Wound-Induced Hair Neogenesis Model

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. 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. IntroductionA rich choreography of cell types and molecular signals accounts for the development and regeneration of mammalian skin. Studying these processes will inform future stem cell‒based therapies and other regenerative medicine interventions. The multilayered epidermis endows the skin with its barrier function and rests on the underlying dermis—rich in extracellular matrix and fibroblasts—that provides skin with its biomechanical properties. Ectodermal appendages, principally hair follicles (HFs) with sebaceous glands, have a particularly complex organization and are rich in epithelial stem cells and specialized mesenchymal niche cells.Summary Points•WIHN is a powerful in vivo assay for studying mechanisms of regeneration after adult skin wounding.•WIHN in transgenic mice permits lineage tracing of specific cell types and evaluation of specific molecular pathways on hair follicle regeneration.•WIHN serves as a preclinical model for evaluating compounds that enhance hair follicle regeneration.•Investigating WIHN-inducing signals in mice might lead to novel therapeutics for activating regenerative healing in human wounds.Historic dogma stated that HFs only develop during embryogenesis and that the number of HFs remains fixed after birth. Yet, nearly 70 years ago, researchers reported wound-induced folliculogenesis in rabbits (Billingham and Russell, 1956Billingham R.E. Russell P.S. Incomplete wound contracture and the phenomenon of hair neogenesis in rabbits' skin.Nature. 1956; 177: 791-792Crossref PubMed Scopus (49) Google Scholar; Breedis, 1954Breedis C. Regeneration of hair follicles and sebaceous glands from the epithelium of scars in the rabbit.Cancer Res. 1954; 14: 575-579PubMed Google Scholar), rodents (Lacassagne and Latarjet, 1946Lacassagne A. Latarjet R. Action of methylcholanthrene on certain scars of the skin in mice.Cancer Res. 1946; 6: 183-188PubMed Google Scholar), and possibly humans (Kligman and Strauss, 1956Kligman A.M. Strauss J.S. The formation of vellus hair follicles from human adult epidermis.J Invest Dermatol. 1956; 27: 19-23Abstract Full Text PDF PubMed Scopus (42) Google Scholar). Unfortunately, the study of this phenomenon was not pursued, and definitive evidence of wound-induced folliculogenesis are lacked for years (Montagna and Dobson, 1967Montagna W. Dobson R. Advances in biology of skin. 9. Pergamon Press, New York1967: 384-391Google Scholar). However, in 2007, Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar rediscovered and confirmed folliculogenesis or wound-induced hair neogenesis (WIHN) in adult mice when they observed completely new HFs developing after large full-thickness skin wounds. Although WIHN rarely is observed in humans in clinical settings, investigating its underlying mechanisms in animal models could potentially be translated into future human therapies. In this paper, we review the current research on WIHN and summarize the techniques for assay standardization and interpretation.Differentiating scarring from regenerationUsually, small full-thickness wounds in adult mice (circular wounds ranging between 3 and 8 mm in diameter) heal with a hairless and adipose-free scar. In distinct contrast, larger full-thickness excisional wounds remarkably heal with new HFs and associated sebaceous glands forming in the center of the wound surrounded by a circular band of hairless scar (Harn et al., 2021Harn H.I. Wang S.P. Lai Y.C. Van Handel B. Liang Y.C. Tsai S. et al.Symmetry breaking of tissue mechanics in wound induced hair follicle regeneration of laboratory and spiny mice.Nat Commun. 2021; 12: 2595Crossref PubMed Scopus (17) Google Scholar; Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar; Lim et al., 2018Lim C.H. Sun Q. Ratti K. Lee S.H. Zheng Y. Takeo M. et al.Hedgehog stimulates hair follicle neogenesis by creating inductive dermis during murine skin wound healing.Nat Commun. 2018; 9: 4903Crossref PubMed Scopus (109) Google Scholar; Nelson et al., 2015Nelson A.M. Reddy S.K. Ratliff T.S. Hossain M.Z. Katseff A.S. Zhu A.S. et al.dsRNA released by tissue damage activates TLR3 to drive skin regeneration.Cell Stem Cell. 2015; 17: 139-151Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar; Wang et al., 2021Wang G. Sweren E. Liu H. Wier E. Alphonse M.P. Chen R. et al.Bacteria induce skin regeneration via IL-1beta signaling.Cell Host Microbe. 2021; 29: 777-791.e6Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) (Figure 1a and b). Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar found new HFs consistently regenerating in the center of 1 cm by 1 cm square excisional wounds in mice. By postwounding day (PWD) 10, such wounds heal to a significant degree by means of contraction, with the remainder of the area (approximately 0.25 cm2) forming a dermal scar covered with new epidermis. The day of scab detachment (SD), called SD0, normally occurs around PWD10‒12 in wild-type mice (Figure 1a). Occasionally, the wound does not fully close, leaving a small scab at the center of the wound for a week or more after other mice have healed. These mice with a highly abnormal SD should be removed from consideration. By PWD14, new epithelial hair placodes form in the scar center (Figure 1a), and they can be detected on the basis of positive staining for keratin 17 (K17) or WNT pathway marker LEF1 (Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar), among other markers. From PWD14 onward, hair placodes undergo progressive maturation into HFs, traversing through a hair germ and peg stage. New hair placodes continue to form for several days. Typically, this process of HF neogenesis terminates by PWD30, and no new hair placodes are evident beyond that. In addition, similar to normal HFs, neogenic HFs possess a sebaceous gland and grow numerous hair fibers during repetitive HF cycling that follows typical phases of anagen, catagen, and telogen (Figure 1a). Importantly, once neogenic HFs reach mature anagen, they secrete BMP that stimulates the conversion of surrounding myofibroblasts into new lipid-laden adipocytes (Plikus et al., 2017Plikus M.V. Guerrero-Juarez C.F. Ito M. Li Y.R. Dedhia P.H. Zheng Y. et al.Regeneration of fat cells from myofibroblasts during wound healing.Science. 2017; 355: 748-752Crossref PubMed Scopus (284) Google Scholar) (Figure 1a).Determining whether HF neogenesis has occurred requires careful examination and ideally whole-mount preparations of the healed wound. Because excisional wounds in mice undergo significant contraction, their hair-bearing edges often become distorted, such that on histology, it can appear as if pre-existing HFs are surrounded by areas of scar (Figure 1b). Several checks should be done to confirm HF neogenesis. First, healed wounds should be evaluated on whole mount, and they should show (i) a clear hair-bearing edge, (ii) a hairless band of scar, and (iii) HF germs in the center (Figure 1b). Second, histologically, it is critical to show immature, developing HFs that have embryonic-like morphology and still lack sebaceous glands. Pre-existing HFs that are mistaken for neogenic HFs have mature morphology with fully formed sebaceous glands and often contain one or several club hairs from earlier hair cycles. True neogenic HFs that are in their first morphogenetic anagen phase lack club hairs.Another critically important feature of neogenic hairs is their lack of pigment (Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar; Plikus et al., 2017Plikus M.V. Guerrero-Juarez C.F. Ito M. Li Y.R. Dedhia P.H. Zheng Y. et al.Regeneration of fat cells from myofibroblasts during wound healing.Science. 2017; 355: 748-752Crossref PubMed Scopus (284) Google Scholar) (Figure 1a). In rare instances, isolated pigmented hairs can form; however, the presence of pigmented hairs throughout the wound likely indicates pre-existing HFs that moved into the wound owing to contraction. Recent publications showing pigmented hairs after wounding likely do not represent true WIHN (Mascharak et al., 2021Mascharak S. desJardins-Park H.E. Davitt M.F. Griffin M. Borrelli M.R. Moore A.L. et al.Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring.Science. 2021; : 372PubMed Google Scholar). Although normally, melanocytes do not migrate into the center of the wound and do not repopulate the new HFs, pigmentation of neogenic HFs can occur by experimental manipulation of melanocyte stem cells by modulating their EDNRB signaling (Takeo et al., 2016Takeo M. Lee W. Rabbani P. Sun Q. Hu H. Lim C.H. et al.EdnrB governs regenerative response of melanocyte stem cells by crosstalk with Wnt signaling.Cell Reports. 2016; 15: 1291-1302Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).Experimental variables that affect WIHNMouse strain effectsMouse genetic strain impacts WIHN capacity. For example, C57BL/6J mice have greater WIHN than those in C57BL/6NJ background. For WIHN experiments to be reliable, all mice ideally should be of the same genetic background. If feasible, experimental mice should be from the same litter or descendants from the same breeders. In addition, a random-number table is recommended to divide experimental mice into groups (Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar; Wang et al., 2017Wang X. Chen H. Tian R. Zhang Y. Drutskaya M.S. Wang C. et al.Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF.Nat Commun. 2017; 814091Google Scholar).Proper controls must be considered when studying WIHN in transgenic mice. In conditional gene loss-of-function models, a littermate Creneg;Floxed allele+/+ mouse is recommended as the control for the Cre+;Floxed allele+/+ experimental mouse, and in the case of inducible Cre models, the same inducing agent (such as tamoxifen) treatment should be performed in both control and experimental mice. A more stringent control to be considered is Cre+;Flox+/neg mouse, albeit the drawback of this choice is that certain genes can show dose-dependent phenotypes. In gain-of-function models, for example, in a tet-ON controlled gene activation system, littermate rtTAneg;tetO-X mice (where X is any transgene) are an important control for rtTA+;tetO-X experimental mice (Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar). The subsequent inducing agent (such as doxycycline) exposure should be identical in the two groups of mice. Because WIHN has significant variability (in terms of the number of neogenic HFs), we recommend starting with a minimum of 10 mice in each of the mouse groups.In addition, even though male and female mice do not appear to show significant differences in WIHN, a similar sex composition is recommended between experimental groups.Age of mouseThe HF regeneration capacity of mice declines, although it does not disappear with age (Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar). Generally, older mice need a larger wound size to regenerate new HFs than do younger ones. Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar compared HF neogenesis between older mice (e.g., aged 7 weeks) with a 1.5 × 1.5 cm square wound and younger mice (aged 3 weeks) with a 1 × 1 cm square wound. The numbers of regenerated HFs were greater in mice aged 3 weeks (40 ± 26, range = 10‒102) than in mice aged 7–8 weeks (30 ± 28, range = 0‒91) or mice aged 10 months (36 ± 26, range = 0‒70) (Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar). Therefore, when WIHN is compared between two groups of mice, they should be of the same age.The hair cycle should also be controlled between experimental and control mice. Wounds should preferably be done in telogen skin, for example, in mice aged 3 weeks (first postnatal telogen) or 7 weeks (second postnatal telogen). Mouse weight should also be taken into consideration. For mice aged 3 weeks, the normal weight is around 10 g; a variance of below or over 0.5 g is acceptable. Outliers should be removed.Environmental factorsCommensal microbiomes modulate the host’s biological activity. WIHN capacity correlates with bacterial quantity and diversity in otherwise genetically matched mice (Wang et al., 2021Wang G. Sweren E. Liu H. Wier E. Alphonse M.P. Chen R. et al.Bacteria induce skin regeneration via IL-1beta signaling.Cell Host Microbe. 2021; 29: 777-791.e6Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Specifically, mice housed in specific pathogen-free facilities (SPF) have greater WIHN efficacy than germ-free mice (Wang et al., 2021Wang G. Sweren E. Liu H. Wier E. Alphonse M.P. Chen R. et al.Bacteria induce skin regeneration via IL-1beta signaling.Cell Host Microbe. 2021; 29: 777-791.e6Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In addition, increased cage-changing frequency reduces WIHN, which suggests that environmental control in vivarium settings is essential for the WIHN assay (Wang et al., 2021Wang G. Sweren E. Liu H. Wier E. Alphonse M.P. Chen R. et al.Bacteria induce skin regeneration via IL-1beta signaling.Cell Host Microbe. 2021; 29: 777-791.e6Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). These results may explain why genetically identical mouse strains regenerate significantly varying HF numbers among independent laboratories (Harn et al., 2021Harn H.I. Wang S.P. Lai Y.C. Van Handel B. Liang Y.C. Tsai S. et al.Symmetry breaking of tissue mechanics in wound induced hair follicle regeneration of laboratory and spiny mice.Nat Commun. 2021; 12: 2595Crossref PubMed Scopus (17) Google Scholar; Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar; Lim et al., 2018Lim C.H. Sun Q. Ratti K. Lee S.H. Zheng Y. Takeo M. et al.Hedgehog stimulates hair follicle neogenesis by creating inductive dermis during murine skin wound healing.Nat Commun. 2018; 9: 4903Crossref PubMed Scopus (109) Google Scholar; Nelson et al., 2015Nelson A.M. Reddy S.K. Ratliff T.S. Hossain M.Z. Katseff A.S. Zhu A.S. et al.dsRNA released by tissue damage activates TLR3 to drive skin regeneration.Cell Stem Cell. 2015; 17: 139-151Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar; Wang et al., 2017Wang X. Chen H. Tian R. Zhang Y. Drutskaya M.S. Wang C. et al.Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF.Nat Commun. 2017; 814091Google Scholar).Because microbes affect WIHN, standard sterilized tools should be used for wounding to minimize experimental bias and generate reproducible results. Moreover, it is advantageous to control cage-changing frequencies during experiments. Hence, housing the mice in the same animal facility during the investigation is essential, and the use of littermates as controls is ideal.Wounding procedureThe wounding assay should be performed under a ventilated biosafety hood on fully anesthetized mice. WIHN assay is typically performed in the lumbar region of dorsal skin (Figure 2a). Mice should be shaved using clippers in this area several days before the wounding procedure to allow for grooming and to prevent freshly clipped hair fragments from getting into the wound bed. The precise area to be excised should be outlined using a nontoxic skin pen, and then sterile scissors should be used to create a full-thickness wound of desired dimensions and geometry (Figure 2a). After wounding, mice should receive analgesia, such as buprenorphine, and health checks should be done frequently (daily for the first week and then every other day until the end of the experiment). Mice that show significant deviations in early healing events (formation of scab, time to SD) (Figure 1a) should be removed from the experiment.Figure 2WIHN 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because damage levels dictate double-stranded RNA (dsRNA) release and therefore regenerative capacity (Nelson et al., 2015Nelson A.M. Reddy S.K. Ratliff T.S. Hossain M.Z. Katseff A.S. Zhu A.S. et al.dsRNA released by tissue damage activates TLR3 to drive skin regeneration.Cell Stem Cell. 2015; 17: 139-151Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), surgical technique is an important factor in WIHN. Consistent surgical approaches in wound creation are necessary to minimize variation. For example, halting cuts with dull surgical instruments may create more damage and induce greater WIHN than smooth cuts with sharp surgical instruments. Care should be taken to create clean and exact skin excisions. Nelson et al., 2015Nelson A.M. Reddy S.K. Ratliff T.S. Hossain M.Z. Katseff A.S. Zhu A.S. et al.dsRNA released by tissue damage activates TLR3 to drive skin regeneration.Cell Stem Cell. 2015; 17: 139-151Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar showed that even small unintentional perpendicular cuts (fringe cuts) to the wound edge significantly increased the number of regenerated HFs, suggesting that WIHN depends on geometric wound characteristics.After wounding, it is allowable to house 3‒5 mice of the same sex from the same litter together for better survival.Wound size and geometryFor efficient WIHN, we recommend performing a 1 cm2 square wound in mice aged 3 weeks and then increasing wound size to 2.25 cm2 square in mice that are aged ≥7 weeks. Wounds should be open to the air and allowed to heal without topical ointments (such as antibacterial cream) or surgical dressings. Typically, the scab falls off on complete wound re-epithelialization, and this event immediately precedes the initiation of the first neogenic hair placode. In certain instances, such as in transgenic mice with overactivated Hedgehog signaling (Lim et al., 2018Lim C.H. Sun Q. Ratti K. Lee S.H. Zheng Y. Takeo M. et al.Hedgehog stimulates hair follicle neogenesis by creating inductive dermis during murine skin wound healing.Nat Commun. 2018; 9: 4903Crossref PubMed Scopus (109) Google Scholar), HF neogenesis can occur in wounds that are smaller than 1 cm2 in size; however, as a general rule, 1 cm2 size is required as a minimum threshold for WIHN activation. If the mice show limited WIHN, it is recommended to increase the wound size, change the mouse breeders, or use mice with a different genetic background.Notably, the final size of the healed wound rather than the initial wounding size strongly correlates with HF neogenesis. For example, 1 cm2 wounds in younger mice and 2.25 cm2 wounds in older mice both repair with WIHN, and both yield approximately 0.25 cm2 nascent scar immediately after re-epithelialization owing to wound contraction increasing with age (Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar). Hence, a critical WIHN assay should measure the wound bed/scar size as a parameter in conjunction with the numbers of neogenic hair follicles.Methods for WIHN quantificationNeogenic HFs can be visualized and quantified by noninvasive imaging of healed wounds with confocal scanning laser microscopy (CSLM) (Figure 2a and b) (Fan et al., 2011Fan C. Luedtke M.A. Prouty S.M. Burrows M. Kollias N. Cotsarelis G. Characterization and quantification of wound-induced hair follicle neogenesis using in vivo confocal scanning laser microscopy.Skin Res Technol. 2011; 17: 387-397Crossref PubMed Scopus (15) Google Scholar; Kim et al., 2019Kim D. Chen R. Sheu M. Kim N. Kim S. Islam N. et al.Noncoding dsRNA induces retinoic acid synthesis to stimulate hair follicle regeneration via TLR3.Nat Commun. 2019; 10: 2811Crossref PubMed Scopus (37) Google Scholar; Nelson et al., 2015Nelson A.M. Reddy S.K. Ratliff T.S. Hossain M.Z. Katseff A.S. Zhu A.S. et al.dsRNA released by tissue damage activates TLR3 to drive skin regeneration.Cell Stem Cell. 2015; 17: 139-151Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar; Wang et al., 2021Wang G. Sweren E. Liu H. Wier E. Alphonse M.P. Chen R. et al.Bacteria induce skin regeneration via IL-1beta signaling.Cell Host Microbe. 2021; 29: 777-791.e6Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The accuracy of CSLM (in terms of its ability to detect all regenerated HFs) is comparable with that of whole-mount staining methods (such as with K17 and alkaline phosphatase [ALP]). As a noninvasive technique, CSLM can be used to track the dynamics of HF regeneration, such that the same wound can be reimaged at predefined intervals (such as every 2 days), and new HFs can be quantified with their distribution pattern recorded and analyzed. CSLM can also be used to quantify neogenic HFs in healed wounds at the experimental endpoint. This approach is especially useful if tissue samples are required to be preserved intact for additional experiments, such as histology or cell isolation for single-cell RNA-sequencing experiments. When WIHN is evaluated with CSLM (either on live mice or on dissected tissues samples), it is essential that periwound skin is carefully shaved and stretched flat so that imaging chamber of the CSLM device (such as Vivascope) can be placed flat because folded skin or areas of new hair growth can result in optical distortions and can make HF quantification unreliable (Figure 2b).Alternatively, WIHN can be confirmed and quantified with histology and hair placode and/or HF marker staining. Histologically, epithelial placodes that express K17 appear as early as PWD14. Placodes overlie dermal condensates that eventually mature into ALP+ dermal papillae. Staining for K17 and ALP can also be performed on whole-mount preparations of healed wounds, and in this modification, these markers can be used to reliably quantify new HFs (Gay et al., 2013Gay D. Kwon O. Zhang Z. Spata M. Plikus M.V. Holler P.D. et al.FGF9 from dermal γδ T cells induces hair follicle neogenesis after wounding.Nat Med. 2013; 19: 916-923Crossref PubMed Scopus (193) Google Scholar; Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar; Wang et al., 2017Wang X. Chen H. Tian R. Zhang Y. Drutskaya M.S. Wang C. et al.Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF.Nat Commun. 2017; 814091Google Scholar). This method can be used as early as PWD17 and requires separation of wound epidermis for K17 staining and wound dermis for ALP staining (Harn et al., 2021Harn H.I. Wang S.P. Lai Y.C. Van Handel B. Liang Y.C. Tsai S. et al.Symmetry breaking of tissue mechanics in wound induced hair follicle regeneration of laboratory and spiny mice.Nat Commun. 2021; 12: 2595Crossref PubMed Scopus (17) Google Scholar; Ito et al., 2007Ito M. Yang Z. Andl T. Cui C. Kim N. Millar S.E. et al.Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.Nature. 2007; 447: 316-320Crossref PubMed Scopus (733) Google Scholar; Wang et al., 2017Wang X. Chen H. Tian R. Zhang Y. Drutskaya M.S. Wang C. et al.Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF.Nat Commun. 2017; 814091Google Scholar).Moreover, if downstream transcriptomic analysis is required, we recommend combining at least three healed wound samples (one per mouse) for one run of bulk RNA sequencing and at least 15 wound samples for one single-cell RNA-sequencing run, aiming to isolate at least 100,000 live cells. Pooling of more wound tissues might be needed depending on the cell isolation or RNA isolation efficiency. Notably, it is essential to carefully dissect the wound scar to remove normal skin edge with normal HFs because the inclusion of this can obscure the results owing that normal HF cells/genes might be confused for neogenic HF cells/genes.ConclusionWe have described the key experimental variables that might affect WIHN in Table 1. Standardizing wounding procedures and postwounding mouse care will minimize inherent variability in WIHN results. In addition, carefully choosing mice on the basis of their genetics and age and controlling the housing environment should further increase the reliability of the WIHN assay. Following these guidelines will help researchers entering this field to standardize the WIHN technique.Table 1Essential Factors that Affect WIHNFactorsNotesMouse genetic backgroundUse littermate controls for experimentsMouse ageOlder mice exhibit less WIHNHair cycleTelogen (mice aged 3 we