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Application of R406 in promotion of somatic cell reprogramming, and reprogramming culture medium and method forpromotion of somatic cell reprogramming

A technology of reprogramming and culture medium, applied in cell culture active agents, applications, animal cells, etc., can solve the problems of limiting the clinical application of stem cells, unclear molecular mechanism, low efficiency, etc., to promote chemical reprogramming and increase H2S level. , the effect of improving efficiency

Active Publication Date: 2021-06-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is currently inefficient, time-consuming, and the molecular mechanism is still unclear, which limits the clinical application of stem cells

Method used

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  • Application of R406 in promotion of somatic cell reprogramming, and reprogramming culture medium and method forpromotion of somatic cell reprogramming
  • Application of R406 in promotion of somatic cell reprogramming, and reprogramming culture medium and method forpromotion of somatic cell reprogramming
  • Application of R406 in promotion of somatic cell reprogramming, and reprogramming culture medium and method forpromotion of somatic cell reprogramming

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047]The chemical reprogramming system includes three stages: Stage 1, Stage 2 and Stage 3. Specifically, after Stage 1 ends, cells begin to express marker genes in the early stage of reprogramming, such as Sall4; after Stage 2 ends, cells begin to express stem cell marker genes, such as Oct4; after Stage 3 ends, mature stem cells are formed. Therefore, somatic cells were sequentially cultured in different media (Stage 1 medium, Stage 2 medium, and Stage 3 medium) in order to search for small molecules that promote chemical reprogramming. Each stage goes through 12 days, and takes 36 to 40 days in total.

[0048] Among them, Stage 1 medium includes 78% DMEM, 10% KSR, 10% FBS, 1% NEAA, 1% P / S, 0.055mM β-ME, 20ng / mL bFGF, 0.5mM VPA, 20μM CHIR99021, 10μM 616452, 5μM Parnate, 50μM Forskolin, 0.5μM AM580, 5μM EPZ004777 and 250μM vitamin C (Vc); Stage 2 medium contains 78% DMEM, 10% KSR, 10% FBS, 1% NEAA, 1% P / S, 0.055mM β-ME, 20ng / mL bFGF, 0.5mM VPA, 10μM CHIR99021, 10μM 616452,...

Embodiment 2

[0062] Through the concentration gradient test, it is found that 1 μM is the optimal concentration of R406 (such as figure 2 shown); in addition, we also confirmed that R406 promotes the expression of the typical early pluripotency gene Sall4, and specifically plays a role in the early stage of reprogramming ( image 3 shown). The above experimental results show that the effect of R406 is stage-specific, and the optimal use time is the early stage of reprogramming (d0-d12), and the optimal concentration is 1 μM.

Embodiment 3

[0064] Since Sall4, Cdh1, Epcam and Esrrb are stem cell marker genes, Cdh1 and Epcam are also marker genes of mesenchymal-epithelial transition (MET). In order to further verify the role of R406 in promoting reprogramming, we performed real-time fluorescent quantitative PCR (RT-qPCR) experiments to detect the expression of these genes. The results showed that the addition of R406 at the early stage of reprogramming significantly increased the expression of pluripotent genes.

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Abstract

The invention discloses application of R406 in promotion of somatic cell reprogramming and a reprogramming culture medium and method thereof. The molecular formula of the R406 is shown in the specification, when the R406 is actually applied, the R406 can be prepared into a reprogramming culture medium for application, namely, the SYK inhibitor R406 is added into a basic culture medium to prepare the reprogramming culture medium. The R406 inhibits a Syk-Cn-NFAT signal channel and weakens direct binding of NFATc1 to glycine, serine and threonine metabolism related genes, so that expression of the genes is promoted, the content of metabolites in metabolism of glycine, serine and threonine and a downstream cysteine metabolic pathway is increased, and the intracellular H2S level is increased. H2S accumulated in cells regulates and controls mitochondrial oxidative phosphorylation activity and redox steady state, and finally promotes chemical reprogramming.

Description

technical field [0001] The invention belongs to the field of somatic cell reprogramming, and specifically relates to an application of R406 in promoting somatic cell reprogramming, a reprogramming medium and a method thereof. Background technique [0002] Stem cells can currently be obtained in the following ways: [0003] 1) Separation from blastocysts. Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst stage of embryonic development. They are pluripotent and can proliferate indefinitely. They are powerful tools for studying self-renewal, development and disease. However, the acquisition of embryonic stem cells involves ethical issues, and there are immune rejection issues in clinical applications. [0004] 2) Nuclear transfer. Transplantation of somatic cell nuclei into enucleated oocytes eliminates somatic features and has the potential to develop into a complete individual. But the technique is difficult to operate and also involves et...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N15/867C12N5/10
CPCC12N5/0656C12N15/86C12N5/0696C12N2501/727C12N2740/15043C12N2800/107C12N2501/603C12N2501/602C12N2501/604C12N2501/606C12N2510/00
Inventor 祝赛勇王卫云
Owner ZHEJIANG UNIV
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