Method for efficiently obtaining beating functional cardiomyocytes from mesenchymal stem cells

A technology for cardiomyocytes and pluripotent stem cells, which is applied in animal cells, vertebrate cells, cell culture active agents, etc., can solve problems such as inability to meet, and achieve the effects of good stability, good clinical application prospects, and strong operability.

Inactive Publication Date: 2016-09-21
NANJING CHILDRENS HOSPITAL AFFILIATED TO NANJING MEDICAL UNIV
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Problems solved by technology

Unable to meet the needs of in vitro tissue engineering myocardial research

Method used

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  • Method for efficiently obtaining beating functional cardiomyocytes from mesenchymal stem cells

Examples

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Embodiment 1

[0020] Example 1 mRNA reprogramming mesenchymal stem cells and cardiomyocyte differentiation

[0021] (1) Obtain and identify pluripotent stem cells from umbilical cord mesenchymal stem cells by using mRNA reprogramming method:

[0022] Oct4, Sox2, Klf4, cMyc, Lin28 were used as mRNA reprogramming factors, according to the molar ratio of 3:1:1:1:1, human NUFF fibroblasts (Stemgent) were used as 2.5×10 4 / cm 2 Inoculate on 0.2% Gelatin-coated six-well plate (for reprogramming feeder cells) and T75 culture flask (for collecting NUffconditioned Pluriton reprogramming medium), and the medium is NUFF special medium. On the second day, the isolated mesenchymal stem cells were divided into 2.5×10 3 / cm 2 Planted on NUFF cells, the medium is DMEM / F12+10% FBS. On day 3, start reprogramming (d0), medium change to Pluriton TM Medium, add 200ng / ml B18R, put in 37℃, 5%CO 2 in the incubator. After 2 hours, start mRNA transfection, add mRNA reprogramming factors, RNAimaxs and Opti-MEM...

Embodiment 2

[0028] Example 2 Screening experiments on the effects of different concentrations of activin A and different concentrations of BMP4 on the differentiation efficiency of cardiomyocytes

[0029] The present invention adopts different concentrations of activin A (5ng / ml, 10ng / ml, 20ng / ml, 50ng / ml and 100ng / ml) and different concentrations of bone morphogenetic protein 4 (BMP4) (5ng / ml, 10ng / ml, 20ng / ml) / ml and 50ng / ml) to induce cardiomyocytes, the induction culture method is the same as in Example 1, and the study found that the matrix sandwich method was adopted, and the concentration was 50ng / ml activin A and 10ng / ml BMP4 and 10ng / ml basic fibroblasts were added. Cell growth factor (bFGF) can achieve the optimal induction effect, and the differentiation efficiency of cardiomyocytes is above 80%.

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Abstract

The invention discloses a method for efficiently obtaining beating functional cardiomyocytes from mesenchymal stem cells. The method comprises the following steps: reprogramming the mesenchymal stem cells into pluripotent stem cells through mRNA reprogramming, and then adding an activin A and a bone morphogenetic protein 4 by adopting a matrix sandwich method to obtain the beating functional cardiomyocytes with differentiation efficiency of more than 80%. The differentiation efficiency of cardiomyocytes can be greatly improved by the method provided by the invention. The in vitro multiplication capacity of the cardiomyocytes is limited in the prior art, so the requirement for establishing the myocardium of tissue engineering in vitro cannot be met. The method provided by the invention can provide important experimental data for the research of cell therapy of ischemic cardiovascular diseases, and has a good clinical application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological cells, and in particular relates to a method for efficiently obtaining beating functional cardiomyocytes from mesenchymal stem cells. Background technique [0002] In the field of cardiac surgery, the various patches and tubes currently used have no growth ability and cannot assist heart function. Infection, thrombosis, damage, etc. will occur in the long run. The application of tissue engineering technology provides a new idea and method for the research and development of new heart repair materials. The construction of tissue-engineered blood vessels or myocardial patches by tissue engineering methods can overcome the shortcomings of the above-mentioned patches and achieve the ideal purpose of surgical reconstruction of congenital heart disease. One of the most important issues limiting the research of tissue engineered myocardium is the lack of a large number of functional and beating cardi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/0657C12N2501/115C12N2501/155C12N2501/16C12N2506/1392
Inventor 武开宏李琳徐飞莫绪明
Owner NANJING CHILDRENS HOSPITAL AFFILIATED TO NANJING MEDICAL UNIV
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