Targeted gene silencing in human embryonic stem cells using cell-penetrating peptide PepFect 14

Abstract

BackgroundHuman embryonic stem (hES) cells serve as an invaluable tool for research and future medicine, but their transfection often leads to unwanted side effects as the method itself may induce differentiation. On the other hand, RNA interference (RNAi)-based targeted gene silencing is a quick, cost-effective, and easy-to-perform method to address questions regarding the function of genes, especially when hypomorphic knockdowns are needed. Therefore, effective transfection method with minimal side effects is essential for applying RNAi to hES cells. Here, we report a highly promising approach for targeted gene silencing in hES cells with siRNA complexed with cell-penetrating peptide PepFect 14 (PF14). This strategy provides researchers with efficient tool for unraveling the functions of genes or addressing the differentiation of pluripotent stem cells.MethodsWe present a method for delivery of siRNA into hES cells with cell-penetrating peptide PF14. Accordingly, hES cells were transfected in ROCK inhibitor containing medium for 24 h right after EDTA passaging as small cell clumps. Fluorescently labeled siRNA and siRNAs targeting OCT4 or beta-2-microglobulin (B2M) mRNA sequences were used to evaluate the efficiency of transfection and silencing. Analyses were performed at various time points by flow cytometry, RT-qPCR, and immunofluorescence microscopy.ResultsEffective downregulation of OCT4 in 70% of treated hES cells at protein level was achieved, along with 90% reduction at mRNA level in bulk population of cells. The applicability of this low-cost and easy-to-perform method was confirmed by inducing silencing of another target not associated with hES cell pluripotency (B2M). Furthermore, we discovered that downregulation of OCT4 induces neuroectodermal differentiation accompanied by reduced expression of B2M during early stage of this lineage.ConclusionsThe results demonstrate PF14 as a promising tool for studying gene function and regulatory networks in hES cells by using RNAi.Electronic supplementary materialThe online version of this article (10.1186/s13287-019-1144-x) contains supplementary material, which is available to authorized users.

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“…[80] Ervin et al have used PF14 as a way to safely transfect human embryonic stem cells (hES) in culture. [27] To probe the delivery of nucleic acid, an Alexa Fluor 568 conjugated siRNA is Urgard et al have also explored the use of PF14 to transfect micro RNA (miRNA) into cultured cells. [28] They investigate the delivery of MiR-34a as a potential therapy for cancer, since MiR-34a has been shown to act as a tumor suppressor.…”