The Conversion Of Novel Mouse Embryonic Stem Cells Under Different Chemically Defined Conditions

As more attentions paid on the induction of mouse embryonic stem cells for these years,research progress is also changing rapidly.Chemically defined culture medium was used in this experiment for studing the relationship between different embryonic stem cell lines.The experiment consists of two parts:The first part is about the affects of four growth factors to the induction of novel mouse advanced embryonic stem cells(ASCs);the second part is about the study of conversion of ASCs to EpiSCs like stem cell(AFSCs)and trophectoderm stem cells(TSCs).Here,novel EpiSCs like stem cell(AFSCs)established in our lab was used by material.Four growth factors(ActivinA,BMP4,CHIR99021,mLif)were test in derivation of novel advanced embryonic stem cell(ASCs)from AFSCs.The GOF-GFP positive clones of AFSCs were found in medium with four growth factors(ABCL group)for a few days,and also built up stable novel advanced stem cell lines(ASCs).We found a few GOF-GFP positive clones in other three groups ACL(no added B group),ABC(no added Lgroup),ABL(no added C group),but need to further research for building stem cell lines.RT-qPCR results also showed Oct4,Sox2,and Nanog were increased expression with culture days in ABCL,particularly,in day 6,Nanog is higher express than other groups.Note,BCL group(no added Activin A)is to promote differentiation of epidermal direction;the role of BMP4 is inhibiting nerve-related genes;the role of Lif is mainly to inhibit primitive endoderm and promote pluripotent gene expression.Next,ASCs and ESCs were cultured in EpiSCs(called AF medium)and trophectoderm stem cell(TSC medium)conditions,respectively.AP staining result showed ASCs and ESCs was heterogeneous when they cultured in AF and TSC medium.However,proliferation and stable passage happened in ASCs-AF,and ASCs-TSC group,and differentiation were observed in ESCs two group.The immunofluorescence staining data and RT-qPCR data also consisted with it.When ASCs culture in AF medium or TSC medium,both conversed to stable AFSCs,but failed to trophectoderm stem cell(TSCs).The results of karyotype analys is showed that the karyotypes of cells of ASCs,ASCs-AF and ASCs-TSC were normal.In summary,the four growth factors induced AFSCs develop to ASCs;ASCs is different with ESCs,could conversed to AFSCs when they cultured in AF medium or TSC medium.It is provide a model to investigate questions of conversion of ESCs to different stem cell in chemical defined medium.