Derivation Of Pig Induced Pluripotent Stem Cells And Establishment Of Embryonic Stem Cells

Since pigs are important animal models because of their similar anatomic and physiological features with human,study of their embryonic stem cells(ESCs)and induced pluripotent stem cells(iPSCs)is increasingly making itself the mainstream research in the field.To date,however,neither porcine ES cell lines nor germline-competent iPS cell lines have been established.This fact is impeding researches on cell fate determination,drug discovery,cell therapies and genetic engineering in large animals.To get high-quality porcine iPSCs,we derived pig iPSCs by two methods with piggyBac(P8)transposon and episomal plasmids respectively.These iPS cells were positive for alkaline phosphatase(AP),expressed the pluripotent markers,OCT4,SOX2,NANOG,KLF4 and c-MYC.They had normal karyotype,and formed teratomas that consisted of tissue types from entoderm,mesoblast and ectoderm in SCID mice.Furthermore,by embryo injection or 4-cell embryos aggregation method,we could produce chimeric blastocysts with pig iPS cells in vitro.But we failed to detect any iPSCs-derived cells in piglets which developed from chimeric blastocysts,suggesting that iPSCs we obtained lack the ability to form chimeras.We found the inactivation of endogenous pluripotency genes of iPS cells is a critical reason for the failure to produce chimeras.To visually track endogenous OCT4 expression,we constructed PB and non-PB reporter vectors containing EGFP driven by mouse,human and pig OCT4 promoter,respectively.The results showed that the pig OCT4-EGFP could be activated in somatic cell nuclear transfer(SCNT)embryos,but could not be activated in iPS cells.This confirmed qPCR results revealing the inactivation status of endogenous OCT4 in pig iPS cells.Meanwhile,we found that Trichostatin A(TSA)could activate the silenced OCT4-EGFP in re-cloned embryos by promoting epigenetic reprogramming during somatic nuclear transfer.To furthermore study the expression pattern of pluripotent genes in pig preimplantation embryos,we constructed non-PB reporter vectors containing tdTomato driven by pig SOX2 and REX1 promoter,respectively.The results showed that SOX2 was in a high expression level in SCNT embryos,however,REX 1 was activated weakly.Additional,to identify gene(s)hindering the activation of endogenous pluripotent genes,we constructed a screening system based on CRISPR/Cas library.We found that this system is a very powerful tool.To explore reasons why it failed to establish pig ESCs,we have used pig embryos of different source to isolate ESCs,including parthenogenetic activation(PA)embryos,in vitro fertilization(IVF)embryos,SCNT embryos carrying OCT4-EGFP or SOX2-tdTomato and in vivo embryos.Medium was optimized by screening for various supplements,such as basic fibroblast growth factor(bFGF),Leukemia inhibitory factor(LIF),fetal pig serum(FPS),pig follicular fluid(PFF),and small molecular inhibitors.ES-like cells could be derived in early passages.We found that bFGF and pig fetal serum could benefit the growth of ES-like cells,but pig follicular fluid and small molecular inhibitors used here inhibited the ES-like cell clones formation.Further,expression of OCT4 and SOX2 lost rapidly after formation of ES-like cell clones.In this study,we demonstrated that pig iPS cells could contribute to chimeric blastocysts in vitro,but no chimeric piglet could be obtained.The inactivation of endogenous OCT4 genes of iPS cells was confirmed by using an OCT4-EGFP reproter.We also optimized culture condition for pig pluripotent stem cells and ESC isolation and found that ES-like cells could grow fast in medium supplenmented with bFGF and FPS,while formation of ES-like cell clones was inhibited by PFF and small molecular inhibitors.Our ESCs/iPSCs study could serve as a systematic start point and benefit further researches in this field.