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

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.

Efficient RNA-mediated reprogramming of human somatic cells to naïve pluripotency facilitated by tankyrase inhibition

“…Integration and/or persisting expression of reprogramming factor transgenes is undesirable in principle, and specifically may perturb the naïve PSC state or subsequent differentiation. We therefore focused on producing transgene-free naïve hPSC by transient delivery of non-modified RNAs (Poleganov et al, 2015).…”

Re‐entering the pluripotent state from blood lineage: promises and pitfalls of blood reprogramming

“…To date, only one report has described the successful reprogramming of blood cells using RNA reprogramming strategies [29]. Typical mRNA reprogramming involves several rounds of repeated transfection of the transcribed reprogramming factor mRNAs into target cells.…”

“…Unlike the high efficiency achieved with fibroblast reprogramming, suspending blood cells seem to be refractory to the mRNA reprogramming. To date, only one report has described the successful reprogramming of blood cells using RNA reprogramming strategies [29]. Of note, the study targeted adherent cells residing in the blood (i.e., endothelial or mesenchymal cells).…”

Concise Review: Application of In Vitro Transcribed Messenger RNA for Cellular Engineering and Reprogramming: Progress and Challenges

“…The self‐replication of RNA constructs in cells makes the daily transfection with mRNA unnecessary, but it requires the addition of conditioned medium containing B18R. Recently, Poleganov et al generated reprogramming mRNAs (Oct4, Sox2, Klf4, Lin28, cMyc, and Nanog) using nonmodified nucleosides and combined them with immune reaction circumventing mRNAs coding for E3, K3, and B18R from vaccinia virus and the enhancer microRNA miR302/367 to overcome RNA‐related cytotoxicity during the reprogramming process . Using this approach, nonmodified mRNAs, which are known to induce cellular defense mechanisms, were effectively able to convert fibroblasts into iPSCs with four repeated transfections within 11 days and blood‐outgrowth endothelial progenitor cells were reprogrammed within 10 days with only eight daily transfections.…”