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Aims Direct cardiac reprogramming represents an attractive way to reversing heart damage caused by myocardial infarction because it removes fibroblasts, while also generating new functional cardiomyocytes. Yet, the main hurdle for bringing this technique to the clinic is the lack of efficacy with current reprogramming protocols. Here, we describe our unexpected discovery that DMSO is capable of significantly augmenting direct cardiac reprogramming in vitro. Methods and results Upon induction with cardiac transcription factors- Gata4, Hand2, Mef2c and Tbx5 (GHMT), the treatment of mouse embryonic fibroblasts (MEFs) with 1% DMSO induced ~5 fold increase in Myh6-mCherry+ cells, and significantly upregulated global expression of cardiac genes, including Myh6, Ttn, Nppa, Myh7 and Ryr2. RNA-seq confirmed upregulation of cardiac gene programmes and downregulation of extracellular matrix-related genes. Treatment of TGF-β1, DMSO, or SB431542, and the combination thereof, revealed that DMSO most likely targets a separate but parallel pathway other than TGF-β signalling. Subsequent experiments using small molecule screening revealed that DMSO enhances direct cardiac reprogramming through inhibition of the CBP/p300 bromodomain, and not its acetyltransferase property. Conclusion In conclusion, our work points to a direct molecular target of DMSO, which can be used for augmenting GHMT-induced direct cardiac reprogramming and possibly other cell fate conversion processes.

Pluripotent human embryonic stem cells (hESCs) have the capability of differentiating into different lineages based on specific environmental cues. We had previously shown that hESCs can be primed to differentiate into either neurons or glial cells, depending on the arrangement, geometry and size of their substrate topography. In particular, anisotropically patterned substrates like gratings were found to favour the differentiation of hESCs into neurons rather than glial cells. In this study, our aim is to elucidate the underlying mechanisms of topography-induced differentiation of hESCs towards neuronal lineages. We show that high actomyosin contractility induced by a nano-grating topography is crucial for neuronal maturation. Treatment of cells with the myosin II inhibitor (blebbistatin) and myosin light chain kinase inhibitor (ML-7) greatly reduces the expression level of microtubule-associated protein 2 (MAP2). On the other hand, our qPCR array results showed that PAX5, BRN3A and NEUROD1 were highly expressed in hESCs grown on nano-grating substrates as compared to unpatterned substrates, suggesting the possible involvement of these genes in topography-mediated neuronal differentiation of hESCs. Interestingly, YAP was localized to the cytoplasm of differentiating hESCs. Taken together, our study has provided new insights in understanding the mechanotransduction of topographical cues during neuronal differentiation of hESCs.

Biophysical cues, such as nanotopography, have been shown to be integral for tissue regeneration and embryogenesis in the stem cell niche. Synthetic nanostructures can drive specific cell differentiation, but the sensing mechanisms for nanocues remain poorly understood. Here, we report that nanotopography-induced human mesenchymal stem cell (hMSC) differentiation through cell mechanotransduction is modulated by the integrin-activated focal adhesion kinase (FAK). On 250 nm nanogratings on polydimethylsiloxane, hMSCs developed aligned stress fibers and showed an upregulation of neurogenic differentiation markers. In addition, our mechanistic study confirmed that this regulation was dependent upon actomyosin contractility, suggesting a direct force-dependent mechanism. FAK phosphorylation was required for topography-induced hMSC differentiation while FAK overexpression overruled the topographical cues in determining cell lineage bias. The results indicated that FAK activity had a direct impact on topography-induced gene expression. These findings suggest that hMSC sense and transduce nanotopo-graphical signals through focal adhesions and actomyosin cytoskeleton contractility to induce differential gene expression.

Regulated biophysical cues, such as nanotopography, have been shown to be integral for tissue regeneration and embryogenesis in the stem cell niche. Tissue homeostasis involves the interaction of multipotent cells with nanoscaled topographical features in their ECM to regulate aspects of cell behavior. Synthetic nanostructures can drive specific cell differentiation, but the sensing mechanisms for nanocues remain poorly understood. Here, we report that nanotopography-induced human mesenchymal stem cell (hMSC) differentiation through cell mechanotransduction is modulated by the integrin-activated focal adhesion kinase (FAK). On nanogratings with 250 nm line width on polydimethylsiloxane, hMSCs developed aligned stress fibers and showed an upregulation of neurogenic and myogenic differentiation markers. The observed cellular focal adhesions within these cells were also significantly smaller and more elongated on the nanogratings compared to microgratings or unpatterned control. In addition, our mechanistic study confirmed that this regulation was dependent upon actomyosin contractility, suggesting a direct force-dependent mechanism. The topography-induced differentiation was observed on different ECM compositions but the response was not indicative of a direct ECM-induced hMSC differentiation pathway. FAK phosphorylation was required for topography-induced hMSC differentiation while FAK overexpression overruled the topographical cues in determining cell lineage bias. The results indicated that FAK activity had a direct impact on topography-induced gene expression, and that this effect of FAK was independent of cell shape. These findings suggest that hMSC sense and transduce nanotopographical signals through focal adhesions and actomyosin cytoskeleton contractility to induce differential gene expression.