High-efficiency RNA-based reprogramming of human primary fibroblasts

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the reprogramming of human patient's fibroblasts into iPSCs can be enhanced to an unprecedented efficiency via the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNAs-367/302s delivered as mature miRNA mimics. This synergism is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to primary human fibroblasts. As a result, we have developed a novel, integration- and feeder-free reprogramming approach that generates up to 4,019 TRA-1-60 positive colonies from only 500 starting human primary neonatal fibroblasts and reprograms up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant iPSCs from a variety of human patient's fibroblasts and opens up new horizons for studying the biology of reprogramming. Overall design: Gene expression profiles of induced pluripotent stem cells (iPSCs), derived from neonatal, adult and senescent human fibroblasts, were compared to the primary cell lines诱导多能干细胞（induced pluripotent stem cells，iPSCs）在再生医学领域展现出巨大应用前景，但其临床应用潜力却因体细胞重编程效率低下而受到显著限制。本研究证实，通过编码重编程因子的合成修饰mRNA与以成熟miRNA模拟物形式递送的miRNA-367/302s的协同作用，可将人类患者成纤维细胞重编程为iPSCs的效率提升至前所未有的水平。该协同作用依赖于针对原代人成纤维细胞优化的RNA转染方案与专属培养条件。据此，我们开发了一种全新的无整合、无饲养层重编程技术方案：仅以500株人原代新生儿成纤维细胞为起始细胞，即可获得至多4019个TRA-1-60阳性克隆；同时可使高达90.7%的单独接种细胞完成重编程，并产生多个姐妹克隆。该技术方案可稳定从多种人类患者成纤维细胞中获得临床级iPSCs，为重编程生物学研究开辟了全新视野。整体实验设计：将源自新生儿、成人及衰老人类成纤维细胞的iPSCs的基因表达谱，与对应原代细胞系进行对比分析。