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Method for preparing heart progenitor cells

A technology of cardiac progenitor cells and cells, applied in the field of cell biology, can solve the problems of low efficiency of cardiac progenitor cells, difficult molecular cloning experimental operations of large fragment gene sequences, virus packaging, and rapid chromatin remodeling, etc., to achieve improved cell reorganization The effect of programming efficiency

Active Publication Date: 2020-02-07
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 heart progenitor cells
  • Method for preparing heart progenitor cells
  • Method for preparing heart 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 directly re-programming heart progenitor cells based on endogenous gene transcriptional activation. The method has better operation feasibility and theory creativity. For the first time, through a CRISPR / Cas9 system, endogenous heart development relevant transcription factors GATA4, HAND2, MEF2C, TBX5 and MEIS1 are activated, human foreskin fibroblasts are induced to be re-programmed into the heart progenitor cells having the potential of being disintegrated towards cardiac muscle cells, smooth muscle cells and endothelial cells, a seed cell source is provided for cardiovascular disease model construction, new medicine screening and myocardial regeneration, and besides, a novel apparent cell re-programming theory and connotation are enriched.

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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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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