Abstract

Fibroblasts can be directly reprogrammed via a combination of small molecules to generate induced neurons (iNs), bypassing intermediate stages. This method holds great promise for regenerative medicine; however, it remains inefficient. Recently, studies have suggested that physical cues may improve the direct reprogramming of fibroblasts into neurons, but the underlying mechanisms remain to be further explored, and the physical factors reported to date do not exhibit the full properties of the extracellular matrix (ECM). Previous in vitro studies mainly used rigid polystyrene dishes, while one of the characteristics of the native in-vivo environment of neurons is the soft nature of brain ECM. The reported stiffness of brain tissue is very soft ranging between 100 Pa and 3 kPa, and the effect of substrate stiffness on direct neuronal reprogramming has not been explored. Here, we show for the first time that soft substrates substantially improved the production efficiency and quality of iNs, without needing to co-culture with glial cells during reprogramming, producing more glutamatergic neurons with electrophysiological functions in a shorter time. Transcriptome sequencing indicated that soft substrates might promote glutamatergic neuron reprogramming through integrins, actin cytoskeleton, Hippo signalling pathway, and regulation of mesenchymal-to-epithelial transition, and competing endogenous RNA network analysis provided new targets for neuronal reprogramming. We demonstrated that soft substrates may promote neuronal reprogramming by inhibiting microRNA-615-3p-targeting integrin subunit beta 4. Our findings can aid the development of regenerative therapies and help improve our understanding of neuronal reprogramming. Statement of significance First, we have shown that low stiffness promotes direct reprogramming on the basis of small molecule combinations. To the