Establishment Of Mouse Novel Diploid Parthenogenetic Embryonic Stem Cell Lines

The genome of offspring can originate from both the male and the female,or one of them(androgenesis or parthenogenetic).The inheritance of the latter situation is particularly rare in nature,but it provides an extremely useful insight into the recessive genetic characteristics of the organism.In this study,we have explored the two steps methods for establishing the parthenogenetic diploid pluripotent stem cells in mouse.First,we used chemically defined medium N2B27,added activin A(ActivinA),fibroblast growth factor(bFGF)and serum substitutes(5% KSR)to culture parthenogenetic blastocysts,and then derived the parthenogenetic epiblast like stem cells(hereinafter referred to as Pa-AFSCs).The morphology and features of Pa-AFSCs showed as follows:1.The formed and the results of alkaline phosphatase staining of Pa-AFSCs showed that Pa-AFSCs is similar to that of Epiblast stem cells(EpiSCs).2.Immunofluorescence staining results showed that the cell number of expression of Nanog in Pa-AFSCs reduced than control group embryonic stem cells(ESCs),where the expression of Gata6 in Pa-AFSCs was significantly increased than ESCs.We also detected H3K27me3 which is the mark of an inactive X chromosome.3.Real-time quantitative PCR(RT-qPCR)results showed that Pa-AFSCs share similarities with EpiSCs,expressed Oct4,Nanog,Gata4,Gata6 and Eomes,where the transcription level of Elf5(trophectoderm marker gene)and cMyc(cycle related gene)was significantly higher than EpiSCs.4.Experimental results of teratoma and chimera: no teratoma formation was observed in the Pa-AFSCs group;One 6.5-day chimeric embryo was obtained,with an embryonic chimera rate of 4.2%.5.Growth curve results showed that the growth rate of Pa-AFSCs derived from ICR mice was significantly higher than that of Pa-AFSCs derived from 129 mice.Second,we cultured the Pa-AFSCs in ABCL medium(ActivinA,BMP4,CHIR99021,and LIF)and obtain novel mouse pluripotent stem cell lines,named as Ja-ASCs(Ja-Advanced stem cell).The morphology and AP staining of Ja-ASCs was same as ESCs,and also able to self-renew for more than 40 passages.The spot of H3K27me3 disappeared in Ja-ASCs.RT-qPCR analysis of Ja-ASCs recealed compare to Pa-AFSCs,higher expression of Oct4,Nanog and Klf4,where lower expression of Gata4 and the mesoderm marker gene Sox17.The trophectoderm mark gene Cdx2 was obviously higher than that of control group Pa-AFSCs.The results of chimera experiment showed Ja-ASCs failed to contribute to embryos.In summary,we produced Pa-AFSCs and Ja-ASCs from parthenogenetic blastocysts.In morphology and some features,Pa-AFSC was same as Epi SCs,and Ja-ASCs was more like ESCs,however The reason that Pa-AFSC failed to form teratoma,and no Ja-ASCs contribute to postimplantation chimeric embryo remains to be further studied.