Reprogramming Of Somatic Cells Via TAT-mediated Protein Transduction Of Recombinant Factors

Successful establishment of Induced pluripotent stem cells (iPSCs) is thought to herald the revolutionaries of regenerative medicine, pharmaceutic screening, disease models and basic researches. Before this promising technique could be fulfilled in the therapeutic use, the efficiency of iPSC generation and safety of iPSCs, as the clinical prerequisites, are required to be satisfied.The exploration for technologies of reprograming pluripotency without ectopic integration, aiming at circumventing of transgene reactivation or insertional mutagenesis, has advanced significantly. So far integration-free iPSCs have been derived via excisable (transposon and floxed lentiviral), nonintegrating vectors (plasmid, episomal, and adenovirus vectors), and DNA-free (recombination proteins incorporating cell-penetrating peptide, Sendai virus and Synthetic Modified mRNA) approaches. Among these, generation of iPSCs through recombinant proteins is the exclusive method to date without the use of genetic materials. Despite such advantages, protein based iPS-generations have so far suffered with an extremely low efficiency and a long conversion process, which limit its further application. Nevertheless, protein based iPSCs generation deserves further research.In this study, we explore a simple, genetic materials-free strategy for somatic cell reprogramming based on fusion proteins with protein transduction domain TAT. We purify TAT-fusion proteins of reprogramming factors (Oct4/Sox2/c-Myc/Klf4) with bio-functions from E. coli and explorate the optimized conditions for TAT-RF transductions, including working concentrations, incubation time. Also, staibilities of TAT-RFs in vivo are exaimed. We demonstrate that TAT-RFs could transduce multiple human and mouse cell lines and activate their corresponding reporter genes. In addition, we show that these fusion proteins can substitute their corresponding retrovirus to promote generation of iPSCs in the reprogramming process using ’3virus+1protein’ assays. Based on above results, we produce a protocol for4TAT-RF induced iPS generations:human forskin fibroblast cells are treated with4TAT-RFs (50nM each, incubation time:2h)2days one round for17days in total. Unfortrunately, no iPS-like colony is emerged under this protocol. We hypotheses Transduction of TAT-fusion proteins generates iPS-like colonies from human fibroblast cells and mouse fetal neural progenitor cells with relatively high efficiency and quick dynamics based on the result of alkaline phosphatase staining. Additionally, our work suggests that the relative low level of protein transduction is likely the key barrier to the success of protein-based iPSCs.In conclusion, our study provides new information of using TAT-based protein transduction approach for the generation of clinic relevant and genetic material-free human iPSCs.