The Generation And Optimization Of Mouse And Human IPS Cells From Skin Fibroblasts

Part I Transient folate deficiency in combination with small molecule compounds facilitates the generation of somatic cells-derived pluripotent stem cells in miceObjective To investigate whether folate deficiency, along with novel combination of small molecule compounds (sodium butyrate, A-83-01, CHIR99021and Y-27632), could facilitate reprogramming of somatic cells and eliminate the need for exogenous transcription factors.Methods To reprogramme somatic cell into iPS cells using SKOM, SKO, OK or O with novel combination of small molecule compounds under the condition of folate deficiency.Results Using combination of small molecule compounds under the condition of transient folate deficiency is sufficient to permit reprogramming from mouse embryonic fibroblasts (MEF) in the presence of transcription factors Oct4and Klf4and accelerate the generation of mouse iPS cells. The induced cell lines resembled mouse ES cells regarding proliferation rate, morphology, pluripotency-associated markers and gene expression profile.Conclusion Combining chemical inhibitors and folate deficiency, we have successfully established a protocol which improves the efficiency of iPS cells induction and reduces the carcinogenicity and use of exogenous reprogramming factors. Part II Efficient Reprogramming of HDFa into Induced Pluripotent Stem Cells using Non-Integrating Episomal Vectors in a culture system free of feeder and serumObjective To establish a practical protocol for efficient iPS cells induction from Human Dermal Fibroblasts-Adult (HDFa) using episomal plasmid vectors without feeder, serum and oncogene c-MYC.Methods We transfected HDFa with an episomal vector encoding enhanced green fluorescent protein (EGFP) using the basic NucleofectorTM Kit for primary mammalian fibroblasts and detected the expression of fluorescent signal in24h,1week,2,3,4weeks. HDFa were reprogrammed into iPS cells with an episomal plasmid vector encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28and shRNA for TP53free of feeder, serum and oncogene c-MYC.Results Under fluorescence microscope, green fluorescence of the fibroblasts cells was detected, transfection efficiency was50-60%and the signal gradually weakened.30human iPS cell clones from3×105HDFa were generated in this feeder and serum free culture system.Conclusion Human fibroblasts were successfully transduced with an episomal vector encoding EGFP via electrotransfection and the fluorescent signal gradually weakened, which indicated the ability of reduction for episomal plasmid vectors and met the need of reprogramming. This protocol allows the generation of individualized iPS cells with electrotransfection in a feeder and serum free environment, which paves the road for its wider clinical application in the future.