A method for inducing directional differentiation of pluripotent stem cells into cardiomyocytes

A technology of pluripotent stem cells and cardiomyocytes, applied in the field of induction of directional differentiation of pluripotent stem cells into cardiomyocytes, can solve the problems of complicated differentiation methods, low safety, and arrhythmia, so as to reduce the area of ​​myocardial infarction and improve the differentiation efficiency , low arrhythmia effect

Active Publication Date: 2021-04-30
SUZHOU UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

[0004] At present, the shortcomings of the differentiation technology of cardiomyocytes mainly include the following aspects: (1) the existing differentiation method is still immature, showing poor reproducibility and unstable differentiation efficiency; (2) the differentiation method is complicated, and the required cost Higher; (3) The differentiated cardiomyocytes have low purity, low safety, and poor uniformity; (4) The induced cardiomyocytes are transplanted into animals, and there are problems such as arrhythmia, tumorigenicity, and poor remodeling of the heart structure.

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  • A method for inducing directional differentiation of pluripotent stem cells into cardiomyocytes
  • A method for inducing directional differentiation of pluripotent stem cells into cardiomyocytes
  • A method for inducing directional differentiation of pluripotent stem cells into cardiomyocytes

Examples

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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 2Digest 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 ste...

Embodiment 2

[0040] Example 2 Directed induction of embryonic stem cells to differentiate into cardiomyocytes

[0041] 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.

[0042] 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 RPMI1640+B27-insulin containing 1 μM retinoic acid was used to induce differentiation on the 2nd to 3rd day , cultured with RPMI1640+B27-insulin containing 5 μM Wnt inhibitor on the 4th to 5th day. On the 6th day, only RPMI1640+B27-insulin was used for culture. After the 7th day, the cells were cultured with RPMI1640+B27 every day, and obvious myocardial beati...

Embodiment 3

[0052] Example 3 Culture, passage and directional induction of induced pluripotent stem cells

[0053] In this example, the HES3 cells in Examples 1 and 2 are replaced by the SCCTM-iPSC-1 cell line in hPSCs as the experimental object. For other specific implementation methods, refer to Examples 1 and 2. According to the statistical experiment results of flow cytometry, it is shown that SCCTM - The positive expression of cTnT in the cardiomyocyte control group (DMSO group) induced by iPSC-1 cells was 36.93% ( Figure 4 C), the cTnT positive expression of RA group is 59.85% ( Figure 4 D).

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Abstract

An induction method for improving the directed differentiation of pluripotent stem cells into cardiomyocytes: the pluripotent stem cells subcultured to 4 to 5 generations are induced to differentiate on the 0th to 1st day, and the medium used contains 2 to 15 μM of GSK-3 Inhibitor; use medium containing 0.2-5 μM retinoic acid to induce differentiation on days 2-3; induce differentiation on days 4-5 using medium containing 2-10 μM Wnt inhibitor; The cells are induced to differentiate, and the beating of cardiomyocytes can be observed on the 9th to 10th days; wherein, on the 1st to 6th days, the first induced differentiation medium is used, which includes RPMI-1640 basal medium and B27-insulin; After day 7, use a second induction medium, which includes RPMI-1640 basal medium and B27; or throughout the induction culture process, use CDM3 induction medium or serum-free induction medium in which CDM3 Differentiation induction medium includes RPMI‑1640 basal medium, serum albumin, ascorbic acid, and dual antibodies.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to an induction method for improving directional differentiation of pluripotent stem cells into cardiomyocytes. Background technique [0002] The morbidity and mortality rate of heart disease has ranked first at home and abroad, and it has become one of the diseases that seriously endanger human health. Most patients suffer from myocardial damage due to ischemic or other pathological injuries, resulting in cardiac insufficiency, which seriously threatens life. Stem cells have the characteristics of high self-renewal, proliferation and multi-directional differentiation potential, implantability and reconstruction ability, and have immeasurable medical value and attractive application prospects in the field of regenerative medicine. In recent years, with the in-depth development of stem cell technology, the use of cardiomyocytes differentiated from stem cells has provided the po...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2500/38C12N2501/415C12N2501/998
Inventor 胡士军苗淑梅雷伟赵振奥沈振亚唐明正
Owner SUZHOU UNIV
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