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Somatic cell reprogramming method and application thereof

A somatic cell reprogramming technology, applied in animal cells, vertebrate cells, cell culture active agents, etc.

Inactive Publication Date: 2020-02-18
BIOISLAND LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, how to change cell fate, especially to promote the reprogramming of somatic cells, remains to be further studied

Method used

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  • Somatic cell reprogramming method and application thereof
  • Somatic cell reprogramming method and application thereof
  • Somatic cell reprogramming method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062]Example 1: Ethanolamine, CDP-ethanolamine, and phosphatidylethanolamine can improve the reprogramming efficiency of mouse fibroblasts

[0063] Such as figure 1 As mentioned, in the basal medium containing DMEM, serum supplement N 2 (Concentration is 1%), growth factor bFGF (concentration is 5ng / ml) and Lif (concentration is 2000units / ml), small molecular compound vitamin C (concentration is 50μg / ml) in the culture medium, add ethanolamine (1mg / ml) respectively ml), CDP-ethanolamine (1mg / ml), and phosphatidylethanolamine (1mg / ml) can improve the reprogramming efficiency of mouse embryonic fibroblasts and greatly increase the number of induced pluripotent stem cell clones ( figure 1 A). The pluripotency of the reprogrammed pluripotent stem cells can be identified by immunofluorescence and chimeric mouse methods. The results of immunofluorescence experiments show that the induced pluripotent stem cells obtained in the control group and ethanolamine group all express the p...

Embodiment 2

[0064] Example 2: Ethanolamine can improve the reprogramming efficiency of mouse tail tip fibroblasts, and can improve the reprogramming efficiency in the basal serum-free medium containing KSR.

[0065] Such as figure 2 As mentioned, in the basal medium containing DMEM, serum supplement N 2 (content is 1%), growth factor bFGF (concentration is 5ng / ml) and Lif (concentration is 2000units / ml), small molecular compound vitamin C (concentration is 50μg / ml) in the culture medium that adds ethanolamine (1mg / ml) After culturing mouse tail tip fibroblasts for 10 days, the reprogramming efficiency of mouse tail tip fibroblasts can be improved, and the number of induced pluripotent stem cell clones can be greatly increased ( figure 2 A). In the basal medium DMEM, serum replacement supplement KSR (15%), growth factors bFGF (5ng / ml) and Lif (2000units / ml), small molecule vitamin C (50μg / ml) Adding ethanolamine (1 mg / ml) to the culture medium of mouse tail tip fibroblasts for 10 days...

Embodiment 3

[0066] Example 3: Ethanolamine can promote the mesenchymal-epithelial transition of mouse embryonic fibroblasts.

[0067] Such as image 3 As mentioned, in the basal medium containing DMEM, serum supplement N 2 (content is 1%), growth factor bFGF (concentration is 5ng / ml) and Lif (concentration is 2000units / ml), small molecular compound vitamin C (concentration is 50μg / ml) in the culture medium that adds ethanolamine (1mg / ml) After culturing mouse embryonic fibroblasts for 2 days, it can promote the mesenchymal epithelial transition of mouse embryonic fibroblasts. Using fluorescent quantitative PCR technology, it was found that ethanolamine promotes the expression of epithelial cell marker genes and reduces the expression of mesenchymal cell marker genes ( image 3 A). Using Western technology to find that ethanolamine can increase the expression of epithelial cell marker proteins in mouse embryonic fibroblasts ( image 3 B).

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Abstract

The invention discloses a somatic cell reprogramming method. The somatic cell reprogramming method comprises the following steps of preparing somatic cells from different sources, so that one or moreof Sox2, Klf4, Oct4 and c-Myc can be expressed; preparing a culture medium for programming: adding serum replacement additives, growth factors and micromolecule compounds to a basic culture medium; then, adding compounds to the basic culture medium, and performing uniform mixing reversely; and culturing somatic cells in the culture medium for programming to obtain induction multipotential stem cells. According to the method, the efficiency that the somatic cells are reprogrammed into the induction multipotential stem cells can be greatly improved. The method is used, so that transition of somatic cell mesenchyma into epithelia in the serum-free culture medium can be realized, the reprogramming efficiency of the somatic cells of different sources can be improved, and a technique support isprovided for application of induction of multipotential stem cells to regeneration medicines in the future.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, the present invention relates to a method for reprogramming of somatic cells and its application. Background technique [0002] With the deepening of stem cell research, scientists have discovered that terminally differentiated mammalian cells are also totipotent, and adult cells can be reprogrammed to change their gene expression profiles to change their fate, regain pluripotency or directly transform differentiation into another cell. There are two main methods for early somatic cell reprogramming: nuclear transfer and cell fusion, but these two methods still need to involve embryos, which are greatly restricted in ethics. In 2006, the Japanese Yamanaka laboratory obtained breakthrough research results in the field of reprogramming, and found that mouse fibroblasts can be reprogrammed into pluripotent stem cells by exogenously expressing four transcription factors So...

Claims

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Application Information

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IPC IPC(8): C12N5/074
CPCC12N5/0696C12N2500/38C12N2500/46C12N2500/90C12N2501/115C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2506/1307
Inventor 刘兴国邬毅陈可实
Owner BIOISLAND LAB
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