Functionality and Transduction Condition Evaluation of Recombinant Klf4 for Improved Reprogramming of iPS Cells

The induced pluripotent stem cell (iPSC) technology holds great potential in regenerative therapy. iPSCs could be induced by proteins (piPSC) linked with poly-arginine cell-penetrating peptides (CPPs) without the risk of genomic alteration, athough with extremely low efficiency and delayed reprogramming. We aimed to evaluate the reprogramming potency of purified mouse Klf4 proteins linked with the CPP of HIV transactivator of transcription (TAT) or Drosophila Penetratin protein at the N- or C-terminus. Eukaryotically expressed recombinant Klf4 targeted cell nucleus while the purified proteins localized in the cytoplasmic and peri-nuclear region. However, using a combined transduction of Klf4 protein and retroviruses expressing Oct4, Sox2, and c-Myc (OSM), we found both TAT-and penetratin-linked Klf4 proteins significantly induced mouse iPSC formation at the nanomolar level in 2 to 4 weeks. Klf4 protein with TAT at the N-terminus showed no reprogramming activity while the fusion protein, with Discosoma red fluorescent protein (DsRed) between TAT and Klf4, exhibited significant iPSC induction function. The iPSCs induced by Klf4 protein and retroviral OSM combinations were pluripotent. Using the protein/retroviral OSM reprogramming assay, we further evaluated Klf4 protein transduction conditions and showed that four continued transductions by purified Klf4 proteins are sufficient for effective iPSC induction. Our results demonstrated for the first time that TAT-and Penetratin-linked Klf4 proteins can effectively replace viral Klf4 in reprogramming fibroblasts, and provided a valuable strategy to evaluate recombinant proteins and transduction conditions for the improvement of piPSC induction efficiency.