfer adherence culture, adding a collagen solution coated pore plate into a third muscle stem cell induction culture medium for culturing for 1 day, changing into a muscle stem cell proliferation culture medium for culturing, and amplifying for 2-5 generations; the third muscle stem cell induction culture medium comprises a stem cell basal culture medium, 10-15 mu M glycogen synthase kinase-3 inhibitor CHIR99021 and 5-10 mu M ROCK inhibitor Y-27632; the muscle stem cell proliferation culture medium comprises a cell growth basic culture medium, bovine serum and alkaline fibroblast growth factors, wherein the volume concentration of the bovine serum is 10-20%, and the concentration of the alkaline fibroblast growth factors is 2-5 ng/mL;
absorbing and removing the amplified muscle stem cells from the original muscle stem cell proliferation culture medium, adding a muscle stem cell differentiation culture medium, and inducing differentiation for 6-8 days to obtain mature muscle fibers; the muscle stem cell differentiation culture medium comprises a mammalian cell growth basal culture medium, horse serum, a tyrosine kinase receptor A agonist and/or a prostaglandin synthetase inhibitor, wherein the tyrosine kinase receptor A agonist is amitriptyline, the concentration is 1-5 mu M, the prostaglandin synthetase inhibitor is benzydamine, the concentration is 1-5 mu M, and the volume ratio of the horse serum is 2-5%.
When mature muscle fibers are obtained by the method provided by the invention, the muscle stem cell balls with higher purity are obtained by a suspension culture mode, the muscle stem cell balls are digested into single cells and then are rapidly amplified on a 2D layer, in the process of differentiating the muscle stem cells into the mature muscle fibers, a differentiation medium containing a specific small molecule inhibitor is adopted for culture, the pluripotent stem cells can be rapidly induced to obtain the muscle stem cells and the mature muscle fibers or skeletal muscles, and a proper amount of the muscle stem cells can be obtained at one time for treatment and simulation of a disease model, so that the problem of difficult acquisition and amplification of human muscle stem cells can be well solved.
As a preferred embodiment, the basal medium in the differentiation medium of the muscle stem cells is DMEM medium; the basic culture medium in the first muscle stem cell induction culture medium is mTESR1 culture medium, and the basic culture medium in the second and third muscle stem cell induction culture mediums is E6 culture medium; the basal medium in the proliferation medium of the muscle stem cells is F10 medium.
As a preferred embodiment, the collagen solution is prepared by adding an acetic acid solution to a rat tail collagen I solution.
As a preferred embodiment, the method for amplifying the human pluripotent stem cells to induce differentiation comprises the following steps: human pluripotent stem cells were grown and maintained on matrigel coated well plates in mTeSR1 medium.
As a preferred embodiment, the human pluripotent stem cells are reprogrammed from human blood mononuclear cells using a reprogramming plasmid.
The second purpose of the invention is to protect a culture medium for efficiently inducing the muscle stem cells, which comprises the muscle stem cell differentiation culture medium.
As a preferred embodiment, the method further comprises 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.
The invention also aims to provide a kit for efficiently inducing the muscle stem cells, which comprises the culture medium.
The method for efficiently inducing the muscle stem cells can obtain a considerable amount of muscle stem cells at one time to perform treatment and simulation of a disease model, and solves the problems that the acquisition and the expansion of human muscle stem cells are difficult.
Drawings
FIG. 1 is the expression level of the MF20 marker in muscle fibers obtained 7 days after induction of differentiation in examples and comparative examples;
FIG. 2 is the expression level of a myogenin marker in myofibers obtained 7 days after induction of differentiation in examples and comparative examples;
FIG. 3 is the expression level of dystrophin markers in muscle fibers obtained 7 days after induction of differentiation in examples and comparative examples;
FIG. 4 shows the expression levels of PAX7 and PAX3 markers in the muscle stem cells obtained after the induced differentiation in example 1 and comparative example 7;
FIG. 5 is a view of human peripheral blood mononuclear cells prior to reprogramming under a microscope;
FIG. 6 is a microscopic observation of induced pluripotent stem cells after passage 10 passages after reprogramming;
FIG. 7 is a view of the induced pluripotent stem cells under a 10X microscope when cultured in suspension;
FIG. 8 is the observation of the induced muscle stem cell suspension ball;
FIG. 9 is a microscopic observation of the induced muscle stem cells after pellet digestion and after 1 day of adherent culture;
fig. 10 is a microscopic observation of the muscle fibers obtained after differentiation.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
The reagents used in the examples of the present invention are all commercially available, without specific explanation. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions, such as molecular cloning, as described in Sambrook et al: the conditions described in the laboratory Manual, or according to the manufacturer's recommendations.
Example 1
The embodiment provides a method for obtaining muscle stem cells, which comprises the following steps:
(1) Obtaining pluripotent stem cells: induced pluripotent stem cells (iPS) were reprogrammed on a samer fly electrotransfer apparatus using a reprogramming plasmid (purchased from samer fly) from human blood mononuclear cells of a healthy donor, wherein the induced pluripotent stem cells were grown and maintained on a six-well plate coated with a matrix membrane matrix (i.e., matrigel, purchased from corning) in mTeSR1 medium.
(2) Inducing differentiation: expanding induced pluripotent stem cells to 20 generations to begin induced differentiation, digesting the induced pluripotent stem cells for 5 minutes by using a cell digestive juice (Ackutase, purchased from Canadian Stem cell technology Co., ltd.) until the fusion degree reaches about 70%, then putting the cells into a low-adsorptivity plate for suspension culture, putting the cells on a shaking table on an incubator, and adding a first muscle stem cell induction culture medium (prepared by adding 5 mu M of ROCK inhibitor Y-27632 into mTESR1 culture medium) for culturing for 24 hours; on the third day, the cells are changed into a second muscle stem cell induction medium (prepared by adding 10 mu M glycogen synthase kinase-3 inhibitor CHIR99021 into E6 medium) to be cultured for 6 days, and muscle stem cell spheres are obtained;
then digesting muscle stem cell balls with cell digestive juice (Ackutase) for 5 minutes, centrifuging, transferring to an adherence culture, coating a six-hole plate with a collagen solution, adding a third muscle stem cell induction culture medium (10 mu M glycogen synthase kinase-3 inhibitor CHIR99021 and 5 mu M ROCK inhibitor Y-27632 are added to an E6 culture medium) and culturing for 1 day;
wherein, the collagen solution is prepared as follows: 6.6mL of rat tail collagen I (Collagen I Rat Tail liquid, purchased from Sieimer) is taken, 0.02mM acetic acid solution is added, and the volume is fixed to 50mL to obtain collagen solution.
(3) Proliferation of muscle stem cells: and the next day, replacing the culture medium with a muscle stem cell proliferation culture medium, and amplifying for 2-5 generations. Wherein the muscle stem cell proliferation culture medium contains north american fetal bovine serum with the volume ratio of 20 percent and 2.5ng/mL alkaline fibroblast growth factor (BFGF for short);
(4) Absorbing the amplified muscle stem cells to the original proliferation culture medium, adding a newly prepared muscle stem cell differentiation culture medium, and inducing differentiation for seven days to obtain the final product; wherein, the muscle stem cell differentiation culture medium is: a high-sugar DMEM medium containing 2% by volume of horse serum, 3. Mu.M of Amitriptyline (Amittriptyline) as an agonist of tyrosine kinase receptor A, 2.5. Mu.M of Benzydamine (Benzydamine) as a prostaglandin synthase inhibitor, and 4% by volume of serum replacement (Ultroser Serum Substitute, UG for short).
Example 2
The embodiment provides a method for obtaining muscle stem cells, which comprises the following steps:
(1) Obtaining pluripotent stem cells: induced pluripotent stem cells (iPS) were reprogrammed on a race-track instrument using a reprogramming plasmid (purchased from race-track) from human blood mononuclear cells of a healthy donor, wherein the induced pluripotent stem cells were grown and maintained on a six-well plate coated with a matrix membrane matrix (purchased from corning) in mTeSR1 medium.
(2) Inducing differentiation: expanding induced pluripotent stem cells to 20 generations to begin induced differentiation, digesting the induced pluripotent stem cells for 5 minutes by using a cell digestive juice (Ackutase, purchased from Canadian Stem cell technology Co., ltd.) until the fusion degree reaches about 70%, then putting the cells into a low-adsorptivity plate for suspension culture, putting the cells on a shaking table on an incubator, and adding a first muscle stem cell induction culture medium (prepared by adding 5 mu M of ROCK inhibitor Y-27632 into mTESR1 culture medium) for culturing for 24 hours; on the third day, the culture medium was changed to a second muscle stem cell induction medium (prepared by adding 10. Mu.M glycogen synthase kinase-3 inhibitor CHIR99021 to E6 medium)
Culturing for 6 days to obtain muscle stem cell spheres;
then digesting muscle stem cell balls with cell digestive juice (Ackutase) for 5 minutes, centrifuging, transferring to an adherence culture, coating a six-hole plate with a collagen solution, adding a third muscle stem cell induction culture medium (10 mu M glycogen synthase kinase-3 inhibitor CHIR99021 and 5 mu M ROCK inhibitor Y-27632 are added to an E6 culture medium) and culturing for 1 day;
wherein, the collagen solution is prepared as follows: 6.6mL of rat tail collagen I (Collagen I Rat Tail liquid, purchased from Sieimer) is taken, 0.02mM acetic acid solution is added, and the volume is fixed to 50mL to obtain collagen solution.
(3) Proliferation of muscle stem cells: and the next day, replacing the culture medium with a muscle stem cell proliferation culture medium, and amplifying for 2-5 generations. Wherein the muscle stem cell proliferation culture medium contains north american fetal bovine serum with the volume ratio of 20 percent and 2.5ng/mL alkaline fibroblast growth factor (BFGF for short).
(4) Absorbing the amplified muscle stem cells to the original proliferation culture medium, adding a newly prepared muscle stem cell differentiation culture medium, and inducing differentiation for seven days to obtain the final product; wherein, the muscle stem cell differentiation culture medium is: a high-sugar DMEM medium containing 2% by volume of horse serum, 3. Mu.M of Amitriptyline (Amittriptyline) as an agonist of tyrosine kinase A, and 4% by volume of serum replacement (Ultroser Serum Substitute, UG for short).
Example 3
The embodiment provides a method for obtaining muscle stem cells, which comprises the following steps:
(1) Obtaining pluripotent stem cells: induced pluripotent stem cells (iPS) were reprogrammed on a race-track instrument from human blood mononuclear cells of a healthy donor using an Episome reprogramming plasmid (purchased from race-track), wherein the induced pluripotent stem cells were grown and maintained on a six-well plate coated with a matrix membrane matrix (purchased from corning) in mTeSR1 medium.
(2) Inducing differentiation: expanding induced pluripotent stem cells to 20 generations to begin induced differentiation, digesting the induced pluripotent stem cells for 5 minutes by using a cell digestive juice (Ackutase, purchased from Canadian Stem cell technology Co., ltd.) until the fusion degree reaches about 70%, then putting the cells into a low-adsorptivity plate for suspension culture, putting the cells on a shaking table on an incubator, and adding a first muscle stem cell induction culture medium (prepared by adding 5 mu M of ROCK inhibitor Y-27632 into mTESR1 culture medium) for culturing for 24 hours; on the third day, the cells are changed into a second muscle stem cell induction medium (prepared by adding 10 mu M glycogen synthase kinase-3 inhibitor CHIR99021 into E6 medium) to be cultured for 6 days, and muscle stem cell spheres are obtained;
then digesting muscle stem cell balls with cell digestive juice (Ackutase) for 5 minutes, centrifuging, transferring to an adherence culture, coating a six-hole plate with a collagen solution, adding a third muscle stem cell induction culture medium (10 mu M glycogen synthase kinase-3 inhibitor CHIR99021 and 5 mu M ROCK inhibitor Y-27632 are added to an E6 culture medium) and culturing for 1 day;
wherein, the collagen solution is prepared as follows: 6.6mL of rat tail collagen I (Collagen I Rat Tail liquid (purchased from Sieimer) is added into acetic acid solution with the concentration of 0.02mM, and the volume is fixed to 50mL to obtain collagen solution.
(3) Proliferation of muscle stem cells: and the next day, replacing the culture medium with a muscle stem cell proliferation culture medium, and amplifying for 2-5 generations. Wherein the muscle stem cell proliferation culture medium contains north american fetal bovine serum with the volume ratio of 20 percent and 2.5ng/mL alkaline fibroblast growth factor (BFGF for short).
(4) Absorbing the amplified muscle stem cells to the original proliferation culture medium, adding a newly prepared muscle stem cell differentiation culture medium, and inducing differentiation for seven days to obtain the final product; wherein, the muscle stem cell differentiation culture medium is: a high-sugar DMEM medium containing 2% by volume of horse serum, 2.5 μm prostaglandin synthase inhibitor Benzydamine (Benzydamine), and 4% by volume of serum replacement (Ultroser Serum Substitute, UG for short).
Comparative examples 1 to 6
Comparative examples 1 to 6 each provide a method for obtaining muscle stem cells, which is different from examples 1 to 3 in that the differentiation medium of muscle stem cells is different from that of examples 1 to 3,
the muscle stem cell differentiation medium in comparative example 1 was a high-sugar DMEM medium containing 2% by volume of horse serum and 4% by volume of serum replacement (Ultroser Serum Substitute, UG for short);
the muscle stem cell differentiation medium in comparative example 2 was a high-sugar DMEM medium containing 2% by volume of horse serum, 3 μΜβ1 adrenergic receptor blocker Atenolol (Atenolol), and 4% by volume of serum replacement (Ultroser Serum Substitute, UG for short);
the muscle stem cell differentiation medium in comparative example 3 was a high-sugar DMEM medium containing 2% by volume of horse serum, 5 μm of aescine (Escin), and 4% by volume of serum replacement (Ultroser Serum Substitute, UG for short);
the muscle stem cell differentiation medium in comparative example 4 was a high-sugar DMEM medium containing 2% by volume of horse serum, 3 μm of fepiprazole (Fipexide), and 4% by volume of serum replacement (Ultroser Serum Substitute, UG for short);
the muscle stem cell differentiation medium in comparative example 5 was a high-sugar DMEM medium containing 2% by volume of horse serum, 1 μm Urethane (urea), and 4% by volume of serum replacement (Ultroser Serum Substitute, UG for short);
the muscle stem cell differentiation medium in comparative example 6 was a high-sugar DMEM medium containing 2% by volume of horse serum, 3 μm Miltefosine (Miltefosine), and 4% by volume of serum replacement (Ultroser Serum Substitute, abbreviated UG).
Comparative example 7 traditional induction method
The present comparative example provides a method for obtaining muscle stem cells, which is a conventional method for obtaining muscle stem cells commonly used in the art, and specifically comprises the following steps:
(1) Obtaining pluripotent stem cells: induced pluripotent stem cells (iPS) were reprogrammed on a race-track instrument using a reprogramming plasmid (purchased from race-track) from human blood mononuclear cells of a healthy donor, wherein the induced pluripotent stem cells were grown and maintained on a six-well plate coated with a matrix membrane matrix (purchased from corning) in mTeSR1 medium.
(2) Inducing differentiation: when the density of the pluripotent stem cells reaches about 30%, the culture medium is changed to be induced for three days, wherein the culture medium contains 10 mu M of CHIR99021, 0.5 mu M of LDN193189, 10ng/mL of bFGF, 1% of nonessential amino acid NEAA and 1% of N-2; the DMEM medium containing 10 mu M DAPT, 20ng/mL bFGF, 1% by volume of nonessential amino acid NEAA and 1% by volume of N-2 is changed for induction for two days; the DMEM medium containing 10 mu M DAPT, 10ng/mL HGF, 20ng/mL bFGF and 1% of nonessential amino acid NEAA in volume ratio and 1% of N-2 in volume ratio is replaced for further induction for two days; the culture medium containing 10 mu M DAPT, 10ng/mL HGF, 10% KSR, 0.1mM beta-ME, 1% nonessential amino acid NEAA, 1% N-2 was changed, and the induction was performed for four days, thereby obtaining muscle stem cells.
Analysis of results:
(1) On the last day of induction, immunofluorescent staining experiments were performed on the myofibers obtained in examples 1 to 3 and comparative examples 1 to 6, and the expression of three mature myofiber markers of MF20, dystrophin (dystrychin, dys), myogenin (MyoG) were detected, and then the fluorescence degree was quantified and counted by Image J software, and the results are shown in table 1 and fig. 1 to 3:
TABLE 1 expression of mature myofiber markers in myofibers obtained in examples and comparative examples
From the above results, it was found that no mature muscle fiber markers were detected in the experimental group (i.e., comparative example 2) to which the small molecular compound atenolol was added after 7 days of addition of the differentiation medium, which suggests that the small molecular compound atenolol had a lethal effect on stem cells;
compared with the comparative example 1 without adding the small molecular compound, the expression level of each marker (namely the proportion of positive cells) in the treatment group with adding the aescin, the feproside, the urethane and the miltefosine is reduced to a certain extent, which indicates that the small molecular compound aescin, the feproside, the urethane and the miltefosine have slight differentiation inhibition effect on stem cells;
compared with the comparative example 1 without adding the small molecular compound, the expression quantity of each marker in the treatment group with the small molecular compound amitriptyline and the benzydamine and the treatment group with the small molecular compound amitriptyline and the benzydamine added together are obviously improved, which indicates that the small molecular compound amitriptyline and the benzydamine both used independently or together have an effect of promoting the differentiation of muscle stem cells, and the induction efficiency of the treatment group with the amitriptyline and the benzydamine added simultaneously is improved by 1.2 times compared with the treatment group with the amitriptyline added independently, and the treatment group with the benzydamine added independently has 1.4 times improved efficiency.
(2) The muscle stem cells obtained in step (2) of example 1 and those obtained by the conventional method (comparative example 7) were subjected to immunofluorescence experiments to determine the expression of two muscle stem cell markers, PAX7 and PAX3, with the results shown in fig. 4 and table 2, while ensuring the same initial induction density:
table 2 comparison of the induction efficiency of muscle stem cells by the method described in example 1 and the conventional method
From the above results, it can be seen that the method of the present invention has significantly higher induction efficiency of muscle stem cells than the conventional induction method.
Fig. 5 to 10 are the observation results under the microscope of the cells of example 1 of the present invention at different periods, wherein fig. 5 is the observation result under the microscope of human peripheral blood mononuclear cells before reprogramming, fig. 6 is the observation result under the microscope of induced pluripotent stem cells after passage 10 generations after reprogramming, fig. 7 is the observation result under the microscope when induced pluripotent stem cells are transferred to suspension culture, fig. 8 is the observation result of induced muscle stem cell suspension balls, fig. 9 is the observation result under the microscope after adherence culture for 1 day after digestion of induced muscle stem cell suspension balls, and fig. 10 is the observation result under the microscope of muscle fibers obtained after differentiation, which shows that a large number of mature muscle fibers can be successfully obtained by the method of the present invention.
The inventor obtains through further experiments that when the content of a ROCK inhibitor Y-27632 in a first muscle stem cell induction culture medium is 5-10 mu M, the content of a glycogen synthase kinase-3 inhibitor CHIR99021 in a second muscle stem cell induction culture medium is 10-15 mu M, the content of a glycogen synthase kinase-3 inhibitor CHIR99021 in a third muscle stem cell induction culture medium is 10-15 mu M, the content of a ROCK inhibitor Y-27632 is 5-10 mu M, the volume concentration of bovine serum in a muscle stem cell proliferation culture medium is 10-20%, the concentration of an alkaline fibroblast growth factor BFGF is 2-5 ng/mL, the concentration of tyrosine kinase receptor A agonist amitriptyline in a muscle stem cell differentiation culture medium is 1-5 mu M, the concentration of prostaglandin synthase inhibitor benzydamine is 1-5 mu M, and the volume ratio of horse serum is 2-5%, a proper amount of muscle fibers can be obtained.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A method for efficiently inducing muscle stem cells, comprising the steps of:
expanding human induced pluripotent stem cells until the induced pluripotent stem cells can be differentiated, digesting the cells with cell digestive juice when the cell fusion degree reaches 60-80%, and placing the cells into a low-adsorptivity plate and adding a first muscle stem cell induction culture medium for suspension culture for 1 day; the first muscle stem cell induction culture medium consists of a mTESR1 culture medium and 5-10 mu M of ROCK inhibitor Y-27632;
on the 3 rd day, replacing the culture medium with a second muscle stem cell induction culture medium for culturing for 5-7 days to obtain muscle stem cell spheres; wherein the second muscle stem cell induction culture medium consists of an E6 culture medium and 10-15 mu M glycogen synthase kinase-3 inhibitor CHIR99021;
digesting muscle stem cell balls by using a cell digestive juice, performing centrifugal transfer adherence culture, adding a collagen solution coated pore plate into a third muscle stem cell induction culture medium for culturing for 1 day, changing into a muscle stem cell proliferation culture medium for culturing, and amplifying for 2-5 generations; the third muscle stem cell induction culture medium consists of an E6 culture medium, 10-15 mu M glycogen synthase kinase-3 inhibitor CHIR99021 and 5-10 mu M ROCK inhibitor Y-27632, and the muscle stem cell proliferation culture medium consists of an F10 culture medium, 10-20% bovine serum and 2-5 ng/mL alkaline fibroblast growth factor;
absorbing and removing the amplified muscle stem cells from the original muscle stem cell proliferation culture medium, adding a muscle stem cell differentiation culture medium, and inducing differentiation for 6-8 days to obtain mature muscle fibers; the muscle stem cell differentiation medium consists of a DMEM medium, horse serum with the volume ratio of 2-5%, 3 mu M of tyrosine kinase receptor A agonist amitriptyline and 2.5 mu M of prostaglandin synthase inhibitor benzydamine, or the muscle stem cell differentiation medium consists of a DMEM medium, horse serum with the volume ratio of 2-5% and 3 mu M of tyrosine kinase receptor A agonist amitriptyline, or the muscle stem cell differentiation medium consists of a DMEM medium, horse serum with the volume ratio of 2-5% and 2.5 mu M of prostaglandin synthase inhibitor benzydamine.
2. The method for efficiently inducing muscle stem cells according to claim 1, wherein the collagen solution is prepared by adding an acetic acid solution into a rat tail collagen I solution.
3. The method for efficiently inducing muscle stem cells according to claim 1, wherein the method for amplifying human induced pluripotent stem cells to induce differentiation comprises: human induced pluripotent stem cells were grown and maintained on matrigel coated well plates in mTeSR1 medium.
4. The method of claim 1, wherein the human induced pluripotent stem cells are reprogrammed from human blood mononuclear cells using an Episome reprogramming plasmid.
5. A culture medium, characterized in that the culture medium is the muscle stem cell differentiation culture medium according to claim 1, and the culture medium is used for inducing human induced pluripotent stem cells to obtain muscle stem cells and mature muscle fibers.
6. The culture medium of claim 5, further comprising the first, second, third, and proliferation media of muscle stem cells of claim 1.
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