Small-molecule compound composition capable of efficiently inducing differentiation of human multipotent stem cells into myocardial cells

A small molecule compound and stem cell differentiation technology, applied in artificially induced pluripotent cells, drug combinations, medical preparations containing active ingredients, etc., can solve the problems of low differentiation efficiency, low yield, unsatisfactory efficiency, etc.

Active Publication Date: 2017-07-28
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These induction methods not only require animal-derived serum, but also have low differentiation efficiency and very low yield
In 2007, the Charles Murry laboratory established a monolayer cell differentiation system. By adding the cardiomyocyte differentiation cytokines activin A and BMP4 to the basic differentiation medium composed of RPMI1640 and B27, the differentiation efficiency of 30% was achieved in the human embryonic stem cell line H7. However, the efficiency in the human embryonic stem cell line H9 is not ideal, only less than 5%
[0003] Although after years of development, the method of cardiac differentiation of human embryonic stem cells has been greatly improved, but there is still room for improvement in differentiation efficiency, and the stability of differentiation and consistency in different cell lines also need to be further improved, and more importantly, the cost of differentiation Factors affecting large-scale production such as production and output need to be further improved

Method used

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  • Small-molecule compound composition capable of efficiently inducing differentiation of human multipotent stem cells into myocardial cells
  • Small-molecule compound composition capable of efficiently inducing differentiation of human multipotent stem cells into myocardial cells
  • Small-molecule compound composition capable of efficiently inducing differentiation of human multipotent stem cells into myocardial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0168] Example 1 Screening of small molecule compounds that promote the differentiation of human stem cells into cardiomyocytes

[0169] Using the H9-cTnT-eGFP human embryonic stem cell line as a reporter system, small molecule compounds that can promote the efficient differentiation of stem cells into cardiomyocytes were screened under the induction conditions of cytokine-free and high-density monolayer culture.

[0170] After extensive screening, it was found that the combination of mTOR signaling pathway inhibitor Rapamycin and Wnt pathway promoter CHIR99021 can significantly promote the differentiation of human stem cells into cardiomyocytes.

Embodiment 2

[0171] Example 2 The combination of rapamycin and CHIR99021 can promote the differentiation of human stem cells into cardiomyocytes

[0172] Preliminary screening results:

[0173] The result is as figure 1 A shown. The results showed that the combination of mTOR signaling pathway inhibitor-Rapamycin and Wnt pathway promoter CHIR99021 could significantly improve the differentiation efficiency of human embryonic stem cells (H9 cell line) into cardiomyocytes, up to 86%. The cardiomyocyte differentiation rate obtained by using CHIR99021 alone is very low, only about 15%, and the cardiomyocyte differentiation efficiency obtained by using rapamycin alone is almost zero.

[0174] Optimization Results:

[0175] After human embryonic stem cells were treated with CHIR (10nM), the inventors added and withdrew rapamycin according to the specified time points after optimizing the treatment time window of rapamycin (10nM). According to the characteristics of monolayer differentiation and...

Embodiment 3

[0179] Enrichment and purification of cardiomyocytes induced by the compound of Example 3

[0180] After 15 days of differentiation, the cardiomyocyte culture medium was replaced with glucose-free DMEM / F12 medium plus 10% (v / v) fetal bovine serum (FBS) and 1mM lactose was added as the only carbon source in the culture, which was changed every two days solution and cultured continuously for 7 days. The proportion of cTnT positive cardiomyocytes was detected by flow cytometry.

[0181] The results showed that culturing the cardiomyocytes derived from stem cells in 1mM lactose (the only carbon source) medium for 7 days could make the purity of cardiomyocytes reach 99.9% ( figure 2 A), statistically found that 1 stem cell input can produce 24 cardiomyocytes, and only half of the culture medium and added factors need to be consumed.

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Abstract

The invention discloses a small-molecule compound composition capable of efficiently inducing differentiation of human multipotent stem cells into myocardial cells. Specifically, the small-molecule compound composition provided by the invention comprises (i) a mTOR signal pathway inhibitor, (ii) a Wnt pathway promoter and (iii) an optional pharmaceutically-acceptable vector. The small-molecule compound composition provided by the invention can efficiently induce differentiation of multipotent stem cells into myocardial cells, wherein the differentiation rate of myocardial cells is as high as 86% after primary screening, and the differentiation rate of myocardial cells is as high as 98.3% after optimization.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, the present invention relates to a combination of small molecule compounds for efficiently inducing the differentiation of human pluripotent stem cells into cardiomyocytes. Background technique [0002] Myocardial disease is one of the major diseases with the highest morbidity and mortality in modern society. Myocardial damage caused by diseases such as myocardial ischemia is irreversible cell death, and there is still no effective treatment. Since the discovery of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPS) (collectively referred to as hPSCs), human pluripotent stem cell-derived cardiomyocytes have become a new hope in the field of cardiomyopathy treatment. In addition to cell therapy, stem cell-derived cardiomyocytes can also be used in important aspects such as therapeutic drug screening for cardiomyopathies and detection of drug cardio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/0735A61K35/34A61P9/00
CPCA61K35/34C12N5/0657C12N2501/04C12N2506/02C12N2506/45C12N2501/415A61K31/436A61K31/506A61P9/00
Inventor 荆清邱晓旭刘洋张一凡
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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