Establishment Of Human Induced Pluripotent Stem Cell Line

In this experiment,eight exogenous transcription factors of Oct4,Sox2,c-Myc,Klf4,Nanog,Lin28,Rarg and Lrh1 were transfected into human embryonic fibroblasts(hEF)by electrotransfection to reprogram them into human induced pluripotent stem(hiPS)cells.The hiPS cells were then inoculated into the culture medium with mouse feeder cells and the medium without feeder cells respectively.Eight hiPS cell lines were successfully established in four different hiPS cell media and four cell lines with feeder cells and four cell lines without feeder cells,named as hiPSABCL,hiPS-2iLGOY,hiPS-E8 and hiPS-C2 L respectively.With hES-t2 iLGOY cell line and hES-E8 cell line as control groups,the characteristics of these four hiPS cell lines cultured in feeder-free medium were detected and identified.Four hiPS cell lines keep a stable proliferation in vitro for a long time.The hiPS-E8 cell line showed flat cell morphology and the other three cell lines showed as ellipsoidal cell morphology;the result of alkaline phosphatase staining showed negative for hiPS-E8 cell line and positive for the other three.The pluripotent gene Oct-4,Sox2 and Nanog were expressed in all of the hiPS-ABCL,hiPS-2iLGOY,hiPS-E8 and hiPS-C2 L cells and the expression levels were lower than the control groups,while the pluripotent gene Zic2 showed higher expression level than control groups.The teratoma tissue can be formed in SCID mouse.However,the karyotype of hiPS-ABCL cells without feeder cells during serial passages was unstable and unable to differentiate into PGCLCs.Although the technology of hiPS cell lines establishment has developed relatively mature,there are still many unknowns and inefficiencies in the reprogramming process,the new hiPS establishment and culture system in this study will provide a theoretical and technical basis for future hiPS research.