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A method for preparing cardiac progenitor cells

A technology of cardiac progenitor cells and mesenchymal stem cells, which is applied in the field of preparation of cardiac progenitor cells to achieve the effect of high positive rate and low immunogenicity

Active Publication Date: 2020-05-01
GMU MEDICAL DRUG DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biggest challenge of protein induction is how large molecular weight and complex structural reprogramming proteins cross the "firewall" of the cell membrane and enter the nucleus to play a role

Method used

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  • A method for preparing cardiac progenitor cells
  • A method for preparing cardiac progenitor cells
  • A method for preparing cardiac progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 The preparation of the required solution of the present invention

[0052] (1) Ni column washing buffer: 20mM Tris-HCl, 0.15M NaCl, 20mM imidazole, pH8.0;

[0053] (2) Ni column elution buffer: 20mM Tris-HCl, 0.15M NaCl, 200mM imidazole, pH8.0;

[0054] (3) Recombinant protein dissolution buffer: 50mM Tris, 150mM NaCl, 10% glycerol (v / v), pH8.0;

[0055] (4) Mesenchymal stem cell culture medium: 45ml MSC basal medium (Cyagen), 5ml FBS, adding final concentrations of 0.1mM non-essential amino acids, 2mM L-glutamine, 50nM β-mercaptoethanol; can be used for human bone marrow culture of mesenchymal stem cells or human umbilical cord mesenchymal stem cells;

[0056] (5) Cardiac progenitor cell reprogramming medium (CRM medium): on the basis of human bone marrow mesenchymal stem cell medium with 5% (v / v) fetal bovine serum, mainly added with a final concentration of 10ng / ml bFGF, 3ng / ml Activin A, 6nM Chir99021, 0.5% (g / L) BSA;

[0057] (6) Cardiac progenitor...

Embodiment 2

[0062] Prokaryotic expression of each reprogramming factor of embodiment 2

[0063] The main purpose of this embodiment is to obtain the following reprogramming factors: Gata4, Tbx5, Nkx2.5, AF9, Hand2, Mef2C, Mesp1, Baf60c, UTX.

[0064] The prokaryotic expression method of each reprogramming factor comprises steps:

[0065] (1) Insert the coding gene sequence of each reprogramming factor into the expression vector pSmart-I (Tiandirenhe Company, China) to obtain the recombinant expression vector;

[0066] Specifically, the nucleotide sequence of the coding gene of Gata4 is shown in Gene ID 2626 of NCBI;

[0067] The nucleotide sequence of the coding gene of Tbx5 is shown in Gene ID 6910 of NCBI;

[0068] The nucleotide sequence of the coding gene of Nkx2.5 is shown in Gene ID 1482 of NCBI;

[0069] The nucleotide sequence of the coding gene of AF9 is shown in Gene ID 4300 of NCBI;

[0070] The nucleotide sequence of the coding gene of Hand2 is shown in Gene ID 9464 of NCB...

Embodiment 3

[0087] Example 3 Transduction of each reprogramming factor and monitoring of transduction efficiency

[0088] The main purpose of this example is to transduce the reprogramming factors obtained in Example 2 into target cells (initial reprogramming objects).

[0089] In this example, the combination of low-molecular chitosan and various low-molecular chitosan derivatives is used as a transduction carrier. Specifically, the low-molecular chitosan is chitosan with a molecular weight of 5000, and the low-molecular chitosan The chitosan derivative is N-carboxybutyl chitosan, and the mass ratio of the former to the latter is 1:1. When in use, the transduction carrier can be configured as an aqueous solution of the transduction carrier with a pH of 7.4.

[0090] The freeze-dried powder of each reprogramming factor obtained in Example 2 was dissolved in a buffer (50 mM Tris, 150 mM NaCl, 10% v / v glycerol, pH 8.0) to obtain a buffer for each reprogramming factor.

[0091] When in use...

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Abstract

The invention belongs to the field of biopharmaceutical research, and particularly relates to a method for preparing cardiac progenitor cells. The method comprises the following steps: transducing a reprogramming factor into an initial reprogramming object cell, wherein the reprogramming factor is selected from cardiac transcription factors and epigenetic regulation factors; preferably the reprogramming factor can be selected from one of or a combination of a plurality of (i) Gata4, ( / i) (i) Tbx5, Nkx2.5, AF9, Hand2, Mef2C, Mesp1, ( / i) (i) Baf60c and UTX ( / i). By preparing the cardiac progenitor cells with the method, the cardiac progenitor cells can be obtained after about 8 to 10 days, the obtained cardiac progenitor cells are all (i) Isl+ / Tbx5+(i) double positive cells, the positive rate is high, and the immunogenicity is low.

Description

technical field [0001] The invention belongs to the field of biomedical research, and in particular relates to a method for preparing cardiac progenitor cells. Background technique [0002] Cardiovascular disease is an important public health problem in my country, and its high incidence, younger onset, and high disability rate are becoming increasingly serious. Although experiments have shown that human heart cells have a certain regeneration ability, this self-regeneration ability is not enough to repair the loss of a large number of heart cells caused by pathological factors such as ischemia. Therefore, the regeneration and repair of damaged cardiac tissue is an urgent problem to be solved in the research of cardiovascular translational medicine. [0003] Cardiac Progenitor Cells (CPCs) are the origin cells of heart development, which can self-renew and differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells. They are potential seed cell sources for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2506/1353C12N2506/1369
Inventor 余细勇李晓红吴岳恒杨翔宇
Owner GMU MEDICAL DRUG DEV CO LTD
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