Autophagy-based stem cell myocardial cell induced differentiation method and application thereof

A technology for inducing differentiation and cardiomyocytes, applied in the field of biomedicine, can solve the problems of complicated and cumbersome operations, unstable differentiation efficiency, time-consuming and expensive, etc., and achieve the effects of improving efficiency, improving induction and differentiation efficiency, and shortening induction time.

Pending Publication Date: 2020-11-03
HUNAN NORMAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0021] The shortcomings of this technology are reflected in: the operation is complex and cumbersome, and it takes more time and money
Differentiation efficiency is unstable and affected by factors such as the state of END-2 cells

Method used

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  • Autophagy-based stem cell myocardial cell induced differentiation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] refer to figure 1 , a method for inducing differentiation of cardiomyocytes from stem cells based on autophagy, comprising the following steps:

[0041] Step 1, preparation of autophagy inducer:

[0042] Rapamycin was dissolved in DMSO, prepared as a 200µM stock solution, and stored at -20°C. Rapamycin was added to the culture medium when the cells were treated to make the final concentration 20nM, and the solvent DMSO (0.1‰) was used as a control, and it was added every time the medium was changed.

[0043] Step 2, stem cell culture: Mouse embryonic stem cells were inoculated on a culture dish treated with 0.1% Gelatin, containing 15% fetal bovine serum, 2 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 10 4 U / ml mouse LIF Medium, 100 U / ml penicillin and streptomycin in DMEM high-glucose medium, at 37°C, the volume fraction is 5% CO 2 cultured in a cell culture incubator.

[0044] Step 3. Stem cell induction (first stage): Take embryonic stem cells in loga...

Embodiment 2

[0049] refer to figure 1 , a method for inducing differentiation of cardiomyocytes from stem cells based on autophagy, comprising the following steps:

[0050] Step 1, preparation of autophagy inducer:

[0051] KU0063794 was dissolved in DMSO, prepared as a 10mM stock solution, and stored at -20°C. KU0063794 was added to the culture medium to make the final concentration of 1µM when the cells were treated, and the solvent DMSO (0.1‰) was used as a control, and was added every time the medium was changed.

[0052] Step 2, stem cell culture: Mouse embryonic stem cells were inoculated on a culture dish treated with 0.1% Gelatin, containing 15% fetal bovine serum, 2 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 10 4 U / ml mouse LIF Medium, 100 U / ml penicillin and streptomycin in DMEM high-glucose medium, at 37°C, the volume fraction is 5% CO 2 cultured in a cell culture incubator.

[0053] Step 3. Stem cell induction (first stage): Take embryonic stem cells in logari...

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Abstract

The invention discloses an autophagy-based stem cell myocardial cell induced differentiation method and an application thereof. The method comprises the following steps of step 1, preparing an autophagy inducer wherein, the autophagy inducer dissolves in DMSO to prepare mother liquor, the mother liquor is stored at 20 DEG C, a solvent DMSO with the concentration being 0.01% is served as a controlgroup, and the mother liquor is added once when liquor is changed; step 2, stem cell culture; step 3, a first stage for stem cell induction, wherein a myocardial differentiation inducer is added, andcomprises the autophagy inducer prepared in the step 1 and vitamin C; step 4, a second stage for stem cell induction, wherein after good embryoid bodies are obtained through suspension culture, embryoid bodies are transferred into a cell culture plate for adherent culture, the induced culture solution is the same as that in the first stage, the liquor is exchanged each day or every two days according to the cell growth condition, and as time goes on, spontaneous rhythmic jump of the embryoid bodies is observed. The method improves the induced differentiation efficiency and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to an autophagy-based method for inducing differentiation of stem cell cardiomyocytes and an application thereof. Background technique [0002] Embryonic stem cells (ES or ESC cells for short): are a type of cells isolated from early embryos (before the gastrula stage) or primitive gonads, which have the characteristics of unlimited proliferation, self-renewal and multi-directional differentiation in vitro . Whether in vitro or in vivo, ES cells can be induced to differentiate into almost all cell types in the body. [0003] Induced pluripotent stem cells (IPS or IPSC cells for short): Through the induction of exogenous transcriptional regulatory factors and other methods, adult cells are reprogrammed into pluripotent cells like embryonic stem cells (ES cells), called induced pluripotent stem cells. Induced pluripotent stem cells are cells similar to embryonic stem cells, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2506/45C12N2500/38C12N2501/999
Inventor 李涛程冠昌李彦明
Owner HUNAN NORMAL UNIVERSITY
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