A method for in vitro differentiation of induced pluripotent stem cells into ventricular myocytes

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 problems of unidentified ventricular muscle, inability to achieve directional differentiation of different cardiomyocytes, and low efficiency of cardiomyocyte differentiation.

Active Publication Date: 2018-01-19
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Abstract
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Problems solved by technology

[0008] 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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  • A method for in vitro differentiation of induced pluripotent stem cells into ventricular myocytes
  • A method for in vitro differentiation of induced pluripotent stem cells into ventricular myocytes
  • A method for in vitro differentiation of induced pluripotent stem cells into ventricular myocytes

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Embodiment 1B

[0103] Example 1 The role of BMP / Smad1 / 5 / 8 signaling pathway in inducing myocardial precursor cells to differentiate into ventricular myocytes

[0104] 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 was ...

Embodiment 2

[0114]Example 2 Differentiation of Induced Pluripotent Stem Cells into Ventricular Myocytes in Vitro (Technical Scheme 1)

[0115] 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

[0116] Example 3 Differentiation of induced pluripotent stem cells into ventricular myocytes in vitro (technical scheme two)

[0117] 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 D...

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Abstract

The present invention provides a method for inducing pluripotent stem cells to differentiate into ventricular myocytes in vitro, which is to maintain, amplify, and cultivate pluripotent stem cells in vitro, and in the middle stage of myocardial differentiation of pluripotent stem cells, the mesoderm cells or myocardial precursor cells develop into In the period of cardiomyocyte differentiation, substances that can directly or indirectly activate the Smad1 / 5 / 8 signaling pathway are added to the medium to make the stem cells differentiate into ventricular myocytes. Utilizing the method of the present invention, ventricular myocytes with biological activity and function have been successfully obtained, which not only reveals the regulatory mechanism in the differentiation process of myocardial precursor cells into ventricular myocytes, but also differentiates human ventricular myocytes to cell transplantation for the treatment of myocardium. Infarction and cardiac toxicology analysis and the development of cardiac-related drugs have a wide range of applications.

Description

technical field [0001] 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 [0002] 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 lost the ability to proliferate and divide, and cannot repair necrotic tissue through regeneration of cardiomyocytes, so the decline in cardiac function caused by such diseases is irreversible. Although the use of drugs can increase myocardial contractility and improve the pumping capacity of the heart, the increased burden on the heart may actually worsen the condition. Replacing dead cells w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00C12N5/071A61K35/545A61P9/00A61K35/28A61K35/34
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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