Integrating scRNA-seq and mRNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts. Integrating scRNA-seq and mRNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts

The mechanisms governing nail stem cell (NSC) differentiation are coupled directly with their ability to orchestrate digit regeneration. Recently, onychofibroblasts (OFs), specialized mesenchymal cells residing beneath the nail matrix in the dermis, have emerged as potential regulators of nail differentiation. However, due to limited sample sources, the cellular properties and transcriptome information of OFs remain largely unexplored. In this study, we isolated human OFs and characterized their mesenchymal stem cell-like phenotypes. To delineate the molecular features of human OFs, we conducted mRNA-seq analysis on three samples of OFs and control fibroblasts from human nail units. Our analysis identified 294 genes that were upregulated in OFs compared to surrounding fibroblasts. Through integrated analysis with scRNA-seq data from human nail units, we confirmed that BMP4, secreted by OFs, is a pivotal signal involved in mesenchymal-epithelial interactions and nail development. Furthermore, we demonstrated that BMP4 derived from OFs mediates the in vitro differentiation of NSCs using a co-culture model. Gene set enrichment analysis highlighted the involvement of the TGF-beta pathway in the differentiation of nail epithelial cells. Consequently, we validated the effect of exogenous BMP4 on the activation of the SMAD-dependent pathway and NSC differentiation, along with the rescue effect of BMP receptor inhibition. Taken together, transcriptome analysis reveals the differentiation-inducing influence of OFs on human NSCs, with the BMP4/BMPR/SMADs signaling pathway playing a key role in this process. These findings establish a connection between the dermal microenvironment and NSC differentiation, suggesting that OFs, in conjunction with NSCs, may hold promise for the development of novel therapies targeting nail and digit defects, even severe limb amputation. Overall design: To elucidate the molecular features of onychofibroblasts and explore their role in coordinating nail development. We then performed mRNA-seq analysis on three human onychofibroblasts samples and three control human fibroblast samples derived from the proximal nail fold.