ACTIVIN A,bFGF,BMP4 And LIF Induced Mammalian Stem Cell Conversion

Mammalian pluripotent stem cells in original divided to embryonic stem cells(ESCs),adult stem cells(ASCs)and induced pluripotent stem cells(iPSCs).These stem cell be able to self renew and with development potential.The pluripotency and developmental potential of these cells depend on culture system that include essential nutrition and exogenous factors or small molecule inhibitors.As we know,since ESCs have ability to differentiate into kind of tissues,and make it to be used for clinical application.In fact,one important step to capture right medium for ESCs differentiated to particular tissues is always focus point for researcher.In this study,we explored new way to establish right medium for conversion of ESCs to progenitor stem cell.First,we used pervious established AFSCs culture system to derive AFSCs from seven genetic background mice.We found F1 embryos from pure Sv129 and ICR,and heterozygotes of ICR × GOF/GFP,C57BL/6 × ICR mice could give rise to AFSCs.These AFSCs showed similar colony morphology,gene expression and developenmtal potential as AFSCs derived from Sv129 × GOF/GFP.Moreover,AFSCs derived from C57BL/6 × GOF/GFP were heterogeneous with flat epithelial cells and dome-shaped round colonies.We did not establish AFSCs from F1 embryos of pure C57BL/6 and NOD mice,despite of using KSR.Second,we studied the function of Activin A,bFGF,BMP4 and LIF on conversion of AFSCs.Our results demonstrated that culture system bFGF + BMP4 + LIF + N2B27(named FBLM)and Activin A + BMP4 + N2B27(named ABM)induced conversion of AFSCs,and stem cells were named FBLSCs and ABSCs respectively.FBLSCs and ABSCs maintained self-renewal and with developmental potential.FBLSCs tended to contribute to embryonic mesoderm in chimeras,which indicated that FBLSCs were mesoderm-like stem cells.ABSCs being able to passage over 50,co-expressed pluripotent gene Oct4 and mesoderm gene T,and showed properties of neural mesodermal progenitors(NMP),lateral plate mesoderm(LPM)and extraembryonic cells.They contributed to caudal epiblast,brain,spinal cord,somite,yolk sac and placenta.Since both FBLSCs and ABSCs showed property of germ layer progenitor cells,they were named as multipotent progenitor stem cells(refered as MPSCs).Finally,we tested combination of cytokins LIF and GSK3 inhibitor CHIR(medium was named CLM)to induce primed hESCs that were similar to AFSCs to particular progenitor stem cell.Results indicated that hESCs were induced to self-renewal primitive nerve cell like stem cells(PNLSCs).Comparing with LIF,WNT signaling(CHIR)was essential for this conversion process.PNLSCs decreased developmental potential compared to hESCs.PNLSCs showed bias to nerve cells and cranial placode when they underwent a spontaneous and directed differentiation respectively,which indicated PNLSCs were cranial placode progenitor cells.In summary,we showed the essential function of Activin A + bFGF,bFGF + BMP4 + LIF and CHIR + LIF cocktail in chemical defined medium on conversion of mouse AFSCs and human ESCs.These findings demonstrate that capturing right medium provide a potent experimental resource for studying development of mammal gastrulation embryos.