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Method for inducing ipsc to be differentiated into myocardial cells

A cardiomyocyte and cell technology, applied in the biological field, can solve the problems of long differentiation time and low differentiation efficiency, and achieve the effect of short differentiation time and high differentiation efficiency

Pending Publication Date: 2022-05-24
武汉百翼生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the above-mentioned current methods for inducing iPSCs to differentiate into cardiomyocytes take a long time to differentiate, and the differentiation efficiency is still low.

Method used

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  • Method for inducing ipsc to be differentiated into myocardial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] A method for inducing the differentiation of iPSCs into cardiomyocytes

[0024] S1 ipsc cell culture: Use matrigel working solution diluted 1:150 to completely cover the petri dish, and let stand at 37°C overnight. The iPSC clones are cultured in the Matrigel-coated petri dish until the degree of incubation reaches 70% to 80%. When culturing, remove the stem cell culture medium, then wash 1-3 times with PBS solution without calcium and magnesium, subculture with 0.5mmol / L EDTA, shake the culture flask gently during culture, so that EDTA covers all the cell surface, wait for the cell layer Slightly loose, when the "film" phenomenon can be observed with the naked eye, pour out the EDTA, continue to act for 2-3 minutes, shake gently, the cell layer can fall off from the bottle wall in flakes with the remaining EDTA, when it is passed through a microscope When it is found that the cells shrink and become round, and the intercellular space increases, the digestion should be ...

Embodiment 2

[0031] A method for inducing the differentiation of iPSCs into cardiomyocytes

[0032] S1 ipsc cell culture: use 1:200 diluted matrigel working solution to completely cover the petri dish, and let stand at 37°C overnight. The iPSC clones are cultured in the Matrigel-coated petri dish until the degree of rotation reaches 70% to 80%. During the culture, the stem cell culture medium was aspirated, then washed 1-3 times with PBS solution without calcium and magnesium, and subcultured with 0.5mmol / L EDTA. Gently shake the culture flask during culture, so that EDTA covers all the cell surface, wait for the cell layer Slightly loose, when the "film" phenomenon can be observed with the naked eye, pour out the EDTA, continue to act for 2-3 minutes, shake gently, the cell layer can fall off from the bottle wall in flakes with the remaining EDTA, when it is passed through a microscope When it is found that the cells shrink and become round, and the intercellular space increases, the dige...

Embodiment 3

[0039] A method for inducing the differentiation of iPSCs into cardiomyocytes

[0040] S1 ipsc cell culture: Use matrigel working solution diluted 1:250 to completely cover the petri dish, and let stand at 37°C overnight. The iPSC clones are cultured in the Matrigel-coated petri dish until the degree of rotation reaches 70% to 80%. When cultured, the stem cell culture medium was aspirated, then washed 1-3 times with calcium and magnesium-free PBS solution, and subcultured with 0.5 mmol / L EDTA. Gently shake the culture flask during culture to make EDTA cover all cell surfaces, and wait for the cell layer Slightly loose, when the "film" phenomenon can be observed with the naked eye, pour out the EDTA, continue to act for 2-3 minutes, shake gently, the cell layer can fall off from the bottle wall in flakes with the remaining EDTA, when it is passed through a microscope When it is found that the cells shrink and become round, and the intercellular space increases, the digestion sh...

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Abstract

The invention discloses a method for inducing ipsc to be differentiated into myocardial cells, and relates to the technical field of biology. The method comprises the following steps: S1, carrying out ipsc cell culture; and S2, inducing the ipsc to be differentiated into myocardial cells. The method comprises the following steps: culturing ipsc by using a stem cell culture medium until the ipsc grows to 80%, culturing by using a mixed culture medium, inducing the ipsc to differentiate into mesoderm precursor cells, culturing by using an inhibitor culture medium, differentiating the cells into myocardial-like precursor cells, and culturing by using a maintenance culture medium to obtain myocardial cells. The mixed culture medium is a basic culture medium containing Chir99021 and atractylenolide II, and the basic culture medium is an RPMI 1640 culture medium containing a B27 additive and LAA2P; the inhibitor culture medium is a basic culture medium containing Wnt-C59; the maintenance culture medium is a basic culture medium. The method for inducing the ipsc to be differentiated into the myocardial cells has the advantages that the differentiation time is short (jumping myocardial cells can be differentiated in 3 days), and the differentiation efficiency is high (the differentiation efficiency reaches up to 85% in 7 days).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for inducing ipsc to differentiate into cardiomyocytes. Background technique [0002] At present, the last resort to treat heart failure, the terminal stage of heart disease, is transplantation. However, many patients die while waiting due to a severe shortage of donor resources. The advent of the iPSC-CM approach brings new hope to this status quo, with the main long-term goal of transforming the clinical practice of obtaining myocardial tissue from a donor for the treatment of damaged hearts. [0003] The gene regulatory network related to the development of the early embryonic cardiovascular system is the theoretical basis for myocardial differentiation. In the past 10 years, the research devoted to the differentiation of stem cells into cardiac muscle mostly adopts stage-specific activation or inhibition of different signaling pathways, and achieves the purpose of fina...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/074
CPCC12N5/0657C12N2506/45C12N2501/727C12N2501/999C12N2501/415C12N2500/30
Inventor 胡俊程亚婷欧阳佳李颖陈志毅苏雨晴袁天立林宝怡
Owner 武汉百翼生物科技有限公司
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