Method for preparing pluripotent cardiovascular precursor cells and maintaining their cardiovascular differentiation ability

A precursor cell, pluripotent stem cell technology, applied in non-embryonic pluripotent stem cells, artificially induced pluripotent cells, animal cells, etc., can solve problems such as regulation and little understanding of molecular basis

Active Publication Date: 2018-07-31
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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Problems solved by technology

[0005] However, simple and reproducible, efficient transformation of hPSCs into homogeneous CVPCs without cell sorting and under trophoblast-free / serum-free conditions remains challenging, and the regulation and molecular basis of self-renewal and differentiation decisions of CVPCs remain challenging. Still poorly understood, the stable expansion of HPSC-derived CVPCs in vitro to maintain their long-term self-renewal and directed differentiation potential into cardiovascular cells remains a major challenge in the field

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  • Method for preparing pluripotent cardiovascular precursor cells and maintaining their cardiovascular differentiation ability
  • Method for preparing pluripotent cardiovascular precursor cells and maintaining their cardiovascular differentiation ability
  • Method for preparing pluripotent cardiovascular precursor cells and maintaining their cardiovascular differentiation ability

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[0085] The present invention also includes the cell group containing cardiovascular precursor cells obtained by the aforementioned preparation method. In the cell group, the number of cardiovascular precursor cells accounts for more than 70% of the total number of cells, preferably more than 80% of the total number of cells; more preferably more than 85% of the total number of cells (expressed as surface markers SSEA1 or MESP1 positive cells count).

[0086] Once a cell population containing cardiovascular precursor cells has been obtained, the identification of cardiovascular precursor cells can be achieved using RT-PCR and other gene expression analysis techniques.

[0087] Cardiovascular precursor cells can be identified or isolated or enriched using methods known to those skilled in the art. More classical methods such as fluorescence-activated cell sorting (FACS) or magnetic cell sorting, that is, the use of antibodies against specific surface molecules of cardiovascular...

Embodiment 1

[0193] Example 1. Efficient transformation of human pluripotent stem cells (hPSCs) into homogeneous CVPCs

[0194] To induce hPSCs towards a cardiovascular fate, the inventors screened important signal transduction pathways during mesoderm formation and subsequent cardiac differentiation, including Wnt, GSK3, FGF, BMP and Activin / Nodal. In order to make the conditions uniform, the inventors used a single cell-based differentiation method on the basis of a monolayer culture system, and added the Rho kinase inhibitor Y27632 in the first day of differentiation to improve the survival of the cells. After a series of systematic attempts, the inventors found that the joint addition of 25ng / ml BMP4, 50μg / ml AA and 3μM GSK3 inhibitor CHIR99021 (CHIR) (the mixture of these combined reagents is named CIM) can effectively remove the The undifferentiated human embryonic stem cell line H9 was converted into a homogeneous cell population, most of which (88.9 ± 1.8%) expressed the recently i...

Embodiment 2

[0197] Example 2. Long-term maintenance of self-renewal of hPSC-derived CVPCs

[0198] Proliferation and lineage determination of CVPCs are regulated by a delicate microenvironment and complex levels of multiple signal transduction pathways, including Wnt, FGF, BMP, Notch, Hedgehog, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF ), MEK, retinoic acid (RA), and Activin / Nodal. Therefore, under specific conditions in vitro, it is generally difficult to maintain the self-renewal and expansion of CVPCs. The inventors hypothesized that if the conditions that induce CVPC differentiation are concertedly eliminated, they should be able to maintain basal state self-renewal and continued proliferation. To verify it, the inventors selected 9 signaling pathway inhibitors together with the GSK3 inhibitor CHIR (CHIR99021) (Table 3) to verify their role in stimulating CVPC expansion. Clonogenicity was not observed in H9-derived CVPCs on day 3 of differentiat...

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Abstract

The present invention relates to methods for preparing pluripotent cardiovascular precursor cells and maintaining their cardiovascular differentiation ability. For the first time, a method for efficiently inducing the differentiation of pluripotent stem cells into pluripotent cardiovascular precursor cells was revealed, including induction with ascorbic acid, bone morphogenic protein 4 and glycogen synthase kinase 3 inhibitors. The present invention also provides a method for stably cultivating multipotential cardiovascular precursor cells, including using BMP signaling pathway inhibitors, Activin / Nodal signaling pathway inhibitors and glycogen synthase kinase 3 signaling pathway inhibitors to enable stable growth and passage. The multipotential cardiovascular precursor cells obtained by the present invention can be further differentiated into some cells of cardiovascular lineage such as cardiomyocytes, vascular smooth muscle cells or vascular endothelial cells, which can be used for the treatment of heart diseases and research on myocardial regeneration, drug cardiovascular cytotoxicity Testing, cardiac drug development.

Description

technical field [0001] The invention belongs to the field of cell biology; more specifically, the application relates to methods for inducing pluripotent stem cells to derive pluripotent cardiovascular precursor cells and maintaining the cardiovascular differentiation ability of cardiovascular precursor cells. Background technique [0002] Human pluripotent stem cells (hPSCs), including human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), because they can proliferate indefinitely in vitro and have the ability to differentiate The ability to form the major cell types of the heart provides a whole new opportunity for cardiovascular research, drug development and safety testing, and cell therapy-based regenerative medicine. However, in order to avoid the tumorigenicity of direct transplantation, such cells must be induced to differentiate into tissue precursor cells and tissue cells. Therefore, in the past ten years, researchers have made unremitting e...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735C12N5/074C12N5/077C12N5/071
Inventor 杨黄恬曹楠梁贺
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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