Technology Optimization Of Induced Pluripotent Stem Cell From Mouse By Small Molecule Compound

The main technologies of somatic cell reprogramming include: nuclear transfer,induced pluripotent stem cells(i PS)and cell fusion.One of the most interesting is i PSC technology established by Shinya Yamanaka in2006.This technology has greatly accelerated the application of stem cell technology from the laboratory to the clinic.However,Retroviral vectors are used in the i PS induction process,which may lead to tumors.In order to solve these problems,Chinese scientists have invented the chemically induced pluripotent stem cells(Ci PS)technology.Ci PS technology make up for the defects of i PS.But,This technology uses five small molecule combinations(VPA,CHIR99021,E-616452,Tranylcypromine,Forskolin,and DZNep)to generate over 40 days.In the process,it was also discovered that an intermediate state called Extraembryonic endoderm(XEN).This intermediate cell is a very important stage in chemical reprogramming.In this study,we optimized the production efficiency of intermediate XEN cells based on previous research methods.The main results showed that the first stage of induction was optimized by extending the culture time of 10% FBS+10% KSR medium(supplemented with small molecule VPA,CHIR99021,E616452,Tranylcypromine,Forskolin,AM580 and EPZ044777),and the induction period was 8-12 days Mouse fibroblasts(Mouse fibroblast,MEF)can form smaller clones of XEN cells.XEN marker genes Sall4 and Sox17 gradually increase with the extension of10%FBS+10%KSR culture time.In addition,we optimized the small molecule combination in the first stage.After replacing AM580 with CH55,the number of XEN cell clones increased by 3 times.Next,we optimize the processing time of the second stage.We found that using 10% FBS+10% KSR culture medium in the first8 days of the second stage,the volume of the XEN cell clones mass increased.And the use of serum-free N2B27 culture medium 4 days after the second stage will help the expression of 2-Cell genes Zscan4 and Tcstv1 and early pluripotency gene Oct4.Ci PSCs were finally obtained stably in the third stage through the optimization of the first two stages of chemical induction,The shortest induction period of the method we established was 24 days.The methylation analysis results showed that the methylation level of the Oct4 promoter region of Ci PSCs was similar comparaed with ESC.Next,we look for small molecules that can continue to optimize our induction system.Through the screening of potential small molecule compounds that promote reprogramming,we found that resveratrol and metformin can increase the efficiency of Ci PSCs-induced intermediate XEN cell clone generation.On the basis of the optimized Ci PSCs induction method we have established,adding 1.5 ?M resveratrol to the small molecule combination in the first stage of the induction method can significantly increase the number of XEN cells and the expression of XEN marker gene Sall4.Adding 4.5 ?M resveratrol in the second stage of reprogramming significantly increased the expression of 2-Cell marker genes Zscan4 and Tcstv3,while the expression of Dppa3 gene was significantly reduced.The addition of different concentrations of metformin in the two phases of reprogramming showed a promoting effect on XEN cell cloning,but it was not significantly different from the control.In summary,this study established an optimized stable induction system for Ci PSCs and shortened the induction period to 24 days.The addition of resveratrol and metformin to the induction system promoted the generation of XEN cell clones.In the future,this study provides theoretical and technical support for the establishment of more stable and efficient Ci PSCs cells.