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The nail unit and hair follicle are both hard keratin-producing organs that share various biological features. In this study, we used digital spatial profiling and single-cell RNA sequencing to define a spatially resolved expression profile of the human nail unit and hair follicle. Our approach showed the presence of a nail-specific mesenchymal population called onychofibroblasts within the onychodermis. Onychodermis and follicular dermal papilla both expressed Wnt and bone morphogenetic protein signaling molecules. In addition, nail matrix epithelium and hair matrix showed very similar expressions profile, including the expression of hard keratins and HOXC13, a transcriptional regulator of the hair shaft. Integration of single-cell RNA sequencing and digital spatial profiling data through computational deconvolution methods estimated epithelial and mesenchymal cell abundance in the nail- and hair-specific regions of interest and revealed close transcriptional similarity between these major skin appendages. To analyze the function of bone morphogenetic proteins in nail differentiation, we treated cultured human nail matrix keratinocytes with BMP5, which are highly expressed by onychofibroblasts. We observed increased expressions of hard keratin and its regulator genes such as HOXC13. Collectively, our data suggest that onychodermis is the counterpart of dermal papilla and that BMP5 in onychofibroblasts plays a key role in the differentiation of nail matrix keratinocytes.

Studies on the etiopathogenesis of keloids mostly have focused on fibroblasts and their dysfunction. In this study, two cutting-edge technologies, single-cell RNA sequencing and spatial transcriptomics, were applied to uncover the underlying pathophysiology of keloids. Keloid tissue samples and normal skin control data were analyzed as well as those of patient-matched keloid and normal mature scar. Single-cell RNA sequencing revealed cellular heterogenicity such as fibroblasts, endothelial cells (ECs), and myofibroblasts within the keloids. Spatial transcriptomics results showed that disease-associated fibroblasts were enriched in the deeper keloid areas, mostly located around the spots with endothelial transcripts. Mesenchymal activation was observed in keloid ECs, characterized by dysregulation of TGF-β/SMAD signaling. Colocalization of mesenchymal and vascular markers through multiplex immunofluorescence suggested mesenchymal activation of keloid ECs. Cell‒cell interaction analysis identified a significant network between keloid fibroblasts and ECs, and this cellular crosstalk was supported by colocalization analysis of spatial transcriptomics. This study depicted the cellular landscape of keloids at a single-cell resolution as well as the integration of single-cell and valuable spatial data of keloids using spatial transcriptomics and multiplex immunofluorescence technologies. Our findings suggested a potential role of fibrovascular communication and mesenchymal activation of ECs that might be involved in the keloid pathogenesis.