Method for rapidly differentiating pluripotent stem cells into skeletal muscle cells and skeletal muscle cells

A technology of pluripotent stem cells and skeletal muscle cells, applied in the field of stem cells, can solve the problems of long time period, influence, and long time of induction of skeletal muscle cells, and achieve the effect of shortening the induction cycle

Inactive Publication Date: 2019-12-13
张文胜
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main technical problems of directed differentiation of induced pluripotent stem cells are low efficiency, long time period, low purity of cells, and incomplete functionality of the generated cells. These problems affect the use of directed differentiated cells for drug screening and toxicity testing reliability
Induction of skeletal muscle cells from human pluripotent stem cells takes a long time and is inefficient

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] S1. Human pluripotent stem cells were cultured on matrigel-coated culture plates, and the medium was a special medium for pluripotent stem cells.

[0018] S2. The human pluripotent stem cells cultured in S2 are made into a single cell suspension, and after centrifugation and resuspension, they are added to a culture plate for cultivation to form embryoid bodies.

[0019] S3, after cultivating the embryoid bodies obtained in step S2 for 24h, import transcription factors to improve the transcription efficiency of cells.

[0020] S4. Culture the cells in S3 until the confluence is 95%.

[0021] Specifically, the degree of confluence refers to the degree of confluence of the adhesion and arrangement state between cells when the cells proliferate in the bottle, and a confluence of more than 80% is conducive to the transfection of the target gene.

[0022] S5. Introduce Mydo1 and Myogenin genes, specifically, import the expression vectors containing Mydo1 and Myogenin genes,...

Embodiment 2

[0026] S1. Human pluripotent stem cells were cultured on matrigel-coated culture plates, and the medium was a special medium for pluripotent stem cells.

[0027] S2. The human pluripotent stem cells cultured in S2 are made into a single cell suspension, and after centrifugation and resuspension, they are added to a culture plate for cultivation to form embryoid bodies.

[0028] S3, after cultivating the embryoid bodies obtained in step S2 for 24h, import transcription factors to improve the transcription efficiency of cells.

[0029] S4. Culture the cells in S3 until the confluence is 95%.

[0030] Specifically, the degree of confluence refers to the degree of confluence of the adhesion and arrangement state between cells when the cells proliferate in the bottle, and a confluence of more than 80% is conducive to the transfection of the target gene.

[0031] S5. Introduce Mydo1, Pax3 and Myogenin genes, specifically, introduce expression vectors containing Mydo1 and Myogenin g...

Embodiment 3

[0035] S1. Human pluripotent stem cells were cultured on matrigel-coated culture plates, and the medium was a special medium for pluripotent stem cells.

[0036] S2. The human pluripotent stem cells cultured in S2 are made into a single cell suspension, and after centrifugation and resuspension, they are added to a culture plate for cultivation to form embryoid bodies.

[0037] S3, after cultivating the embryoid bodies obtained in step S2 for 24h, import transcription factors to improve the transcription efficiency of cells.

[0038] S4. Culture the cells in S3 until the confluence is 95%.

[0039] Specifically, the degree of confluence refers to the degree of confluence of the adhesion and arrangement state between cells when the cells proliferate in the bottle, and a confluence of more than 80% is conducive to the transfection of the target gene.

[0040] S5. Introduce Desmin and Mef2c genes, specifically, introduce an expression vector containing Desmin and Mef2c genes, an...

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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, and aims to solve the problems of long time and low efficiency of inducing the skeletal muscle cells by the pluripotent stem cells. The method specifically comprises the step of introducing an expression vector containing at least one potentialgene into the pluripotent stem cells in a culture medium so as to directionally differentiate the pluripotent stem cells into the skeletal muscle cells. At different stages for inducing the directional differentiation of human pluripotent stem cells into skeletal muscle cells, the specific induction of expression of one or more different combination genes of Mef2b, Pax3, Pax7, Mydo1, Pitx1, Myogenin, Mef2c, Mrf4 and Desmin is carried out, the functional skeletal muscle cells with purity as high as 80% can be obtained, and an induction period is substantially shortened.

Description

technical field [0001] 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 skeletal muscle cells. Background technique [0002] The unlimited proliferation ability and pluripotency of human embryonic stem cells provide the possibility to obtain a large number of all cell types in the human body. However, due to the acquisition of human embryonic stem cells from different individuals and ethical restrictions, it is very difficult to obtain differentiated cells from human embryonic stem cells for personalized treatment and drug screening. The technique of inducing pluripotent stem cells from human somatic cells avoids the above-mentioned limitations of human embryonic stem cells, making it possible to generate human induced pluripotent stem cells from somatic cells for each individual person, which makes these cells ideal for disease models, The best choice for drug scree...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N15/867C12N5/10
CPCC07K14/47C07K14/4702C12N5/0658C12N15/86C12N2501/60C12N2506/45C12N2740/15043
Inventor 张文胜李鹏吴倩鑫
Owner 张文胜
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