Generatioin Of Induced Pluripotent Stem Cells From Adult Human Dermal Fibroblasts

Objective:The generation of induced pluripotent stem cells (iPSCs) from somatic cells demonstrated that adult mammalian cells can be reprogrammed to a pluripotent state by the enforced expression of a few embryonic transcription factors. Human iPSCs raise the possibility of clinical application of autologous stem cell-based therapies without immune rejection or ethical concerns, and provide a unique platform for studying genetic diseases in vitro.Methods:Currently, a number of different approaches have been devised to shuttle reprogramming factors into somatic cells. Such as various virus vectors, plasmids, purified recombinant proteins, modified RNA molecules. Besides, some small-molecule compounds have facilitated the generation of iPSCs, for instance, vitamin C can accelerate gene expression and promote the transition of pre-iPSC colonies to a fully reprogrammed state, while VPA may upregulate the ES-specific genes and downregulate the MEF- specific genes.In the current study, we sought to generate iPSCs from adult human dermal fibroblasts (HDFs) by the retrovirus-mediated transfection of four transcription factors, namely Oct4, Sox2, c-myc, and K1f4. We added vitamin C (Vc) in combination with valproic acid sodium (VPA) to the medium during subsequent culture conditions. By means of live staining using the TRA-1-60/TRA-1-81 antibody, we picked up the positive clones into a new well by Pasteur pipettes. The iPS cell clones were characterized through morphology, alkaline phosphatase staining and immunocytochemistry for pluripotent cell markers.Results:(1) With Lipofectamine 2000 transfection, the virus production has been properly qualified. (2) The derived iPSC-like clones retained the homogeneous features of human ESCs (hESCs) through multiple passages, as evaluated by morphology. And the exogenous gene (GFP) became silenced upon acquisition of pluripotency, while TRA-1-60 and TRA-1-81 were expressed by living cells staining in those derived clones. The iPSC-like clones were picked up for further characterization by alkaline phosphatase staining and immunoflurescence staining for pluripotent cell markers, showing positive results.Conclusions:Based on the preliminary results, the iPSCs have been successfully generated from HDFs, which provides promising cell sources for the future application in autologous stem cell-based gene therapy.