Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

Abstract

Direct generation of skeletal muscle cells from human pluripotent stem cells (hPSCs) would be beneficial for drug testing, drug discovery, and disease modelling in vitro. Here we show a rapid and robust method to induce myogenic differentiation of hPSCs by introducing mRNA encoding MYOD1 together with siRNA-mediated knockdown of POU5F1 (also known as OCT4 or OCT3/4). This integration-free approach generates functional skeletal myotubes with sarcomere-like structure and a fusion capacity in several days. The POU5F1 silencing facilitates MYOD1 recruitment to the target promoters, which results in the significant activation of myogenic genes in hPSCs. Furthermore, deep sequencing transcriptome analyses demonstrated that POU5F1-knockdown upregulates the genes associated with IGF- and FGF-signaling and extracellular matrix that may also support myogenic differentiation. This rapid and direct differentiation method may have potential applications in regenerative medicine and disease therapeutics for muscle disorders such as muscular dystrophy.

Induction of human pluripotent stem cells into kidney tissues by synthetic mRNAs encoding transcription factors

“…Our strategy uses syn-mRNAs encoding for TFs (syn-TFs) for the forced expression of TFs. As we have also shown in our earlier work 17–20,59 , syn-TF-based differentiation of hPSCs is footprint-free, because mRNAs are directly translated into proteins to function and are not converted into DNAs, which could cause unfavorable genome modification 60 . The forced expression of combinations of TFs directly manipulates the gene regulatory network, which is most likely the reason for the faster and more efficient differentiation of hPSCs into NPCs and kidney cells compared to growth factor- and small molecule-based methods.…”

“…In a previous study, we demonstrated that silencing POU5F1 (also known as OCT4, OCT3/4) accelerated MYOD1-mediated myogenic differentiation of hPSCs 17 . To test whether suppressing POU5F1 expression is also beneficial for kidney differentiation, we transfected siRNA targeting POU5F1 (siPOU5F1) with syn-HNF1A and determined the efficiency of differentiation.…”

“…Recently, we have demonstrated that synthetic mRNAs can be transfected efficiently (>90%) in hPSCs 17,18 . We have also reported that synthetic mRNAs encoding TFs can differentiate hPSCs towards neurons, myocytes, and lacrimal gland epithelial-like cells 17–20 . Due to its non-mutagenic feature, this synthetic mRNA-based technology may be suitable for possible future clinical applications.…”

Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors

“…In previous studies by others, more than 2 weeks were necessary just to induce PDX1 + /NKX6.1 + cells. We have previously reported differentiation methods for neurons [ 13 ], skeletal muscle cells [ 17 ], and lacrimal gland epithelium-like cells [ 15 ] in a short period of time by introducing synthetic mRNAs encoding specific transcription factors required for the target mature cells. In this study, we have attempted to shorten the period of time required for the induction of pancreatic endocrine cells from human pluripotent stem cells with the same differentiation method using PDX1 and NKX6.1 synthetic mRNAs.…”

“…Differentiation by synRNAs holds many desirable features for further clinical application, because it allows rapid, efficient, and footprint-free differentiation of human pluripotent stem cells [ 16 ]. We have also reported that the forced silencing of pluripotency master regulator POU5F1 (also known as OCT4, OCT3/4) by siPOU5F1 facilitates synRNA-based hPSC differentiation [ 17 ].…”