Method for preparing cardiac progenitor cells

A technique of cardiac progenitor cells, cells

Active Publication Date: 2021-07-23
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, large fragments of gene sequences are difficult to perform molecular cloning experiments and virus packaging, and cannot directly access endogenous gene loci for rapid chromatin remodeling
Therefore, the efficiency of generating cardiac progenitor cells by exogenous transgene-induced cell reprogramming is low

Method used

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  • Method for preparing cardiac progenitor cells
  • Method for preparing cardiac progenitor cells
  • Method for preparing cardiac progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Using the SAM system to induce simultaneous expression of GATA4, HAND2, MEF2C and TBX5 in human foreskin fibroblasts

[0088] (1) Cardiac development-related transcription factors GATA4, HAND2, MEF2C, and TBX5 were selected as reprogramming factors to construct sgRNA-MS2 fusion expression vectors for each gene.

[0089] (2) The day before lentivirus infection of cells, take well-grown human foreskin fibroblasts and inoculate them in a 75cm 2 Cell culture flasks were brought to a cell density of 30-40% at the time of infection.

[0090] (3) Dilute dCas9-VP64 and MS2-p65-HSF1 lentiviral particles at 1:10000 with DMEM medium containing 8 μg / mL polybrene. The cell culture medium was discarded, the cells were gently washed twice with PBS, and 15 mL of DMEM medium containing lentiviral particles was added to infect human foreskin fibroblasts. Human foreskin fibroblasts stably expressing dCas9-VP64 and MS2-p65-HSF1 were screened by Blasticidin S HCL and Hygromycin ...

Embodiment 2

[0096] Example 2: The SAM system induces the formation of cardiac progenitor cells

[0097] (1) Two days before the cells were infected with sgRNA lentiviruses of GATA4, HAND2, MEF2C and TBX5, the DMEM culture medium without Blasticidin S HCL and Hygromycin B was replaced.

[0098] (2) Before cell seeding, pipette 312 μL of Matrigel hESC-qualified Matrix and dilute it in 25 mL of pre-cooled serum-free DMEM / F-12 medium, then take an appropriate amount of diluted Matrigel hESC-qualified Matrix and add them to laser confocal culture dishes and 60 mm culture medium respectively. dish and 100mm petri dish, mix gently, and place at room temperature for 1h.

[0099] (3) Take human foreskin fibroblasts that grow well and stably express dCas9-VP64 and MS2-p65-HSF1 at 4000 cells / cm 2 Inoculated on Matrigel hESC-qualified Matrix-coated Petri dishes.

[0100] (4) Take out the virus liquid from -80°C, put it on ice to dissolve, and then dilute it with DMEM culture medium containing 8 μg / ...

Embodiment 3

[0109] Example 3: Expression of fibroblast markers gradually down-regulated during cell reprogramming

[0110] (1) First, the expression of fibroblast markers FSP1 and α-SMA was analyzed by flow cytometry.

[0111] (2) Two days before the cells were infected with sgRNA lentiviruses of GATA4, HAND2, MEF2C and TBX5, the DMEM culture medium without Blasticidin S HCL and Hygromycin B was replaced.

[0112] (3) Before cell seeding, pipette 312 μL of Matrigel hESC-qualified Matrix and dilute it in 25 mL of pre-cooled serum-free DMEM / F-12 medium, then take an appropriate amount of diluted Matrigel hESC-qualified Matrix and add them to laser confocal culture dishes and 60 mm culture medium respectively. In a dish, mix gently, and place at room temperature for 1 hour.

[0113] (4) Take human foreskin fibroblasts that grow well and stably express dCas9-VP64 and MS2-p65-HSF1 at 4000 cells / cm 2 Inoculated on Matrigel hESC-qualified Matrix-coated Petri dishes.

[0114] (5) Take out the ...

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Abstract

The invention provides a method for direct reprogramming of cardiac progenitor cells based on transcriptional activation of endogenous genes. The method has good operational feasibility and theoretical innovation. For the first time, the endogenous cardiac development-related transcription factors GATA4, HAND2, MEF2C, TBX5 and MEIS1 are activated by the CRISPR / Cas9 system to induce the reprogramming of human foreskin fibroblasts with tropism Cardiac progenitor cells with differentiation potential of cardiomyocytes, smooth muscle cells and endothelial cells provide seed cell sources for the construction of cardiovascular disease models, new drug screening and myocardial regeneration, and enrich the theory and connotation of new epigenetic cell reprogramming.

Description

technical field [0001] The invention belongs to the field of cell biology, in particular to a method for preparing cardiac progenitor cells. Background technique [0002] Cardiomyocytes are terminally differentiated cells with limited regenerative capacity. After acute myocardial infarction, endogenous cardiomyocytes have limited regenerative capacity and cannot effectively prevent the progression of cardiac disease. Stem cell-based regenerative medicine has opened up a new way for myocardial regeneration. Researchers began to use cell reprogramming technology to induce fibroblasts to transform into cardiac progenitor cells, providing a rich source of seed cells for myocardial regeneration. [0003] In 2010, Ieda et al. reported for the first time that mouse cardiac fibroblasts were directly reprogrammed into cardiomyocyte-like cells by carrying three transcription factors GATA4, MEF2C, and TBX5 related to cardiac development. Song et al. added HAND2 to GATA4, MEF2C, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N9/22C12N15/867C12N5/10
CPCC12N5/0657C12N9/22C12N15/86C12N15/907C12N2740/15043
Inventor 余细勇王江林张灵敏
Owner GUANGZHOU MEDICAL UNIV
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