Tang Yong, Lin Chih-Jen, Tian X Cindy
Center for Regenerative Biology, Department of Animal Science, University of Connecticut, 1390 Storrs Road, Storrs, CT 06269, USA.
Cell Reprogram. 2011 Apr;13(2):99-112. doi: 10.1089/cell.2010.0072.
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.
诱导多能干细胞（iPSC）技术在再生治疗中具有巨大潜力。与聚精氨酸细胞穿透肽（CPP）相连的蛋白质（piPSC）可诱导产生iPSC，且无基因组改变风险，尽管效率极低且重编程延迟。我们旨在评估与HIV转录激活因子（TAT）或果蝇穿膜肽蛋白的CPP在N端或C端相连的纯化小鼠Klf4蛋白的重编程能力。真核表达的重组Klf4靶向细胞核，而纯化的蛋白定位于细胞质和核周区域。然而，通过Klf4蛋白与表达Oct4、Sox2和c-Myc（OSM）的逆转录病毒联合转导，我们发现TAT和穿膜肽相连的Klf4蛋白在2至4周内均能在纳摩尔水平显著诱导小鼠iPSC形成。N端带有TAT的Klf4蛋白无重编程活性，而在TAT和Klf4之间带有盘基网柄菌红色荧光蛋白（DsRed）的融合蛋白则具有显著的iPSC诱导功能。由Klf4蛋白和逆转录病毒OSM组合诱导产生的iPSC具有多能性。利用蛋白/逆转录病毒OSM重编程检测方法，我们进一步评估了Klf4蛋白转导条件，结果表明纯化的Klf4蛋白连续转导四次足以有效诱导iPSC。我们的结果首次证明，TAT和穿膜肽相连的Klf4蛋白在重编程成纤维细胞时可有效替代病毒Klf4，并为评估重组蛋白和转导条件以提高piPSC诱导效率提供了有价值的策略。
