Wound-Induced Hair Neogenesis Model

Abstract

Skin wounds in adult mammals typically heal with a fibrotic scar and fail to restore ectodermal appendages, such as hair follicles or adipose tissue. Intriguingly, new hair follicles regenerate in the center of large full-thickness wounds of mice in a process called wound-induced hair neogenesis (WIHN). WIHN is followed by neogenesis of dermal adipose tissue. Both neogenic events reactivate embryonic-like cellular and molecular programs. The WIHN model provides a platform for studying mammalian regeneration, and findings from this model could instruct future regenerative medicine interventions for treating wounds and alopecia. Since Ito et al. rediscovered WIHN 15 years ago, numerous investigators have worked on the WIHN model using varying wounding protocols and model interpretations. Because a variety of factors, including environmental variables and choice of mouse strains, can affect the outcomes of a WIHN study, the purpose of this article is to provide an overview of the experimental variables that impact WIHN so that experiments between laboratories can be compared in a meaningful manner.

Figure 1.. Wound healing and regeneration.

(a) Timeline of WIHN. Representative images of healing wounds are shown on PWD3, 5, 7, and 10. SD occurs around PWD10–12, and this day is termed SD0. Neogenic hair placodes with mesenchymal condensates start to appear as early as PWD14 and only occur in the center of the wound scar. WIHN is followed by neogenesis of dermal adipose tissue. The newly regenerated HFs commonly but not always lack pigmentation. (b) Differentiating scarring from regeneration. Small wounds heal with hairless and fatless scar tissue, whereas large wounds heal with hair and adipocyte regeneration in the wound center. HF, hair follicle; PWD, postwounding day; SD, scab detachment; WIHN, wound-induced hair neogenesis.

Figure 2.. WIHN assay procedure.

(a) Workflow of WIHN investigation. Create a 1 cm² square full-thickness wound on the back of a mouse aged 3 weeks (2.25 cm² square wound for a mouse aged 7–8 weeks). The wound then heals at around PWD10–12. Collect the healed wound tissue on PWD17–24 for subsequent WIHN quantification. (b) WIHN quantification by noninvasive CSLM. Left: a bright-field image of the targeted area shows healed wound tissue in the center, which is surrounded by wound edges where neogenic HFs are not detectable. Dashed outline circles the wound edge. Middle: a scanned confocal image of the healed wound with clearly visible HFs in the center. Right: a magnified image from the middle and an example of HF counting. Each + indicates one HF. (c) Whole-mount HFN assay. Representative images of K17 and ALP staining of the healed wounds for WIHN quantification. Each dot represents an HF. ALP, alkaline phosphatase; CSLM, confocal scanning laser microscopy; HF, hair follicle; HFN, hair follicle neogenesis; K17, keratin 17; PWD, postwounding day; WIHN, wound-induced hair neogenesis.