Inducing culture medium for inducing fibroblast to trans-differentiate into cardiac muscle cells and application of inducing culture medium

A technology of fibroblasts and induction medium, which is applied in the direction of cell culture active agents, embryonic cells, tissue culture, etc., and can solve problems such as hidden dangers in system security, high technical barriers, and efficiency that needs to be improved

Active Publication Date: 2016-08-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the method of overexpression of exogenous genes has high technical barriers, the expression vector may be inserted into the genome, there are hidden dangers in the safety of the introduction system, and the efficiency needs to be improved

Method used

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  • Inducing culture medium for inducing fibroblast to trans-differentiate into cardiac muscle cells and application of inducing culture medium
  • Inducing culture medium for inducing fibroblast to trans-differentiate into cardiac muscle cells and application of inducing culture medium
  • Inducing culture medium for inducing fibroblast to trans-differentiate into cardiac muscle cells and application of inducing culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) The preparation steps of mouse embryonic fibroblasts (MEFs) are as follows:

[0036] 1) Pretreatment of the culture vessel: Cover the bottom wall of the culture vessel with 0.2% gelatin, place it at room temperature for 30 minutes, suck out the 0.2% gelatin, dry it at room temperature and set aside.

[0037] 2) After injecting about 0.5 mL of avertin to a mouse on the 13.5th day of pregnancy for anesthesia, execute the mouse by neck dislocation, and immerse it in 75% alcohol for disinfection for 5 minutes.

[0038] 3) Wipe the abdomen with 75% ethanol, cut the skin and pull the skin back to expose the abdominal wall. Cut the abdominal wall to expose the uterus. Move the uterus to a 100mm dish and wash it three times with 10mL PBS.

[0039] 4) Cut open the embryo sac with scissors, and move the embryos to a petri dish.

[0040] 5) Carefully remove the head and viscera of the embryo, transfer the torso of the embryo to a penicillin vial, and wash three times with 2 m...

Embodiment 2

[0049] In order to explore the potential role of small molecules in changing cell fate, more than 10 small molecule compounds and their combinations that have been proven to have an impact on reprogramming were systematically analyzed and screened, including

[0050] HDAC inhibitors: NaB(N), VPA(V), TSA(A);

[0051] DNMT inhibitors: RG108(R), 5-AZA(5);

[0052] G9a inhibitor: BIX-01294(B);

[0053] Ezh2 inhibitors: DZNep (D), GSK126 (G);

[0054] LSD1 inhibitor: phencypromine (T);

[0055] AC inhibitor: Forskolin (F);

[0056] GSK3 inhibitor: CHIR99021(C);

[0057] MEK inhibitor: PD032590(P);

[0058] ALK5 inhibitors: A-83-01(8), E616452(6), SB431542(S).

[0059] The experimental process is as follows figure 1 As shown, 1000 MEFs cells prepared in Example 1 were inoculated on a 96-well plate, and different small molecule combinations were added to the medium from the second day, and then the medium was changed every four days, and detected on the 16th day, by statistica...

Embodiment 3

[0063] Inoculate 20,000 TTFs cells prepared in Example 1 on a 35mm cell culture dish, add different small molecule combinations to the medium from the second day, then change the medium every four days, detect on the 16th day, and count the formation of clones Count and qPCR analysis to determine the screening effect of different small molecule combinations. Such as Figure 4 As shown, small molecules combined with 6TCF to treat TTFs can also obtain a-Actinin-positive cardiomyocyte-like cells and Tuj1-positive neuron-like cells.

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Abstract

The invention discloses an inducing culture medium for inducing fibroblast to trans-differentiate into cardiac muscle cells, a method and an application of the inducing culture medium. The inducing culture medium comprises a basic culture medium and an inducing small molecular assembly which is 6TCFOW or SCFOV, wherein 6 is E61541, T is tranylcypromine, C is CHIR99021, F is forskolin, O is Dorsomorphin, W is IWR-I, S is SB431542, and V is valproic acid. The inducing culture medium can trans-differentiate the fibroblast into the cardiac muscle cells which have normal cardiac muscle cell specific molecular tags and a normal cardiac muscle function, so that a new way is provided for solving the cell source problem of the regenerative medicine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an induction medium for inducing fibroblasts to transform into cardiomyocytes and application thereof. Background technique [0002] Most multicellular organisms develop from totipotent fertilized eggs. The fertilized egg formed by the combination of sperm and egg undergoes step-by-step lineage differentiation and finally develops into a mature individual. Among them, the determination of cell fate is the result of the synergy between thousands of exogenous signals and endogenous factors. This process is complex and difficult to control. In the rapid development of life science in recent years, one of the most striking achievements is that people can change the fate of cells through exogenous pathways. By overexpressing lineage-specific regulatory factors, people can not only dedifferentiate adult cells into embryonic stem cells, but also realize the process of transdifferentiation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/073
CPCC12N5/0657C12N2501/415C12N2501/70C12N2501/71C12N2501/727C12N2501/73C12N2506/02
Inventor 郭国骥韩晓平李侠岑燕红张芬
Owner ZHEJIANG UNIV
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