CN110564676A - method for rapidly differentiating pluripotent stem cells into skeletal muscle cells and skeletal muscle cells - Google Patents
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Abstract
the invention relates to the field of stem cells, in particular to a method for rapidly differentiating pluripotent stem cells into skeletal muscle cells, aiming at solving the problems of long time and low efficiency of inducing the skeletal muscle cells by the pluripotent stem cells. Specifically, the method comprises introducing an expression vector containing at least one potential gene into a pluripotent stem cell in a culture medium, thereby directionally differentiating the pluripotent stem cell into a skeletal muscle cell. By specifically inducing the expression of one or more different combined genes of Mef2b, Pax3, Pax7, Mydo1, Pitx1, Myogenin, Mef2c, Mrf4 and Desmin at different stages of inducing the directional differentiation of human pluripotent stem cells to skeletal muscle cells, the functional skeletal muscle cells with the purity of up to 80 percent can be obtained, and the induction period is obviously shortened.
Description
Technical Field
the invention relates to the field of stem cells, in particular to a method for rapidly differentiating pluripotent stem cells into skeletal muscle cells and the skeletal muscle cells.
Background
the characteristics of unlimited proliferation capacity and pluripotency of human embryonic stem cells provide the possibility of obtaining all cell types of human bodies in large quantities. However, due to the acquisition of human embryonic stem cells from different individuals and ethical limitations, it is very difficult to obtain differentiated cells from human embryonic stem cells for personalized therapy and drug screening. The technology of inducing human-derived pluripotent stem cells from human somatic cells circumvents the above-mentioned limitations of human embryonic stem cells, making it possible to generate human-derived pluripotent stem cells from somatic cells for each individual person, which makes these cells the best choice for disease models, drug screening, toxicity testing, and personalized cell therapy.
The efficient and rapid differentiation of human pluripotent stem cells into different types of high-purity somatic cells is crucial to drug screening, toxicity testing and personalized cell therapy of stem cells. The main technical problems of the prior art for inducing the directional differentiation of pluripotent stem cells are low efficiency, long time period, low cell purity and incomplete functionality of the generated cells. The induction of skeletal muscle cells from human pluripotent stem cells is long and inefficient.
Disclosure of Invention
Therefore, the present invention provides a method for rapidly differentiating pluripotent stem cells into skeletal muscle cells and skeletal muscle cells, so as to rapidly differentiate skeletal muscle cells from pluripotent stem cells.
The technical purpose of the invention is realized by the following technical scheme: a method for rapid differentiation of pluripotent stem cells into skeletal muscle cells, comprising introducing an expression vector comprising at least one potent gene into pluripotent stem cells in a culture medium, thereby directing differentiation of the pluripotent stem cells into skeletal muscle cells.
Further, the potential genes comprise one or more of Mef2b, Pax3, Pax7, Mydo1, Pitx1, Myogenin, Mef2c, Mrf4 and Desmin.
Further, comprising: s1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a special culture medium for the pluripotent stem cells; s2, preparing the human pluripotent stem cells cultured in the S1 into single cell suspension, centrifuging and resuspending the single cell suspension, and adding the single cell suspension into a culture plate for culture to form an embryoid body; s3, culturing the embryoid body obtained in the step S2 for 24h, and introducing a transcription factor; s4, culturing the cells in the S3 until the confluency is 95%; s5, introducing Mydo1 and Myogenin genes, and continuously culturing to ensure that pluripotent stem cells are directionally differentiated to obtain skeletal muscle cells.
Further, comprising: s1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a special culture medium for the pluripotent stem cells; s2, preparing the human pluripotent stem cells cultured in the S1 into single cell suspension, centrifuging and resuspending the single cell suspension, and adding the single cell suspension into a culture plate for culture to form an embryoid body; s3, culturing the embryoid body obtained in the step S2 for 24h, and introducing a transcription factor; s4, culturing the cells in the S3 until the confluency is 95%; s5, Mydo1, Pax3 and Myogenin genes are introduced, and the pluripotent stem cells are directionally differentiated by continuous culture to obtain skeletal muscle cells.
Further, comprising: s1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a special culture medium for the pluripotent stem cells; s2, preparing the human pluripotent stem cells cultured in the S1 into single cell suspension, centrifuging and resuspending the single cell suspension, and adding the single cell suspension into a culture plate for culture to form an embryoid body; s3, culturing the embryoid body obtained in the step S2 for 24h, and introducing a transcription factor; s4, culturing the cells in the S3 until the confluency is 95%; s5, introducing Desmin and Mef2c genes, and continuously culturing to ensure that pluripotent stem cells are subjected to directional differentiation to obtain skeletal muscle cells.
Further, comprising: s1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a special culture medium for the pluripotent stem cells; s2, preparing the human pluripotent stem cells cultured in the S1 into single cell suspension, centrifuging and resuspending the single cell suspension, and adding the single cell suspension into a culture plate for culture to form an embryoid body; s3, culturing the embryoid body obtained in the step S2 for 24h, and introducing a transcription factor; s4, culturing the cells in the S3 until the confluency is 95%; s5, Pax7 and Mef2c genes are introduced, and culture is continued to allow pluripotent stem cells to undergo directed differentiation, thereby obtaining skeletal muscle cells.
the invention also provides a technical scheme that: a skeletal muscle cell prepared by the above method.
The technical scheme of the invention is different from the prior art: by specifically inducing the expression of one or more different combined genes in Mef2b, Pax3, Pax7, Mydo1, Pitx1, Myogenin, Mef2c, Mrf4 and Desmin at different stages of inducing the directional differentiation of human pluripotent stem cells to skeletal muscle cells, the functional skeletal muscle cells with the purity of up to 80 percent can be obtained, and the induction period is obviously shortened.
Detailed Description
in order to make the objects, technical solutions and advantages of the present invention more apparent, the following embodiments further describe the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
One embodiment of the present application provides a method for rapid differentiation of pluripotent stem cells into skeletal muscle cells, comprising introducing an expression vector comprising at least one potent gene into pluripotent stem cells in a culture medium, thereby directing differentiation of the pluripotent stem cells into skeletal muscle cells. Potential genes comprise one or more of Mef2b, Pax3, Pax7, Mydo1, Pitx1, Myogenin, Mef2c, Mrf4 and Desmin. By specifically inducing the expression of the different combined genes at different stages of inducing the directional differentiation of the human pluripotent stem cells to the skeletal muscle cells, the functional skeletal muscle cells with the purity of up to 80 percent can be obtained, and the induction period is obviously shortened.
The method of the present application for the rapid differentiation of pluripotent stem cells into skeletal muscle cells is illustrated by the following specific examples:
The first embodiment is as follows:
S1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a culture medium special for the pluripotent stem cells.
S2, preparing the human pluripotent stem cells cultured in the S2 into single cell suspension, centrifuging, resuspending, and adding into a culture plate for culture to form an embryoid body.
s3, after culturing the embryoid body obtained in the step S2 for 24h, introducing a transcription factor to improve the transcription efficiency of the cell.
S4, the cells in S3 were cultured until the confluency was 95%.
Specifically, the confluency means the degree of confluency in an adhesion and arrangement state among cells when the cells proliferate in a flask, and the degree of confluency is 80% or more, which is advantageous for transfection of a target gene.
s5, introducing Mydo1 and Myogenin genes, specifically, introducing an expression vector containing Mydo1 and Myogenin genes, and continuously culturing to ensure that pluripotent stem cells are subjected to directional differentiation to obtain skeletal muscle cells.
In example one, an expression vector containing Mydo1 and Myogenin genes was prepared as follows: the lentiviral expression plasmid is digested by restriction enzyme to obtain an empty vector. The Mydo1 gene and the Myogen gene are respectively inserted into an empty body to obtain plasmids containing the Mydo1 gene and the Myogen gene. Then transfecting the T cells with plasmids containing Mydo1 and Myogen, culturing the transfected T cells and collecting culture supernatant to obtain an expression vector carrying Mydo1 and Myogen.
Mydo1 and Myogenin genes are introduced into pluripotent stem cell iPS together, so that the differentiation efficiency of human iPS cells can be greatly improved, and the purity of the obtained skeletal muscle cells is as high as 80%.
example two:
S1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a culture medium special for the pluripotent stem cells.
S2, preparing the human pluripotent stem cells cultured in the S2 into single cell suspension, centrifuging, resuspending, and adding into a culture plate for culture to form an embryoid body.
S3, after culturing the embryoid body obtained in the step S2 for 24h, introducing a transcription factor to improve the transcription efficiency of the cell.
s4, the cells in S3 were cultured until the confluency was 95%.
Specifically, the confluency means the degree of confluency in an adhesion and arrangement state among cells when the cells proliferate in a flask, and the degree of confluency is 80% or more, which is advantageous for transfection of a target gene.
S5, Mydo1, Pax3 and Myogenin genes are introduced, specifically, an expression vector containing Mydo1 and Myogenin genes is introduced, and the pluripotent stem cells are cultured continuously to undergo directed differentiation to obtain skeletal muscle cells.
in example one, an expression vector containing Mydo1, Pax3, and Myogenin genes was prepared as follows: the lentiviral expression plasmid is digested by restriction enzyme to obtain an empty vector. Mydo1, Pax3 and Myogen genes are respectively inserted into an empty carrier to obtain plasmids containing Mydo1, Pax3 and Myogen genes. Then transfecting the plasmid containing Mydo1, Pax3 and Myogen to a T cell, culturing the transfected T cell and collecting a culture supernatant to obtain an expression vector carrying Mydo1, Pax3 and Myogen.
Mydo1, Pax3 and Myogenin genes are introduced into pluripotent stem cell iPS together, so that the differentiation efficiency of human iPS cells can be greatly improved, and the purity of the obtained skeletal muscle cells is as high as 80%.
Example three:
S1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a culture medium special for the pluripotent stem cells.
s2, preparing the human pluripotent stem cells cultured in the S2 into single cell suspension, centrifuging, resuspending, and adding into a culture plate for culture to form an embryoid body.
S3, after culturing the embryoid body obtained in the step S2 for 24h, introducing a transcription factor to improve the transcription efficiency of the cell.
S4, the cells in S3 were cultured until the confluency was 95%.
Specifically, the confluency means the degree of confluency in an adhesion and arrangement state among cells when the cells proliferate in a flask, and the degree of confluency is 80% or more, which is advantageous for transfection of a target gene.
S5, introducing Desmin and Mef2c genes, specifically, introducing an expression vector containing Desmin and Mef2c genes, and continuing to culture to ensure that pluripotent stem cells are subjected to directional differentiation to obtain skeletal muscle cells.
In example one, an expression vector containing the Desmin and Mef2c genes was prepared as follows: the lentiviral expression plasmid is digested by restriction enzyme to obtain an empty vector. The Desmin and Mef2c genes are respectively inserted into an empty carrier to obtain plasmids containing the Desmin and Mef2c genes. Then, the plasmid containing Desmin and Mef2c is used for transfecting T cells, the transfected T cells are cultured, and culture supernatant is collected, so that an expression vector carrying the Desmin and the Mef2c is obtained.
the Desmin and Mef2c genes are introduced into pluripotent stem cell iPS together, so that the differentiation efficiency of human iPS cells can be greatly improved, and the purity of the obtained skeletal muscle cells is as high as 80%.
example four:
s1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a culture medium special for the pluripotent stem cells.
S2, preparing the human pluripotent stem cells cultured in the S2 into single cell suspension, centrifuging, resuspending, and adding into a culture plate for culture to form an embryoid body.
S3, after culturing the embryoid body obtained in the step S2 for 24h, introducing a transcription factor to improve the transcription efficiency of the cell.
S4, the cells in S3 were cultured until the confluency was 95%.
Specifically, the confluency means the degree of confluency in an adhesion and arrangement state among cells when the cells proliferate in a flask, and the degree of confluency is 80% or more, which is advantageous for transfection of a target gene.
S5, introducing Mydo1 and Myogenin genes, specifically, introducing an expression vector containing Mydo1 and Myogenin genes, and continuously culturing to ensure that pluripotent stem cells are subjected to directional differentiation to obtain skeletal muscle cells.
in example one, an expression vector containing Mydo1 and Myogenin genes was prepared as follows: the lentiviral expression plasmid is digested by restriction enzyme to obtain an empty vector. The Mydo1 gene and the Myogen gene are respectively inserted into an empty body to obtain plasmids containing the Mydo1 gene and the Myogen gene. Then transfecting the T cells with plasmids containing Mydo1 and Myogen, culturing the transfected T cells and collecting culture supernatant to obtain an expression vector carrying Mydo1 and Myogen.
mydo1 and Myogenin genes are introduced into pluripotent stem cell iPS together, so that the differentiation efficiency of human iPS cells can be greatly improved, and the purity of the obtained skeletal muscle cells is as high as 80%.
example five:
S1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a culture medium special for the pluripotent stem cells.
S2, preparing the human pluripotent stem cells cultured in the S2 into single cell suspension, centrifuging, resuspending, and adding into a culture plate for culture to form an embryoid body.
S3, after culturing the embryoid body obtained in the step S2 for 24h, introducing a transcription factor to improve the transcription efficiency of the cell.
S4, the cells in S3 were cultured until the confluency was 95%.
specifically, the confluency means the degree of confluency in an adhesion and arrangement state among cells when the cells proliferate in a flask, and the degree of confluency is 80% or more, which is advantageous for transfection of a target gene.
S5, introducing genes Pax7 and Mef2c, specifically, introducing an expression vector containing genes Pax7 and Mef2c, and continuing culturing to ensure that pluripotent stem cells are directionally differentiated to obtain skeletal muscle cells.
In example one, an expression vector containing the Pax7 and Mef2c genes was prepared as follows: the lentiviral expression plasmid is digested by restriction enzyme to obtain an empty vector. The Pax7 and Mef2c genes were inserted into the empty vector, respectively, to give plasmids containing the Pax7 and Mef2c genes. Then, the plasmid containing Pax7 and Mef2c was transfected into T cells, the transfected T cells were cultured and culture supernatant was collected to obtain expression vectors carrying Pax7 and Mef2 c.
The Pax7 and Mef2c genes are introduced into pluripotent stem cell iPS together, so that the differentiation efficiency of human iPS cells can be greatly improved, and the purity of the obtained skeletal muscle cells is as high as 80%.
In another embodiment, the present invention provides a skeletal muscle cell, which can be prepared by any of the above methods, and is not described herein again.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made without departing from the scope of the invention.
Claims (7)
1. A method for the rapid differentiation of pluripotent stem cells into skeletal muscle cells, comprising introducing an expression vector comprising at least one potent gene into pluripotent stem cells in a culture medium, thereby directing differentiation of the pluripotent stem cells into skeletal muscle cells.
2. The method for rapidly differentiating the pluripotent stem cells into skeletal muscle cells according to claim 1, wherein the potential genes comprise one or more of Mef2b, Pax3, Pax7, Mydo1, Pitx1, Myogenin, Mef2c, Mrf4 and Desmin.
3. The method for rapid differentiation of pluripotent stem cells into skeletal muscle cells according to claim 1, comprising:
S1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a special culture medium for the pluripotent stem cells;
S2, preparing the human pluripotent stem cells cultured in the S1 into single cell suspension, centrifuging and resuspending the single cell suspension, and adding the single cell suspension into a culture plate for culture to form an embryoid body;
S3, culturing the embryoid body obtained in the step S2 for 24h, and introducing a transcription factor;
S4, culturing the cells in the S3 until the confluency is 95%;
S5, introducing Mydo1 and Myogenin genes, and continuously culturing to ensure that pluripotent stem cells are directionally differentiated to obtain skeletal muscle cells.
4. the method for rapid differentiation of pluripotent stem cells into skeletal muscle cells according to claim 1, comprising:
S1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a special culture medium for the pluripotent stem cells;
S2, preparing the human pluripotent stem cells cultured in the S1 into single cell suspension, centrifuging and resuspending the single cell suspension, and adding the single cell suspension into a culture plate for culture to form an embryoid body;
s3, culturing the embryoid body obtained in the step S2 for 24h, and introducing a transcription factor;
S4, culturing the cells in the S3 until the confluency is 95%;
S5, Mydo1, Pax3 and Myogenin genes are introduced, and the pluripotent stem cells are cultured continuously to undergo directed differentiation to obtain skeletal muscle cells.
5. The method for rapid differentiation of pluripotent stem cells into skeletal muscle cells according to claim 1, comprising:
S1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a special culture medium for the pluripotent stem cells;
s2, preparing the human pluripotent stem cells cultured in the S1 into single cell suspension, centrifuging and resuspending the single cell suspension, and adding the single cell suspension into a culture plate for culture to form an embryoid body;
s3, culturing the embryoid body obtained in the step S2 for 24h, and introducing a transcription factor;
S4, culturing the cells in the S3 until the confluency is 95%;
S5, introducing Desmin and Mef2c genes, and continuously culturing to ensure that pluripotent stem cells are subjected to directional differentiation to obtain skeletal muscle cells.
6. the method for rapid differentiation of pluripotent stem cells into skeletal muscle cells according to claim 1, comprising:
S1, culturing the human pluripotent stem cells on a culture plate coated with matrigel, wherein the culture medium is a special culture medium for the pluripotent stem cells;
S2, preparing the human pluripotent stem cells cultured in the S1 into single cell suspension, centrifuging and resuspending the single cell suspension, and adding the single cell suspension into a culture plate for culture to form an embryoid body;
S3, culturing the embryoid body obtained in the step S2 for 24h, and introducing a transcription factor;
s4, culturing the cells in the S3 until the confluency is 95%;
S5, Pax7 and Mef2c genes are introduced, and the pluripotent stem cells are cultured continuously to undergo directed differentiation to obtain skeletal muscle cells.
7. A skeletal muscle cell prepared by the method of any one of claims 1 to 6.
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| US20170107487A1 (en) * | 2014-06-10 | 2017-04-20 | President And Fellows Of Harvard College | Methods for differentiation |
| WO2017106932A1 (en) * | 2015-12-23 | 2017-06-29 | Monash University | Cell reprogramming |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170107487A1 (en) * | 2014-06-10 | 2017-04-20 | President And Fellows Of Harvard College | Methods for differentiation |
| WO2017106932A1 (en) * | 2015-12-23 | 2017-06-29 | Monash University | Cell reprogramming |
Non-Patent Citations (5)
| Title |
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| RADBOD DARABI等: "Functional skeletal muscle regeneration from differentiating embryonic stem cells", 《NATURE MEDICINE》 * |
| SABRINA SALANI等: "Generation of skeletal muscle cells from embryonic and induced pluripotent stem cells as an in vitro model and for therapy of muscular dystrophies", 《J. CELL. MOL. MED.》 * |
| YUSAKU KODAKA等: "Skeletal Muscle Cell Induction from Pluripotent Stem Cells", 《STEM CELLS INTERNATIONAL》 * |
| 徐威主编: "《药物细胞生物学》", 31 August 2015 * |
| 郭珊珊等: "干细胞向骨骼肌细胞的诱导分化及其在治疗骨骼肌病变中的应用", 《中国细胞生物学学报》 * |
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