Abstract
IntroductionThe OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells.MethodsWe tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4.ResultsWe found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels.ConclusionsFor the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.
Highlights
The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation
These results demonstrate that increasing synthetic mRNA (synRNA) concentration and mitigating IFN signaling via B18R are not sufficient to permit robust OCT4 expression from synRNA.we analyzed the immune response pathways to identify alternative potential small-molecule candidates that could potentially block the intra-cellular immune response pathway and stabilize OCT4 expression from synRNA (Figure 2a)
Interaction between pathogen-associated molecular patterns, including single-stranded RNA (ssRNA) [30], leads to activation of the mitogen-activated protein kinases (MAPKs) and the inhibitor B (IB) kinase (IKKa and IKKb), which subsequently turn on nuclear factor-kappa-B (NF-B), via phosphorylating IBa [31]
Summary
The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. The most promising method for reprogramming human somatic cells back into a pluripotent state - referred to as induced pluripotent stem cells - uses viruses to deliver the reprogramming factors (OCT4, SOX2 combined with KLF4 and cMYC or with NANOG and LIN28) into human somatic cells [13,14]. As these viruses randomly integrate into the genome, insertional mutagenesis is an important safety concern [15,16,17]. The most important of these delivered reprogramming factors is OCT4, as recent research has demonstrated that OCT4, in combination with certain small molecules, can itself induce a somatic cell to reprogram to pluripotency without requiring assistance from the other factors [25]
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