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Method for promoting the differentiation of pluripotent stem cells into cardiomyocytes

A technology for pluripotent stem cells and cardiomyocytes, which is applied in artificially induced pluripotent cells, biochemical equipment and methods, embryonic cells, etc., to solve the problem of low maturity, promote maturation, and arrange neatly

Active Publication Date: 2020-10-09
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In-depth study of the role of RA in the process of induction of cardiomyocytes from pluripotent stem cells is conducive to promoting the maturation of cardiomyocytes. This study has not yet been reported.

Method used

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  • Method for promoting the differentiation of pluripotent stem cells into cardiomyocytes
  • Method for promoting the differentiation of pluripotent stem cells into cardiomyocytes
  • Method for promoting the differentiation of pluripotent stem cells into cardiomyocytes

Examples

Experimental program
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Embodiment 1

[0036] Cultivation and subculture of embryonic stem cells of embodiment 1

[0037] In this example, the HES3 cell line in hPSCs was used as the experimental object. The HES3 cells were grown on Matrigel-coated culture dishes and diluted with 1:200 DMEM / F12 basal medium for use. Then the HES3 cells were planted on matrigel, and the mTeSR1 or E8 medium was used for cell culture, and the medium was changed every day. When the HES3 cell density reaches about 80% and the cell clone is large enough, the cells are subcultured.

[0038] When subcultured, wash twice with DPBS to remove dead cell debris on the surface, add 0.1mol / L EDTA and place at 37°C, 5% CO 2 Digest for 7 minutes in a constant temperature incubator. After the digestion time is up, observe the gaps between the adherent cells under the inverted microscope but have not yet separated from each other. The cell colonies are observed to be opaque and whitish with the naked eye. Suck off the digestion solution, add the st...

Embodiment 2

[0039]Example 2 Directed induction of embryonic stem cells to differentiate into mature cardiomyocytes

[0040] The HES3 cells that had been cultured and subcultured to passage 4-5 were washed with DPBS, digested with 0.1 mol / L EDTA for 7 minutes, and washed with 10 4 / cm 2 Inoculate the culture dish at a density of 37°C, 5% CO 2 Cultivate in the incubator for 4 days, and the cell density reaches more than 85%, and replace with RPMI1640+B27-insulin induction medium.

[0041] Retinoic acid treatment group (RA group): RPMI1640+B27-insulin containing 5 μM CHIR99021 was used to induce differentiation on the 0th to 1st day, and cultured with RPMI1640+B27-insulin on the 2nd to 3rd day; Days were cultured with RPMI1640+B27-insulin containing 5 μM Wnt inhibitor. On the 6th day, only RPMI1640+B27-insulin was used for culture. After the 7th day, cultured with RPMI1640+B27 every day, obvious myocardial beating can be observed on the 9th to 10th day (D9~D10) of induced differentiation...

Embodiment 3

[0046] Example 3 Culture, passage and directional induction of induced pluripotent stem cells

[0047] In this example, the HES3 cells in Examples 1 and 2 were replaced with the SCCTM-iPSC-1 cell line in hPSCs as the experimental object, and other specific implementation methods refer to Examples 1 and 2.

[0048] The action potential changes of ventricular myocytes were recorded by patch clamp. On the 30th day, the cardiomyocytes were digested with 0.25% trypsin for 5 minutes, single cells were plated, and adhered to the wall for 3 days, and then detected, including the beating frequency, action potential amplitude, ascending speed and hyperpolarized diastolic potential of cardiomyocytes. According to the experimental results, the results of cardiomyocytes induced by SCCTM-iPSC-1 cells are as follows: Figure 4 as shown, Figure 4 A, B, and C are the test results of cardiomyocytes in the DMSO group, the test results of cardiomyocytes in the RA group, and the comparison char...

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Abstract

The invention relates to a method for promoting maturation of myocardial cells differentiated from multipotential stem cells. In the 0-1 day, the multipotential stem cells which are subcultured to bethe 4-5 generation are subjected to induced differentiation, and a culture medium used in the method contains a GSK-3 inhibitor with the concentration being 2-15 mu M / L; in the 2-3 day, the culture medium is used for conducting induced differentiation continuously; in the 4-5 day, a culture medium containing a Wnt inhibitor with the concentration being 2-10 mu M / L is used for conducting induced differentiation; in the 14-20 days, a culture medium containing retinoic acid with the concentration being 0.2-5 mu M / L is used for conducting induced differentiation continuously, later on, the culturemedium is used for conducting induced differentiation culture on the multipotential stem cells, wherein in the 1-6 days, a first induced differentiation culture medium is used and comprises an RPMI-1640 basic culture medium and B27-insulin, after the 7 day, a second induced differentiation culture medium is used and comprises the RPMI-1640 basic culture medium and B27, or in the whole induced differentiation culture process, a CDM3 induced differentiation culture medium or a serum-free induced differentiation culture medium is used, and the CDM3 induced differentiation culture medium comprises the RPMI-1640 basic culture medium, serum albumin, ascorbic acid and double-antibody.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for promoting the differentiation of pluripotent stem cells into cardiomyocytes. Background technique [0002] According to the "China Cardiovascular Disease Report 2015", the mortality rate of cardiovascular disease still ranked first in 2014, and 2 out of every 5 deaths were due to cardiovascular disease. Compared with 1990, the absolute number of cardiovascular disease deaths increased by 46% in 2013; among them, the number of ischemic heart disease deaths increased by 90.9%. Ischemia and subsequent reperfusion caused by coronary artery occlusion can cause irreversible cellular damage, resulting in necrosis of cardiomyocytes and decreased systolic function of the heart. Since adult cardiomyocytes lack regenerative capacity and cannot repair necrotic myocardium, cell transplantation may become an effective therapeutic approach for ischemic heart disease. At pres...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2500/38C12N2501/405C12N2501/415C12N2506/02C12N2506/45
Inventor 赵振奥胡士军苗淑梅雷伟沈振亚陈一欢
Owner SUZHOU UNIV
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