Single-Cell Transcriptomic Identification of a Proliferative Intermediate State During Early Anagen Hair Follicle Regeneration.

Background: Secondary hair germ (SHG)-associated epithelial cells are a key progenitor population that contribute to hair follicle regeneration during early anagen. Although SHG-associated cells become activated during early anagen and contribute to the formation of regenerating follicular structures, the morphologically recognizable SHG compartment becomes less apparent as lower hair follicle structures develop during mid-anagen. However, the transcriptional changes accompanying the transition from SHG-associated states toward lower hair follicle-associated states remain poorly characterized. This study aimed to characterize the transcriptional changes associated with this transition and identify a proliferative intermediate transcriptional state during early anagen. By integrating single-cell transcriptomic analysis with regulatory and cell-cell communication analyses, we sought to improve the understanding of cellular state transitions and microenvironmental changes during early hair follicle regeneration. Methods: Hematoxylin and eosin staining (H&E) was performed to examine histological changes in mouse dorsal skin on days 0, 5, 10, 15, 20, 25, and 30 of the hair cycle. In parallel, previously published single-cell RNA sequencing data from mouse dorsal skin during early anagen were reanalyzed. Hair follicle epithelial cells were extracted and reclustered to identify cell subpopulations with distinct transcriptional characteristics. Cell types were annotated based on canonical marker genes and subpopulation-specific expression patterns. Differential expression analysis, functional enrichment analysis, cell-cycle analysis, developmental potential assessment, trajectory inference, transcription factor regulon analysis, and cell-cell communication analysis were further performed to characterize the relationships among hair germ cells, proliferative lower hair follicle-like cells (PR-loHF), and lower hair follicle cells. Results: Proliferative lower hair follicle-like cells (PR-loHF) were identified as a transcriptionally defined intermediate state associated with the transition from secondary hair germ (SHG)-associated cells toward lower hair follicle-associated cells. PR-loHF exhibited enrichment of biological programs associated with cell-cycle activity, epithelial remodeling, extracellular matrix organization, and developmental regulation. Moreover, compared with SHG-associated and lower hair follicle-associated states, PR-loHF exhibited distinct transcription factor regulon activity and cell-cell communication patterns, further supporting its characterization as a transcriptionally distinguishable intermediate state. Conclusions: PR-loHF represents a proliferative intermediate transcriptional state emerging during the transition from secondary hair germ (SHG)-associated cells toward lower hair follicle-associated states, providing new insights into transcriptional transitions and microenvironmental regulation during early hair follicle regeneration.