Method for inducing minimal embryonic stem cells to differentiate into cardiomyocytes

A technology of embryonic stem cells and cardiomyocytes, which is applied in the field of inducing the differentiation of rat minimal embryonic stem cells into cardiomyocytes, can solve the problems of limited popularization and application of immune rejection, insufficient ethics of cell sources, etc., and achieve rigorous experimental process and verification results reasonably accurate effect

Active Publication Date: 2020-11-06
YANGZHOU UNIV
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Problems solved by technology

At present, a variety of stem cells (embryonic stem cells, bone marrow mesenchymal stem cells) have been successfully induced into cardiomyocytes in vitro, but problems such as insufficient cell sources, ethics, and immune rejection limit their clinical application.

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  • Method for inducing minimal embryonic stem cells to differentiate into cardiomyocytes
  • Method for inducing minimal embryonic stem cells to differentiate into cardiomyocytes

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Embodiment Construction

[0023] The present invention will be further described below in conjunction with the drawings and descriptions of the drawings.

[0024] A method for inducing rat very small embryonic stem cells to differentiate into cardiomyocytes, the specific method comprising:

[0025] (1) Separate the rat tibia and femur, remove the muscle and connective tissue, wash out the bone marrow with PBS, add red blood cell lysate to the washed out bone marrow and place it in the dark for 10 minutes, and obtain the total mononuclear cells (TNCs) after centrifugation, CD45 , CD106, and lin monoclonal antibody (1:200) were incubated at room temperature for 30 minutes, and CD45-Lin-CD106+ cells (very small embryonic stem cells) were obtained by flow cytometry sorting. The sorted minimal embryonic stem cells were cultured in a DMEM medium containing 10% embryonic bovine serum in a 37°C, 5% CO2 incubator. During the proliferation of the very small embryonic stem cells, the medium was changed every thr...

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Abstract

The invention relates to a method for inducing minimal embryonic stem cells to differentiate into cardiomyocytes. The method includes the following steps: (1) obtaining CD45-Lin-CD106+cells (minimal embryonic stem cells) in rat bone marrow through flow cytometry sorting; (2) inducing, by 5-azacytidine, the minimal embryonic stem cells to differentiate; and (3) through qRT-PCR and immunofluorescence, detecting myocardial protein related genes, and validating the process that the 5-azacytidine induces the minimal embryonic stem cells to differentiate. The purpose of the method is to isolate andculture the minimal embryonic stem cells from rat bone marrow tissue; through the adoption of the 5-azacytidine to induce and differentiate, biological characteristics and directed differentiation characteristics can be observed, so that a convenient and fast approach for cell transplantation treatment of clinical bradyarrhythmia can be found. Validation results are reasonable and accurate, and experimental processes are precise and cautious.

Description

technical field [0001] The invention relates to a method for inducing rat very small embryonic stem cells to differentiate into cardiomyocytes, belonging to the field of stem cell differentiation and cultivation. Background technique [0002] Bradyarrhythmia is a group of life-threatening diseases including sick sinus syndrome and atrioventricular block, which is characterized by cardiac pacing or conduction dysfunction. Biological pacemakers composed of somatic gene transfection, cell fusion, or cell transplantation provide a new option for the treatment of bradyarrhythmias. Pure gene biological pacing has uncertainties in transfection targeting and transfection efficiency, so people gradually choose cell biological pacing therapy. Although the cellular biological pacemaker has many advantages, there are two problems, 1) the selection of seed cells is difficult, and 2) the induction method is difficult to choose. At present, a variety of stem cells (embryonic stem cells, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/074
CPCC12N5/0657C12N5/0607C12N2506/03C12N2501/06C12N2509/00
Inventor 孙小林顾翔徐佩李红校朱业左其生李碧春姜江董晓娜韦玮
Owner YANGZHOU UNIV
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