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. 2015 Nov;26(11):751-66.
doi: 10.1089/hum.2015.045. Epub 2015 Oct 1.

Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion

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Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion

Marco Alexander Poleganov et al. Hum Gene Ther. 2015 Nov.

Abstract

mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent and time-consuming and mainly restricted to fibroblasts, thereby demonstrating the need for a simple but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Nonmodified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence, to overcome RNA-related toxicity we combined nonmodified reprogramming mRNAs (OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 [OSKMNL]) with immune evasion mRNAs (E3, K3, and B18R [EKB]) from vaccinia virus. Additionally, we included mature, double-stranded microRNAs (miRNAs) from the 302/367 cluster, which are known to enhance the reprogramming process, to develop a robust reprogramming protocol for the generation of stable iPS cell lines from both human fibroblasts and human blood-outgrowth endothelial progenitor cells (EPCs). Our novel combination of RNAs enables the cell to tolerate repetitive transfections for the generation of stable iPS cell colonies from human fibroblasts within 11 days while requiring only four transfections. Moreover, our method resulted in the first known mRNA-vectored reprogramming of human blood-derived EPCs within 10 days while requiring only eight daily transfections.

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