Induced Pluripotent Stem Cell Production From Neural Stem Cell Using A Single Oct4 Transcriptional Factor

Pluripotent stem cells have both the abilities to unlimitedly self-renew and to differentiate into various functional lineages. Scientists have been making efforts to find effective ways to obtain pluripotent stem cells. In the past five years, generation of pluripotent stem cells using 4 transcriptional factors provided a promising strategy to solve the problems that hamper the acquisition of pluripotent stem cells. However, this strategy needs to be further improved mainly due to low efficiency and safety. To overcome these weakness, we used a fusion protein between a single transcriptional factor, hOct4, and cell penetrating peptides, which is produced using prokaryotic expression systems, to induce human neural stem cells into pluripotent stem cells. The results are as follows:1. We successfully constructed a pET-based prokaryotic expression system consisting of hOct4 cDNA, 11 Arg cell penetrating peptide and His purification tag (hOct4-11R-His). Subsequently, the recombinant plasmid was transferred into Rosetta2(DE3)E.coli. The expression of 43 KDa fusion protein was induced by IPTG (1mM) at 28℃. Finally, hOct4-11R-His from the supernatant part of bacterial lysate was purified by Ni affinity chromatography and identified by Western Blotting.2. The penetrating capacity of Rhodamine-labelled hOct4-11R-His and EGFP-11R-His was investigated using BJ cells and human neural stem cells. The result showed that recombinant proteins effectively enter the tested cells without cytotoxicity at the concentration of 10μg/ml. hOct4-11R-His was demonstrated to have an effective cell penetrating capacity to human neural stem cells and a tropism to nucleus.3. The NSC were treated 12h with the recombinant protein hOct4-11R-His at 10ug/ml in the ReNcell NSC maintain medium supplemented with 1mM valproic acid (VPA),followed by changing to the same media without the recombinant protein, and culturing for 96h before the next cycle of the treatment. After six rounds of treatment, several colonies with iPS-like morphology were observed. Quantitative Real Time-PCR showed increased expression of endogenous Oct4 and Nanog in hOct4-11R-His-treated neural stem cells. Moreover, AP positive cells also appeared in this cell population.The strategy of protein-mediated induced pluripotent stem (iPS) cells production, which is not involved in any genetic modification, avoids the potential tumor-inducing risk resulting from usage of viral vector and is currently the safest way to acquire iPS cells. Our works showed that single transcriptional factors-mediated iPS production is feasible and provided a research platform for further investigation.