Etv5 Regulates Cell Reprogramming And Embryonic Stem Cells Differentiation

Induced pluripotent stem cells(iPSCs)have been placed high hopes on autologous cell replacement therapy and establishment of disease models like Parkinson’s disease ever since its first report.However,the low efficiency at establishing bona fide pluripotency and clonal variation between iPSC lines severely hold back its step towards clinical applications.Based on the fact that SSCs cultured in vitro can be reprogrammed to pluripotent stem cells only by changing the culture conditions,we speculated that there might be reprogramming enhancers among the SSCs TFs.In this study,we screened a pool of TFs which are reported to be crucial for SSCs selfrenew,and found that the efficiency of iPS induction increased significantly when combining Etv5 with Yamanaka factors(Oct4,Sox2,Klf4,and c-Myc,abbreviated as OSKM hereafter),whereas knockdown of Etv5 dramatically decreased the the efficiency of iPS induction.iPSCs derived from OSKME combination(OSKME-iPSCs)express multiple pluripotent marker genes and were competent for differentiation into cells from all three germlayers,and thus were bona fide pluripotent stem cells.We demonstrated that Etv5 promote somatic reprogramming by facilitating mesenchymal-epithelial transition(MET)and mainly through Tet2-miR200s-Zeb1 axis.In mESCs,knockdown of Etv5 didn’t influence the maintenance of mESCs but decreased the global genomic 5hmC level by downregulating Tet2.The embryoid body assay revealed that Etv5 could positively regulate primitive endoderm specification through regulating Gata6 and negatively regulate epiblast specification by inhibiting Fgf5 expression.1.Etv5 promotes somatic reprogramming.We screened SSCs TFs by reference retrieval and found that Etv5 showed highest efficiency of iPS induction when combined with Yamanaka factors.We confirmed that Etv5 mainly functions at the early stage of somatic reprogramming by introducing the overexpression of Etv5 at different timepoint.In addition,RNAi of Etv5 significantly decreased the efficiency of somatic reprogramming.OSKME-iPSCs showed typical mESCs morphology and expressed multiple pluripotent markers like Nanog,Ssea1,Rex1 and Klf2.Bisulfite sequencing of Nanog and Oct4 promoters showed dramatic DNA demethylation when compared OSKME-iPSCs with MEFs.Moreover,OSKME-iPSCs can differentiate into cells of all three germlayers in vitro/vivo.When induced specifically towards germ cell lineage,OSKME-iPSCs exhibited better response to differentiation factors.2.Etv5 promotes MET through Tet2-miR200s-Zeb1 axis.By comparing the proliferation rate and expression of cell cycle regulators of OSKM and OSKME transduced MEFs,we excluded the possibility that Etv5 might promote somatic reprogarmming by facilitating cell proliferation.Instead,immunostaining and flow cytometry analysis of epithelial marker gene CDH1 in reprogramming MEFs revealed that MET was triggered earlier and broader in OSKME tranduced MEFs.qRT-PCR of mesenchymal TFS revealed that knockdown of Etv5 exclusively increased Zeb1 expression.Overexpression of Etv5 in OSKM combination was found to increase the expression of all mi R-200 faminly members.Morever,TET proteins were proposed to directly regulate the expression of miR-200 s,and we confirmed the exclusive contribution of Etv5 to Tet2 expression through manipulation of Etv5 in OSKM induced reprogramming.Together,the results above suggest that Etv5 promotes MET through Tet2-miR200s-Zeb1 axis.3.Etv5 orchestrates the specification of primitive endoderm and epiblast during mESCs differentiation in vitro.Through lentivirus mediated transduction,we established ES cell line stably infected with Etv5 shRNAs.Etv5-KD mESCs showed no obvious difference with control mESCs on AP staining and expression of pluripotent marker genes,except for some compromise in cell proliferation.We confirmed the regulation of Etv5 towards Tet2 at RNA and protein levels through qRT-PCR and Western blot in Etv5-KD mESCs.In addition,Etv5-KD decreased the genomic 5hmC level by downregulating Tet2.Gene ontology(GO)analysis of Etv5-KD RNA-Seq data revealed that genes influenced by Etv5 knockdown were highly enriched in GO terms like MAPK signaling pathway,sensory organ morphogenesis,urogenital system development and angiogenesis.Embryoid body assay revealed that Etv5 could positively regulate primitive endoderm specification through regulating Gata6 and negatively regulate epiblast specification by inhibiting Fgf5 expression.In summary,our findings provide insights into understanding the regulation mechanisms of Etv5 under the context of somatic reprogramming,mESCs maintenance,and differentiation.