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Wound-Induced Hair Neogenesis Model

  • Yingchao Xue
    Affiliations
    Department of Dermatology, Johns Hopkins University, Baltimore, Maryland, USA
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  • Chae Ho Lim
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA

    Department of Cell Biology, New York University School of Medicine, New York, New York, USA
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  • Maksim V. Plikus
    Affiliations
    Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, Irvine, California, USA

    Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, Irvine, California, USA

    Center for Complex Biological Systems, University of California, Irvine, Irvine, California, USA

    NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine, Irvine, California, USA
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  • Mayumi Ito
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA

    Department of Cell Biology, New York University School of Medicine, New York, New York, USA
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  • George Cotsarelis
    Affiliations
    Kligman Laboratories, Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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  • Luis A. Garza
    Correspondence
    Correspondence. Luis A. Garza, Department of Dermatology, Johns Hopkins University, Suite 204 Koch CRBII, Baltimore, Maryland 21231, USA.
    Affiliations
    Department of Dermatology, Johns Hopkins University, Baltimore, Maryland, USA

    Department of Cell Biology, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA

    Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA
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      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.

      Abbreviations:

      ALP (alkaline phosphatase), CSLM (confocal scanning laser microscopy), HF (hair follicle), K17 (keratin 17), PWD (postwounding day), SD (scab detachment), WIHN (wound-induced hair neogenesis)

      Introduction

      A 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 R.E.
      • Russell P.S.
      Incomplete wound contracture and the phenomenon of hair neogenesis in rabbits' skin.
      ;
      • Breedis C.
      Regeneration of hair follicles and sebaceous glands from the epithelium of scars in the rabbit.
      ), rodents (
      • Lacassagne A.
      • Latarjet R.
      Action of methylcholanthrene on certain scars of the skin in mice.
      ), and possibly humans (
      • Kligman A.M.
      • Strauss J.S.
      The formation of vellus hair follicles from human adult epidermis.
      ). Unfortunately, the study of this phenomenon was not pursued, and definitive evidence of wound-induced folliculogenesis are lacked for years (
      • Montagna W.
      • Dobson R.
      ). However, in 2007,
      • Ito 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.
      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 regeneration

      Usually, 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 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.
      ;
      • Ito 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.
      ;
      • Lim 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.
      ;
      • Nelson 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.
      ;
      • Wang G.
      • Sweren E.
      • Liu H.
      • Wier E.
      • Alphonse M.P.
      • Chen R.
      • et al.
      Bacteria induce skin regeneration via IL-1beta signaling.
      ) (Figure 1a and b).
      • Ito 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.
      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 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.
      ), 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 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.
      ) (Figure 1a).
      Figure thumbnail gr1
      Figure 1Wound 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.
      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 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.
      ;
      • Plikus 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.
      ) (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 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.
      ). 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 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.
      ).

      Experimental variables that affect WIHN

      Mouse strain effects

      Mouse 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 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.
      ;
      • Wang 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.
      ).
      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 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.
      ). 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 mouse

      The HF regeneration capacity of mice declines, although it does not disappear with age (
      • Ito 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.
      ). Generally, older mice need a larger wound size to regenerate new HFs than do younger ones.
      • Ito 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.
      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 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.
      ). 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 factors

      Commensal microbiomes modulate the host’s biological activity. WIHN capacity correlates with bacterial quantity and diversity in otherwise genetically matched mice (
      • Wang G.
      • Sweren E.
      • Liu H.
      • Wier E.
      • Alphonse M.P.
      • Chen R.
      • et al.
      Bacteria induce skin regeneration via IL-1beta signaling.
      ). Specifically, mice housed in specific pathogen-free facilities (SPF) have greater WIHN efficacy than germ-free mice (
      • Wang G.
      • Sweren E.
      • Liu H.
      • Wier E.
      • Alphonse M.P.
      • Chen R.
      • et al.
      Bacteria induce skin regeneration via IL-1beta signaling.
      ). In addition, increased cage-changing frequency reduces WIHN, which suggests that environmental control in vivarium settings is essential for the WIHN assay (
      • Wang G.
      • Sweren E.
      • Liu H.
      • Wier E.
      • Alphonse M.P.
      • Chen R.
      • et al.
      Bacteria induce skin regeneration via IL-1beta signaling.
      ). These results may explain why genetically identical mouse strains regenerate significantly varying HF numbers among independent laboratories (
      • Harn 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.
      ;
      • Ito 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.
      ;
      • Lim 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.
      ;
      • Nelson 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.
      ;
      • Wang 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.
      ).
      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 procedure

      The 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 thumbnail gr2
      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.
      Because damage levels dictate double-stranded RNA (dsRNA) release and therefore regenerative capacity (
      • Nelson 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.
      ), 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 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.
      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 geometry

      For 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 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.
      ), 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 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.
      ). 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 quantification

      Neogenic HFs can be visualized and quantified by noninvasive imaging of healed wounds with confocal scanning laser microscopy (CSLM) (Figure 2a and b) (
      • Fan 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.
      ;
      • Kim 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.
      ;
      • Nelson 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.
      ;
      • Wang G.
      • Sweren E.
      • Liu H.
      • Wier E.
      • Alphonse M.P.
      • Chen R.
      • et al.
      Bacteria induce skin regeneration via IL-1beta signaling.
      ). 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 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.
      ;
      • Ito 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.
      ;
      • Wang 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.
      ). 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 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.
      ;
      • Ito 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.
      ;
      • Wang 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.
      ).
      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.

      Conclusion

      We 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 WIHN
      FactorsNotes
      Mouse genetic backgroundUse littermate controls for experiments
      Mouse ageOlder mice exhibit less WIHN
      Hair cycleTelogen (mice aged 3 weeks or 7‒8 weeks)
      Wounding siteLower lumbar dorsal skin
      Wound sizeWIHN requires minimal wound size (1 × 1 cm square wound at age 3 weeks and 1.5 × 1.5 cm square wound at age 7‒8 weeks), and regenerated HF number increases with wound size in mice.
      Wound cuttingEnsure that all surgical tools are sterilized before the wounding and that cuts are neat
      MicrobiomeHouse experimental mice in the same environment and control the cage-changing frequencies between experimental groups
      Abbreviations: HF, hair follicle; WIHN, wound-induced hair neogenesis.

      Conflict of Interest

      GC is on the scientific advisory board of Follica, a company that has licensed intellectual property on wound-induced hair neogenesis originating in his laboratory, at University of Pennsylvania. GC receives royalties as dictated by Penn's patent policy and also receives compensation for sitting on the Scientific Advisory Board (SAB). The remaining authors state no conflict of interest.

      Acknowledgments

      The authors thank Claire Levine for feedback on the manuscript. We thank all of our dedicated colleagues for our discoveries in this field and contributions to promoting the development of skin regeneration studies. Their efforts energized us to write this review. Because of space limitations, we were not able to discuss all of the exciting and relevant articles that have been published. This work was supported by the National Institutes of Health grant R01 AR074846 to LAG; National Institutes of Health grants U01 AR073159 and P30 AR075047 and LEO Foundation grant LF-OC-20-000611 to MVP, and National Institutes of Health grant P30 AR069589 and LEO Foundation grant to GC.

      Author Contributions

      Conceptualization: YX, MVP, LAG; Investigation: YX, CHL; Supervision: MVP, MI, GC, LAG; Visualization: YX, CHL; Writing – Original Draft Preparation: YX; Writing – Review and Editing: CHL, MVP, MI, GC, LAG

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