Study On The Pluripotency Maintenance Of Pig IPS Cells And Hurdles Of Their In Vivo Development

To date no authentic germline-competent embryonic stem cell(ESC)line or induced pluripotent stem cell(iPSC)line has been established for large animals.Elucidation of the causes of this failure in obtaining high quality pig ES/iPS cells may not only broaden the perspective of stem cell knowledge of scientists,but also offer essential clues for eventual establishment of authentic pluripotent stem cells for large animals including humans.Several groups have reported derivation of pig iPSCs,however,no transgene-free pig iPSCs could be induced and no pig iPSCs could be used to reliably produce viable,fertile offspring.Most importantly,whether pig iPSCs have the ability to pass the crucial test of germ-line contribution needed to generate gene-modified piglets through chimera approaches remains to be demonstrated.To a great extent,these defects also restrict the research about pluripotency,differentiation,and development of pig iPSCs,In order to deal with these disadvantages,we first generated pig iPSCs using non-integrating replicating episomal plasmids,excisable piggyBac transposons,Cre/loxP excision of transgenes,and DOX/TMP-inducible systems.Pluripotency was demonstrated by 2i/LIF dependency,robust clonogenicity,reactivation of the X chromosome in female iPSCs,pluripotency gene expression and teratoma formation.The iPSCs could be genetically modified throughout the pig genome using piggyBac transposon based vectors.These pig iPSCs could develop into cloned embryos and chimeric blastocysts in vitro,and participated in the generation of inner cell mass(ICM)and trophectoderm(TE).However,nuclear transfer,early embryo injection,or embryo aggregation methods all failed to produce viable iPSC-derived piglets.From detailed analysis of individual transgenes contained in the episomal vectors,it appeared that all the Yamanaka factors,and particularly OCT4,could hardly be further removed from pig iPSCs.And all pig iPSCs expressed the endogenous OCT4 gene at levels much lower than the integrated exogenous OCT4 jtranscript.CpG sites in the pluripotent endogenous OCT4 gene and imprinted GTL2 gene promoter regions were highly methylated in these iPSCs,almost on the same level with fibroblasts.The majority of differentiated female pig iPSC lines maintained two active X(Xa)chromosomes.Experimental results validated that epigenetic modification abnormalities occur regularly during reprogramming and differentiation.The data of RNA-seq was used to show the transcripts and the intersection of the number of the differentially expressed genes in reciprocal cross fibroblasts,pig iPSCs and differentiated pig iPSCs.The results of the study confirmed that the morphology of differentiated pig iPSCs changed obviously under differentiation medium,However,their gene expression profiles were quite similar to the previous pig iPSCs.The fibroblast-like differentiated pig iPSCs could not compete with the original fibroblasts.In our experiments,although the differentiated pig iPSCs retained high blastocyst rate and implantation rate in vitro,we still failed to produce cloned piglets by nuclear transfer.Through extensive in vivo experiments with pig iPSCs,we have found that incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appeared to be the main reason for the inability of pig iPSCs to form iPSC-derived piglets.These abnormalities in pig iPSCs may include those in DNA methylation,imprinting,and X chromosome inactivation.Elucidation of these abnormalities should help us develop better reprogramming methods and culture conditions for eventual derivation of genuine pig iPSCs as well as ESCs.