Derivation Of Advanced Pluripotent Stem Cells From Post-implantated Mouse Embryons

Mouse epiblast stem cells(EpiSCs)are derived from the epiblast tissue of mouse E5.5-7.5 embryos.The pluripotency of EpiSC is maintained by Activin(Nodal)/Smad and FGF/ERK signaling.Compared with na?ve state of mouse embryonic stem cell(ESC),EpiSC is considered as a primed state.Primed refers to the initiation of lineage specification in gastrula stage epiblast cells and EpiSCs.EpiSCs are very different from ESCs in transcriptomical,epigenetical,metabolical and functional fields.Here EpiSCs was derived from mouse E6.5 epiblast tissue in a chemically defined medium with Activin A and basic fibroblast growth factor(bFGF)(called AF medium)without MEFs or serum.We named the EpiSCs as AF-EpiSCs.Furthermore,AF-EpiSCs were cultured in ABCL medium(Activin A,BMP4,CHIR99021 and LIF)to establish the advanced pluripotent stem cells(called post-ASCs).Final,we assay the pluripotency of post-ASCs in the protein level and molecular level.The results showed in below.1.Establishment of AF-EpiSCsThe AF-EpiSCs that established under AF culture medium have the same characteristics as traditional Epi SCs.AF-EpiSCs has the ability for self-renew and also maintain pluripotency which proof by investigating the cell morphology,cell growth curve,chromosome karyotype,immunofluorescence assay,result of RT-qPCR and in vivo teratoma differentiation.2.Derivation and characterization of post-ASCsWe started with established AF-EpiSCs cell lines with GOF-GFP reporters.When AF-EpiSCs were cultured in ABCL medium for 2 weeks,we detected GOP-GFP positive clones(post-ASCs),which were able to self-renew for more than 20 passages.AP staining result is positive in post-ASCs.Karyotype analysis show that chromosomes of post-ASCs were 40,no abnormalities were observed in derived process.The immunofluorescence staining data and RT-qPCR results showed that post-ASCs expressed the core pluripotent genes such as Oct4,Sox2,Nanog,and Klf4,and the expression level was same as the ESCs.3.Assessment of potential of post-ASCsPost-ASCs able to differentiated into the three germ layers in formed teratomas.We also found that post-ASCs contributed to embryo proper and germline,as well as to the full-term pups.Importantly,post-ASCs contributed to the placenta in chimeric conceptuses.In conclusions,post-ASCs are a novel and distinct self-renewing type of pluripotent stem cell,which exhibit greater stability and potency.However,the reprogramming mechanisms of this high potency in post-ASCs are not known,need to explore in future.