| Mouse pluripotent embryonic stem cells(mESCs)and epiblast stem cells(EpiSCs)were isolated from inner cell mass(ICM)in blastocyst and epiblast from post-implantation embryos respectively.The maintenance of pluripotency of mESCs relies on two inhibitors of Mek1/2 and GSK3(2i),and Leukemia inhibitory factor(LIF),however EpiSCs keep with Activin A(Act A)and basic fibroblast growth factor(bFGF).A novel embryonic stem cell line(AFSCs)was obtained from mouse blastocysts cultured in Act A and bFGF medium(excluding KSR and feeder layer cells)in our lab.In this study,we first investigated the effects of Act A and bFGF on the establishment of mouse early embryo lines and induction of AFSCs cell lines.Secondly,bone morphogenetic protein 4(BMP4),GSK3 inhibitor(CHIR99021)and LIF were used to replace bFGF in AF medium to form AB,AC and AL cultures,respectively.AFSCs were cultured in these three medium to induce a cell line with early specific embryonic characteristics,self-renewal and pluripotent ability.1.Establishment of an epiblast-like stem cell lineMouse blastocysts(E3.5)which collected in 3.5 days after fertilization were cultured in five mediums supplemented with AF(Act A + bFGF),A(Act A),2A(2 × Act A),4A(4 × Act A)and 2F(2 × bFGF).AF/SCs(The source and method of establishing the AFSCs are the same),2A/SCs and 4A/SCs were successfully obtained in AF,2A and 4A medium,and the efficiency was 15%,14.3% and 7.2%,respectively.4A/SCs is similar to AF/SCs in morphology,2A/SCs is heterozygous,both maitain AP staining positive cells.The expression of pluripotency and differentiation genes in 4A/SCs was significantly higher than 2A/SCs.2.Effect of Act A on AF/SCs cell linesThe AF/SCs cell lines were cultured in six mediums with different concentrations of Act A and bFGF(Act A,2 × Act A,4 × Act A,bFGF,2 × bFGF,4 × bFGF).Six cell lines were obtained,namely AF-A/SCs,AF-2A/SCs,AF-4A/SCs,AF-F/SCs,AF-2F/SCs and AF-4F/SCs.AP staining showed that there were more positive cells in Act A group than in bFGF group.In addition,AF-4A/SCs higher expressed Nanog,Gata4 and Cdx2 than other groups,but the results of AF-4A/SCs chimerism showed that there were no labeled cells in the embryonic and extraembryonic structures,and they did not have the ability to contribute to embryos.3.Effect of Act A and BMP4 on AF/SCs cell linesThe BMP4,CHIR99021,and LIF were added to AF/SCs instead of bFGF respectively,named AF-AB/SCs,AF-AC/SCs and AF-AL/SCs.AF-AB/SCs and AF-AL/SCs were similar to AF/SCs in clonal morphology,and AP was positive.Nanog,Gata6 and T were highly expressed in AF-AB/SCs and AF-AL/SCs.Chimera preparation technology proved when AF-AB/SCs and AF-AL/SCs were injected into 8-cell embryos and cultured for 44 hours in vitro,both cells were found to contribute both to ICM and to trophectoderm(TE);furthermore we found AF-AB/SCs contributed to the extraembryonic tissues,but failed to embryo in E6.5 chimeras,while AF-AL/SCs did not contribute to embryos and extraembryonic tissues.Transcriptome analysis showed that there were different gene expression patterns in AF/SCs,AF-AB/SCs and AF-AL/SCs.Developmental regulatory genes,some differentially expressed genes are enriched in extra-embryonic tissue development and pluripotency signaling pathways are upregulated in AF-AB/SCs.In conclusion,Act A has an ability to support and maintain self-renewal of stem cells indepently.Act A and BMP4 can transform AF/SCs into Mesoderm-like stem cells.Due to the culture conditions applied to derive AF/SCs were essentially the same as those used for propagating human pluripotent stem cells,our findings provide new insights into the study of differentiation of human embryo stem cells. |
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