Yoshioka Naohisa, Dowdy Steven F
Department of Cellular & Molecular Medicine, UCSD School of Medicine, La Jolla, California, United States of America.
PLoS One. 2017 Jul 27;12(7):e0182018. doi: 10.1371/journal.pone.0182018. eCollection 2017.
We previously devised a polycistronic, synthetic self-replicating RNA (srRNA) to generate human induced Pluripotent Stem Cells (iPSCs) that simultaneously expresses four reprogramming factors (4F). However, while the best 4F srRNA efficiently generated iPSCs from young fibroblasts, it was inefficient on adult human fibroblasts (>50 years). To increase the iPSC generation efficiency, we included additional reprogramming factors. We found that a single transfection of a five factor (5F) srRNA, containing OCT4, KLF4, SOX2, GLIS1 and c-MYC, robustly generated iPSCs from adult human fibroblasts aged 54 to 77 and from a 24 year old cardiomyopathy patient donor. Interestingly, 5F-srRNA induced LIN28A, which was one of the original reprogramming factors. 5F-srRNA also accelerated the generation of iPSCs by seven days compared to 4F-srRNAs. Further improvements include phosphatase treatment to remove 5' phosphate and use of Lipofectamine MessengerMAX that increased transfection efficiency to ~90%. Together, these improvements enabled us to efficiently generate iPSCs from human fibroblasts using 5F-srRNA while eliminating both puromycin selection and feeder cells.
我们之前设计了一种多顺反子合成自复制RNA（srRNA），用于生成同时表达四种重编程因子（4F）的人诱导多能干细胞（iPSC）。然而，尽管最佳的4F srRNA能有效地从年轻成纤维细胞中生成iPSC，但对成人成纤维细胞（>50岁）效率低下。为了提高iPSC的生成效率，我们加入了额外的重编程因子。我们发现，单次转染包含OCT4、KLF4、SOX2、GLIS1和c-MYC的五因子（5F）srRNA，能从54至77岁的成人成纤维细胞以及一名24岁心肌病患者供体的细胞中强劲地生成iPSC。有趣的是，5F-srRNA诱导了LIN28A，它是最初的重编程因子之一。与4F-srRNAs相比，5F-srRNA还将iPSC的生成时间加快了七天。进一步的改进包括进行磷酸酶处理以去除5'磷酸基团，以及使用Lipofectamine MessengerMAX，这将转染效率提高到了约90%。总之，这些改进使我们能够使用5F-srRNA从人成纤维细胞中高效生成iPSC，同时无需嘌呤霉素筛选和饲养细胞。
