Hair follicle (HF) regeneration can be achieved in the center of large full-thickness wounds on mouse backs (wound-induced HF neogenesis model, WIHN). Investigations with this model have allowed for the identification of some of the factors limiting the extent of fibrosis, which creates a permissive environment for the reposition of HF. For WIHN, specific subpopulations of cells rather than cell types are permissive to this process. Detailed information on the cellular composition in WIHN is not available. Here, we provide a description of changes in cell numbers of fibroblasts, HF dermal papilla, endothelial cells, keratinocytes (interfollicular epidermis, HF-infundibulum, HF-isthmus, HF-bulge (basal and suprabasal), HF-hair germ) and immune cells (macrophages, monocytes, dendritic cells, T cells (CD4+, CD8+, CD4+/CD8+, regulatory T cells) and neutrophils) based on flow cytometric analysis. We compared unwounded skin with large wounds (1.5 × 1.5 cm) at different time points after wounding. We found that non-immune dermal cells have the largest share in the skin at all time points studied, and that the number of epidermal cells started increasing nine days after wounding, which precede isthmus cells and bulge cells, mirroring the development of hair follicles. Monocytes and neutrophils represent most myeloid cells in wounds and remain in wounds even beyond the inflammatory phase of wound healing. Macrophages can be identified as inflammatory and alternative cells and are also found in wounds even in the late remodeling phase of wound healing. Lastly, we provide information about T cells in large wounds. Most T cells in the wounds were CD8+ at all time points and expressed γδTCR, which was previously thought to be expressed mainly on CD4+. We also report the existence of double positive CD4/CD8. Our study provides a guide in terms of time points suitable for the further study of cell subpopulations aiming to dissect the cellular heterogeneity in WIHN. Our results might set the base for the comparison of WIHN between control mice and animals manipulated to influence HF neogenesis and the full understanding of the responsible actors allowing for HF regeneration.
Keywords: flow cytometry; regeneration; wound-induced hair follicle neogenesis.