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Culture medium and method for increasing somatic cell reprogramming efficiency

A culture medium and reprogramming technology, applied in the biological field, can solve problems such as poor quality, uneven quality, and unreported research on lipid effects

Active Publication Date: 2017-03-08
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many problems to be solved in the process of iPSCs induction. For example, c-Myc among the four reprogramming factors is itself a proto-oncogene, so that iPSCs have certain tumorigenicity; the induction efficiency of iPSCs is low, less than 1 %; and the obtained iPS cell lines are heterogeneous, the quality is not uniform, and most of them are of poor quality (only about 10% of the clones can produce chimeric mice)
[0007] However, most of the studies reported so far focus on the role of transcription factors and small molecule inhibitors in the reprogramming process, while the research on the role of lipids in reprogramming has not been reported yet.

Method used

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  • Culture medium and method for increasing somatic cell reprogramming efficiency
  • Culture medium and method for increasing somatic cell reprogramming efficiency
  • Culture medium and method for increasing somatic cell reprogramming efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Preparation of pluripotent stem cells

[0062] 1. Induction of somatic cell reprogramming using media containing different concentrations of PA

[0063] 1) Prepare somatic cells. Before inducing somatic cell reprogramming, thaw or passage OG2MEF (Oct4drive GFP mouse embryonic Oct4-GFP fibroblasts (purchased from the Institute of Zoology, Chinese Academy of Sciences) were inoculated in a six-well plate, and each well was inoculated with 1-2×10 5 After culturing the cells for 24 h, add 1 ml concentrated TetO-FUW-OSKM and FUW-M2rtTA (purchased from Addgene, product number 20321 and 20342) virus mixture at 1:1, and supplement 0.5 ml MEF cell culture medium (DMEM medium (purchased from Gibco, Cat. No. 11965), containing 10% FBS by volume, and NEAA), placed in a 37° C. incubator to continue culturing. After 24 hours of virus infection, the virus liquid was discarded, washed twice with PBS, replaced with fresh MEF medium and continued to culture until the cel...

Embodiment 2

[0077] Identification and result analysis of the pluripotent stem cells obtained in Example 2

[0078] 1. Phosphatidic acid (PA) effectively promotes reprogramming efficiency

[0079] In order to select the optimal concentration of phosphatidic acid (PA), the inventors added 100 μM, 200 μM, 400 μM, 600 μM and 800 μM PA to the medium when inducing reprogramming, and performed alkaline phosphatase (AP) staining on d14. The statistical results showed that PA can significantly improve the reprogramming efficiency, and the number of AP-positive clones in the PA group was significantly more than that in the control group, and the increase was in a gradient-dependent manner of PA concentration ( figure 2 A). Among them, the effect of flow cytometry analysis and 400μM PA is the most significant. Flow cytometry was used to analyze the proportion of GFP-positive cells on d14. The results showed that the proportion of GFP-positive cells in the 400 μM PA group was significantly hig...

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Abstract

The invention provides a culture medium for induction somatic cell reprogramming, the culture medium includes a culture medium of normal induction somatic cell reprogramme and the phosphatidic acid. The invention also provides a method for induction somatic cell reprogramming, the method includes the culture substrate used in any period of the induction somatic cell reprogramme or any period of the induction somatic cell reprogramme, and adds the phosphatidic acid in the culture substrate. The study found the phosphatidic acid as the most simple lipoid, by adding the phosphatidic acid into the culture substrate of the induction somatic cell reprogramme, the method can obviously increase the reprogramming efficiency of somatic cells, and adds to the lowering of cell apoptosis in the reprogramming process, thereby can obtain large quantity of high quality iPS cells, and builds a foundation for application of PS cells in drug screening and in future reform medicine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a culture medium and a method for improving the reprogramming efficiency of somatic cells. Background technique [0002] Embryonic stem cells (ESCs), as a kind of totipotent stem cells, have developmental totipotency and can differentiate into all organs, tissues and even germ cells, which brings new opportunities for gene and cell therapy and regenerative medicine research. However, embryonic stem cells are mainly produced by the isolation and culture of the inner cell mass at the blastocyst stage, which requires the destruction of embryos in the early stages of development. Therefore, research on human embryonic stem cells has caused ethical controversy due to source issues. In addition, the immune rejection usually caused by allogeneic transplantation also limits the clinical application of ESCs. The emergence of induced pluripotent stem cells (iPSCs) is expected to break through t...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/10C12N15/86
Inventor 蒋远杜明霞胡宝洋
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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