Study On Transformation To Naive Pluripotent State Of PiPSCs By Chemical Small Molecule Compounds

This experiment was conducted to optimize the culture condition which can promote the transformation of piPSCs(Dox-inducible Pig induced pluripotent stem cells)to Naive pluripotency state in vitro by screening the small molecules compounds,and then preliminary investigated the mechanism of optimized culture condition.What’s more,we also studied the influence on pluripotency of piPSCs that cultured with optimized medium under the condition of feeder-free or Dox-free.The results were as follows:1.After 5iL(CHIR99021+PD0325901 +Lif+616452+Forskolin+LPA)was added to piPSCs culture condition in different basic medium,all of the piPSC colonies showed a more dome-shaped morphology,stronger AP activity and significantly improved relative fluorescence intensity of Sox2,Oct4 and H3K4me3 than that of control group.At the same time,it also showed that the relative fluorescence intensity of Nanog,Sox2,Oct4 and H3K4me3 was significantly improved in basic medium of N2B27 than that of other basic medium with 5iL.Furthermore,the N2B27/5iL culture system was optimized as N2B27+2iL+b+L(CHIR99021+PD0325901+Lif +bFGF+LPA).At this optimized culture system,colonies showed a tighter morphology which are more likely to mESCs and a significantly higher relative fluorescence intensity of the Oct4,Nanog,Sox2 and H3K4me3 than piPSCs in N2B27/5iL.2 Subculturing and bio-assay of piPSCs that treated with 2iLbL culture condition.The results showed that the piPSCs cultured in optimized 2iLbL group not only possessed dome-shaped mESC-like morphology but also showed a stronger alkaline phosphatase activity and higher protein expression level of Nanog,Oct4,Sox2,SSEA4,H3K4me3 than in control group.Meanwhile the expression of the naive pluripotency state marker genes Rex-1,Essrb,Eras,Stella,Oct4,Sox2 of piPSCs was significantly improved.What’s more,the deletion of single small molecule compound as LPA,bFGF and Lif had a great influence on morphology of colonies.What’s more,compared to piPSCs in"N2B27+2iL+b+L",the expression of Nanog and Oct4 were significantly reduced in all incomplete groups except that there was no significant difference in the expression of Nanog in the absence group of the bFGF and Oct4 in the LPA deletion group.3 In the condition of feeder-free,colonies in 2iLbL culture condition maintained a dome-shaped mESC-like morphology and a strong AP activity.What’s more,it could tolerate trypsin digestion and then formed monoclone.Meanwhile it also possessed immunofluorescence staining of Oct4,Nanog,Sox2,SSEA-4,Tra-1-81.In Dox-free condition,the morphology of colonies can be maintained in the 2iLbL culture system to a certain extent.All above results showed that we had screened the culture condition of N2B27+2iL+b+L,which could promote the conversion of piPSCs to naive state and all of the compounds worked together to realize the function of it.What is more,the system can also maintained the pluripotency of piPSCs in the condition of feeder-free and morphology of colonies in Dox-free condition.This study can provide not only theoretical basis for the transition from primed to naive pluripotency state in piPSCs but also a rich cellular resource for the subsequent better use of piPSCs.So that it can be better applied in fields such as animal husbandry,clinical and regenerative medicine.