Method for differentiating as ventricular muscle cells by in-vitro induced pluripotent stem cells

A technology of pluripotent stem cells and ventricular myocytes, which is applied in the field of pluripotent stem cell differentiation and cell signal transduction, can solve the problem that the ventricular muscle is not clearly identified, the efficiency of cardiomyocyte differentiation is not high, and the directional differentiation of different cardiomyocytes cannot be realized. question

Active Publication Date: 2017-12-12
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The various methods of using stem cells to differentiate cardiomyocytes have the following defects: the efficiency of inducing cardiomyocyte differentiation is not high, and the obtained cardiomyocytes are a mixed cell population of pacemaker cells, atrial myocytes and ventricular myocytes, which cannot be achieved. Directed differentiation of different cardiomyocytes 4
Although this research result explains for the first time that stem cell myocardial differentiation is a method to induce atrial myocytes and ventricular myocytes, no clear active regulatory molecules have been identified for how to induce ventricular myocyte differentiation

Method used

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  • Method for differentiating as ventricular muscle cells by in-vitro induced pluripotent stem cells

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Experimental program
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Embodiment 1

[0104] Example 1 The role of BMP / Smad1 / 5 / 8 signaling pathway in inducing the differentiation of cardiac precursor cells into ventricular myocytes

[0105] 1 Studies have found that in the process of myocardial differentiation of stem cells, at the stage of determining the differentiation of cardiomyocyte subtypes, adding retinoic acid or its synthesis-required substrates such as vitamin A to the medium can induce stem cells to become atrial myocytes; If retinoic acid inhibitors are added to the medium at this time or vitamin A, the synthetic substrate of retinoic acid, is removed from the medium, stem cells can be directed to differentiate into ventricular myocytes 9 . By RT-PCR reaction, analyze the expression of BMP2 / 4 and its corresponding receptors in the differentiated human embryonic stem cells in the middle stage of differentiation, the results are as follows figure 1 As shown, both ligands and receptors of the BMP pathway are present in cultured cells. Western blot w...

Embodiment 2

[0115]Example 2 Differentiation of Induced Pluripotent Stem Cells into Ventricular Myocytes in Vitro (Technical Scheme 1)

[0116] Spread human embryonic stem cells H7 on a culture dish containing gelatin (Gelatin), add RPMI1640 medium containing B27 (1×concentration) at 37°C CO 2 cultured in a cell culture incubator. The process of myocardial differentiation is Figure 9 As indicated, from day 0 to day 3, Activin A (10 ng / mL), BMP4 (6 ng / mL) and bFGF (6 ng / mL) were added to the medium. At the end of the third day, the medium was changed, and Noggin (300 ng / mL), an inhibitor of BMP2 / 4, was added at the same time. At the end of day 5, the medium was changed to RPMI1640 medium with B27 without vitamin A supplementation. At the same time, Wnt3a inhibitors DKK1 (300ng / mL) and BMP4 (10ng / mL) were added to the medium. At the end of day 8 of differentiation, the medium was changed to one containing only 300 ng / mL DKK1, and at the end of day 10 of differentiation without the addit...

Embodiment 3

[0117] Example 3 Differentiation of Induced Pluripotent Stem Cells into Ventricular Myocytes in Vitro (Technical Scheme 2)

[0118] Spread human embryonic stem cells H7 on a culture dish containing gelatin (Gelatin), add RPMI1640 medium containing B27 (1×concentration) at 37°C CO 2 cultured in a cell culture incubator. The process of myocardial differentiation is Figure 10 From day 0 to day 3, Activin A (10 ng / mL), BMP4 (6 ng / mL) and bFGF (6 ng / mL) were added to the medium as indicated. At the end of the 3rd day, the medium was changed, and Noggin (300 ng / mL), an inhibitor of BMP2 / 4, was added at the same time. At the end of day 5, the medium was changed to RPMI1640 medium with B27 without vitamin A supplementation. At the same time, the Wnt3a inhibitor DKK1 (300 ng / mL) and the retinoic acid receptor RARγ activator BMS961 (0.1 μM, purchased from Tocris) were added to the medium. At the end of day 8 of differentiation, change the medium to one containing only 300 ng / mL DKK...

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Abstract

The invention provides a method for differentiating as ventricular muscle cells by in-vitro induced pluripotent stem cells. The method comprises the following steps: through maintaining, amplifying, and culturing pluripotent stem cells in vitro, in the metaphase of pluripotent stem cell cardiac muscle differentiation, namely the differentiation stage from mesoblastema or cardiac muscle precursor cells to the cardiac muscle cells, adding a substance for directly or indirectly activating an Smad1 / 5 / 8 signal channel to a culture medium, and enabling the stem cells to be directionally differentiated as the ventricular muscle cells. The ventricular muscle cells with the biology activity and function can be successfully obtained by using the method. A regulation mechanism in the differentiation process from the cardiac muscle precursor cells to the ventricular muscle cells is presented, and the human ventricular muscle cells obtained by the differentiation can be extensively applied for the myocardial infarction therapy by the cell transplantation and the cardiac toxicology analysis and the development of cardiac related drugs.

Description

[0001] The present invention is a PCT invention patent application with the international application number PCT / CN2013 / 079811. The priority date is July 23, 2012. The national phase application number is 2013800394581. The title of the invention is "in vitro induction of pluripotent stem cells into ventricular myocytes" Method" is a divisional application of the Chinese patent application. technical field [0002] The invention relates to the fields of differentiation of pluripotent stem cells and cell signal transduction, in particular to a method for inducing differentiation of pluripotent stem cells into ventricular myocytes in vitro. Background technique [0003] In humans and mammals, cardiomyocytes have the ability to divide and proliferate before birth, but this ability rapidly decreases after birth. Adult cardiomyocytes hardly have the ability to divide and proliferate. In the event of heart tissue necrosis such as myocardial infarction, adult cardiomyocytes have l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A61K35/28A61K35/34A61K35/545
CPCC12N5/0657C12N2506/45C12N2506/02C12N2501/155C12N2501/998A61K35/28A61K35/34C12N2501/115C12N2501/16C12N2501/385C12N2501/415A61K35/545A61P9/00C12N2501/40C12N2503/02
Inventor 马跃
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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