Single‐Cell Analysis of Late‐Stage Wound‐Induced Hair Follicle Neogenesis Reveals Keratinocyte Heterogeneity and Enrichment of Infundibular Basal Keratinocytes

ABSTRACT

Scarring alopecia remains a major clinical challenge, characterized by irreversible hair follicle (HF) loss and replacement by scar tissue. Wound‐induced hair follicle neogenesis (WIHN) serves as a model in which HFs can regenerate following skin wounding. In this study, we characterized the cellular landscape associated with differences in regenerative capacity between small wound (SW) and large wound (LW) tissues at a late regenerative stage (post‐wounding day 22, PWD22), a time point at which newly formed hair follicles are already present. Using single‐cell RNA sequencing (scRNA‐seq), we profiled re‐epithelialized wound tissues from SW and LW conditions and identified major cutaneous cell populations, including keratinocytes and fibroblasts. Keratinocytes were further categorized into 8 subpopulations, among which infundibular basal keratinocytes (INFU basal KCs) were increased in LW compared with SW tissues, as validated by immunofluorescence (IF) staining. Pseudotime analysis indicated differences in the temporal expression pattern of the INFU basal KC marker Fst between SW and LW conditions. Cell–cell communication between keratinocytes and fibroblasts showed that extracellular matrix (ECM)–receptor interactions were markedly enriched, while Masson's trichrome staining revealed decreased collagen content in LW. IF staining showed increased COL6A3 expression and decreased FN1 expression in LW tissues. Taken together, our study provides a descriptive single‐cell atlas of late‐stage wound tissues with differing regenerative outcomes, indicating differences in keratinocyte composition, transcriptional states, and intercellular communication. Collectively, our findings offer a resource for understanding cellular heterogeneity associated with WIHN and may inform future studies aimed at elucidating the mechanisms of hair follicle regeneration in cutaneous repair.