Wound-induced Hair Neogenesis Research Articles

Approaches to Study Wound-Induced Hair Neogenesis (WIHN).

Embryonic wound repair proceeds with complete regeneration of the tissue without any scar formation, whereas tissue repair in adults usually results in scars and the tissue does not completely regain its preinjured state. Wound-induced hair neogenesis (WIHN) in adult rodents results in de novo hair follicle formation in the center of large wounds, mimicking regeneration processes seen in fetal tissue. The investigation of WIHN therefore provides a unique quantitative framework for scrutinizing the mechanistic underpinnings of regenerative repair, which can have clinical implications in the context of scarless healing. In this chapter, we present a detailed protocol for inducing wounds that lead to hair neogenesis in laboratory mice and facilitating the identification and characterization of distinct stages in neogenic hair follicle development. Additionally, we present a whole-mount alkaline phosphatase assay to distinguish de novo hair follicles. These protocols can facilitate studies toward obtaining a comprehensive understanding of WIHN and shedding light on the intricate molecular and cellular processes involved in mammalian regenerative repair.

Epidermal–dermal coupled spheroids are important for tissue pattern regeneration in reconstituted skin explant cultures

Tissue patterning is critical for the development and regeneration of organs. To advance the use of engineered reconstituted skin organs, we study cardinal features important for tissue patterning and hair regeneration. We find they spontaneously form spheroid configurations, with polarized epidermal cells coupled with dermal cells through a newly formed basement membrane. Functionally, the spheroid becomes competent morphogenetic units (CMU) that promote regeneration of tissue patterns. The emergence of new cell types and molecular interactions during CMU formation was analyzed using scRNA-sequencing. Surprisingly, in newborn skin explants, IFNr signaling can induce apical-basal polarity in epidermal cell aggregates. Dermal-Tgfb induces basement membrane formation. Meanwhile, VEGF signaling mediates dermal cell attachment to the epidermal cyst shell, thus forming a CMU. Adult mouse and human fetal scalp cells fail to form a CMU but can be restored by adding IFNr or VEGF to achieve hair regeneration. We find different multi-cellular configurations and molecular pathways are used to achieve morphogenetic competence in developing skin, wound-induced hair neogenesis, and reconstituted explant cultures. Thus, multiple paths can be used to achieve tissue patterning. These insights encourage more studies of “in vitro morphogenesis” which may provide novel strategies to enhance regeneration.

What Do We Know About Hair Growth Induced by Wounding and Its Therapeutic Applications?

Many studies have reported the role of hair follicles (HFs) in the wound healing response, and vice versa, the creation of superficial injuries may stimulate hair growth, which has encouraged new treatments for hair loss. To review the phenomenon of wound-induced hair growth and the usefulness of therapeutic procedures based on skin wounding in androgenetic alopecia (AGA). A literature search was conducted to review cases of localized hypertrichosis induced by wounds and the role of microneedling, fractional laser, and scalp threading as monotherapy for AGA. Localized hypertrichosis has been extensively reported after bone fractures, burn injury, chronic venous ulcer, etc. Only 2 cases of wound-induced hair neogenesis in humans have been reported. As monotherapy for AGA, 1 of 3 studies of microneedling, 4 of 6 of fractional lasers, and 2 of 3 studies of scalp threading show good efficacy. Certain types of wounds seem to stimulate localized hair growth in humans, but the underlying mechanism is unclear. Reports on wound-induced HF neogenesis in humans are anecdotal and questions remain as to whether this is a true phenomenon in humans. Further clinical studies are needed before recommending wound-induced hair growth procedures as therapies for AGA.

83 Wnt signaling attenuates mechanotransduction and protects against wound occlusion-mediated abolishment of regeneration

OBJECTIVES/GOALS: Current clinical practice recommends occlusive dressings (e.g., film and hydrocolloid) for wounds with variable regenerative capacities. However, clinical evidence suggests that occlusion may hinder regeneration. Our objective was to test the impact of occlusion on regeneration using animal models. METHODS/STUDY POPULATION: The murine wound-induced hair neogenesis (WIHN) is a well-established model of regeneration characterized by de novo hair follicle (HF) formation in the center of large full-thickness wounds. The quantity of neogenic HFs depends on the robustness of Wnt signaling. Optimal tissue mechanics is also required for WIHN. Utilizing the murine WIHN model, we tested the hypothesis that wound occlusion impedes regeneration. We determined how (i) the timing and duration of wound occlusion impacts WIHN, (ii) Wnt signaling influences the occlusion-induced effects on regeneration and (iii) occlusion alters the tissue mechanics of the wound, which establishes the morphogenetic field needed for WIHN. RESULTS/ANTICIPATED RESULTS: Occlusion completely eliminated WIHN. Only a brief period of occlusion between post-wound days 0-3 or 4-7 was sufficient to abrogate WIHN. Microarray and qPCR of open and occluded wounds demonstrated that occlusion promotes fibrosis by upregulating TGF-β2 and mechanotransduction, a mechanosensitive profibrotic pathway. Recruitment of these potent profibrotic pathways generated a symmetrically rigid wound incapable of de novo HF regeneration. Using transgenic animal models with enhanced Wnt signaling, we determined that the ligand-dependent Wnt signaling protected against the occlusion-induced inhibition of WIHN, as well as the occlusion-induced upregulation of both profibrotic pathways. DISCUSSION/SIGNIFICANCE: In animal models, occlusion promoted fibrosis at the expense of regeneration during acute wound healing. Augmented Wnt signaling protected against this effect. Occluding wounds may reduce regeneration. Further studies are warranted to validate these findings.

CXXC5 Mediates DHT-Induced Androgenetic Alopecia via PGD2.

The number of people suffering from hair loss is increasing, and hair loss occurs not only in older men but also in women and young people. Prostaglandin D2 (PGD2) is a well-known alopecia inducer. However, the mechanism by which PGD2 induces alopecia is poorly understood. In this study, we characterized CXXC5, a negative regulator of the Wnt/β-catenin pathway, as a mediator for hair loss by PGD2. The hair loss by PGD2 was restored by Cxxc5 knock-out or treatment of protein transduction domain-Dishevelled binding motif (PTD-DBM), a peptide activating the Wnt/β-catenin pathway via interference with the Dishevelled (Dvl) binding function of CXXC5. In addition, suppression of neogenic hair growth by PGD2 was also overcome by PTD-DBM treatment or Cxxc5 knock-out as shown by the wound-induced hair neogenesis (WIHN) model. Moreover, we found that CXXC5 also mediates DHT-induced hair loss via PGD2. DHT-induced hair loss was alleviated by inhibition of both GSK-3β and CXXC5 functions. Overall, CXXC5 mediates the hair loss by the DHT-PGD2 axis through suppression of Wnt/β-catenin signaling.

Bioinspired Strategies for Wound Regeneration.

Regeneration allows animals to replace and restore injured tissues. Animal phyla have evolved different regenerative strategies to increase survival advantages. In contrast to the earlier principle that regeneration recapitulates development, recent studies indicate that wound healing in adult mammals is modified by the inflammatory response to injury, and biochemical signaling from immune and other cellular systems may modulate wound reparative responses to achieve successful tissue regeneration. Here we briefly survey different regenerative strategies used by animals across different phyla. We next focus on skin regeneration using the mouse wound-induced hair neogenesis model as an example to show the circumstances required to rebuild a new, morphogenetically competent field in the adult mammalian skin. Parallel investigations in African spiny mice (Acomys sp.) have further shown that skin rigidity can also modulate wound bed properties to facilitate de novo formation of skin appendages. These regenerating, periodically arranged hair primordia emerge using Turing activator/inhibitor principles with activities derived from sources that differ from those used in embryonic development, including the mechanical environment. Thus, a novel combination of biochemical, immunological, and mechanical signaling strategies can work together to achieve successful cutaneous regeneration in adult animals, potentially inspiring novel therapeutic strategies.

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 etal. 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.

Wound-Induced Hair Neogenesis: A Portal to the Development of New Therapies for Hair Loss and Wound Regeneration.

Adult mammals retain the remarkable ability to regenerate hair follicles after wounding. Wound-induced hair neogenesis (WIHN) in many ways recapitulates embryogenesis. The origin of the stem cells that give rise to a nascent hair follicle after wounding and the role of mesenchymal cells and signaling pathways responsible for this regenerative phenomenon are slowly being elucidated. WIHN provides a potential therapeutic window for manipulating cell fate by the introduction of factors during the wound healing process to enhance hair follicle formation.

Topological Distribution of Wound Stiffness Modulates Wound-Induced Hair Follicle Neogenesis.

In the large full-thickness mouse skin regeneration model, wound-induced hair neogenesis (WIHN) occurs in the wound center. This implies a spatial regulation of hair regeneration. The role of mechanotransduction during tissue regeneration is poorly understood. Here, we created wounds with equal area but different shapes to understand if perturbing mechanical forces change the area and quantity of de novo hair regeneration. Atomic force microscopy of wound stiffness demonstrated a stiffness gradient across the wound with the wound center softer than the margin. Reducing mechanotransduction signals using FAK or myosin II inhibitors significantly increased WIHN and, conversely, enhancing these signals with an actin stabilizer reduced WIHN. Here, α-SMA was downregulated in FAK inhibitor-treated wounds and lowered wound stiffness. Wound center epithelial cells exhibited a spherical morphology relative to wound margin cells. Differential gene expression analysis of FAK inhibitor-treated wound RNAseq data showed that cytoskeleton-, integrin-, and matrix-associated genes were downregulated, while hair follicular neogenesis, cell proliferation, and cell signaling genes were upregulated. Immunohistochemistry staining showed that FAK inhibition increased pSTAT3 nuclear staining in the regenerative wound center, implying enhanced signaling for hair follicular neogenesis. These findings suggest that controlling wound stiffness modulates tissue regeneration encompassing epithelial competence, tissue patterning, and regeneration during wound healing.

Single-Cell RNA-seq Analysis Reveals Cellular Functional Heterogeneity in Dermis Between Fibrotic and Regenerative Wound Healing Fates.

BackgroundFibrotic scars are common in both human and mouse skin wounds. However, wound-induced hair neogenesis in the murine wounding models often results in regenerative repair response. Herein, we aimed to uncover cellular functional heterogeneity in dermis between fibrotic and regenerative wound healing fates.MethodsThe expression matrix of single-cell RNA sequencing (scRNA-seq) data of fibrotic and regenerative wound dermal cells was filtered, normalized, and scaled; underwent principal components analysis; and further analyzed by Uniform Manifold Approximation and Projection (UMAP) for dimension reduction with the Seurat package. Cell types were annotated, and cell–cell communications were analyzed. The core cell population myofibroblast was identified and the biological functions of ligand and receptor genes between myofibroblast and macrophage were evaluated. Specific genes between fibrotic and regenerative myofibroblast and macrophage were identified. Temporal dynamics of myofibroblast and macrophage were reconstructed with the Monocle tool.ResultsAcross dermal cells, there were six cell types, namely, EN1-negative myofibroblasts, EN1-positive myofibroblasts, hematopoietic cells, macrophages, pericytes, and endothelial cells. Ligand and receptor genes between myofibroblasts and macrophages mainly modulated cell proliferation and migration, tube development, and the TGF-β pathway. Specific genes that were differentially expressed in fibrotic compared to regenerative myofibroblasts or macrophages were separately identified. Specific genes between fibrotic and regenerative myofibroblasts were involved in the mRNA metabolic process and organelle organization. Specific genes between fibrotic and regenerative macrophages participated in regulating immunity and phagocytosis. We then observed the underlying evolution of myofibroblasts or macrophages.ConclusionCollectively, our findings reveal that myofibroblasts and macrophages may alter the skin wound healing fate through modulating critical signaling pathways.

4‐Aminopyridine promotes cutaneous reinnervation and accelerates wound healing

Skin wound healing is a tightly orchestrated process between epithelial, dermal, neuronal and vascular cells, all working in concert to restore tissue integrity and skin barrier function. Here, we report the therapeutic regenerative effects of FDA‐approved 4‐aminopyridine (4‐AP) to promote skin wound healing by accelerating re‐epithelialization, wound induced hair neogenesis (WIHN), dermal collagen deposition, angiogenesis, and reinnervation. We demonstrate that 4‐AP enhanced wound closure by promoting keratinocyte proliferation and migration as well as collagen deposition through myofibroblast differentiation. 4‐AP treatment increased both epidermal thickness, as well as the number of hair follicles and blood vessels in healed wounds, suggesting better restoration of skin function and architecture compared to simple wound scar formation. Moreover, 4‐AP enhanced reinnervation of healed wounds by promoting Schwann cell (SC) de‐differentiation and secretion of neuromediators (NGF, SP) associated with regeneration. In‐vitro studies using human cells demonstrated that 4‐AP enhanced proliferation and migration of keratinocytes, and SCs and that 4‐AP enhances cellular interactions between neuronal and non‐neuronal cells to further accelerate wound healing. 4‐AP induced secretion of NGF from both SCs and keratinocytes. 4‐AP enhances many of the key attributes of successful wound healing and is a promising therapeutic adjuvant for skin wound healing and tissue regeneration.

GDNF neurotrophic factor signalling determines the fate of dermal fibroblasts in wound-induced hair neogenesis and skin regeneration.

We propose that GDNF, a glial cell line‐derived neurotrophic factor, can promote hair follicle neogenesis and skin regeneration after wounding by directing the fate of dermal fibroblasts. Our hypothesis is largely based on detailed GDNF and receptor analysis during skin regenerative stages, as well as the induction of GDNF receptors after wounding between the pro‐regenerative spiny mouse (genus Acomys) and its less‐regenerative descendant, the house mouse (Mus musculus). To characterize the GDNF‐target cells, we will conduct a series of lineage‐tracing experiments in conjunction with single‐cell RNA and assay for transposase‐accessible chromatin sequencing experiments. The heterogenetic dynamics of skin regeneration have yet to be fully defined, and this research will help to advance the fields of regenerative medicine and biology. Finally, we believe that stimulating the GDNF signalling pathway in fibroblasts from less‐regenerative animals, such as humans, will promote skin regeneration, morphogenesis and scarless wound healing.

Modulating Cellular Responses to Mechanical Forces to Promote Wound Regeneration.

Significance: Skin scarring poses a major biomedical burden for hundreds of millions of patients annually. However, this burden could be mitigated by therapies that promote wound regeneration, with full recovery of skin's normal adnexa, matrix ultrastructure, and mechanical strength. Recent Advances: The observation of wound regeneration in several mouse models suggests a retained capacity for postnatal mammalian skin to regenerate under the right conditions. Mechanical forces are a major contributor to skin fibrosis and a prime target for devices and therapeutics that could promote skin regeneration. Critical Issues: Wound-induced hair neogenesis, Acomys "spiny" mice, Murphy Roths Large mice, and mice treated with mechanotransduction inhibitors all show various degrees of wound regeneration. Comparison of regenerating wounds in these models against scarring wounds reveals differences in extracellular matrix interactions and in mechanosensitive activation of key signaling pathways, including Wnt, Sonic hedgehog, focal adhesion kinase, and Yes-associated protein. The advent of single-cell "omics" technologies has deepened this understanding and revealed that regeneration may recapitulate development in certain contexts, although it is unknown whether these mechanisms are relevant to healing in tight-skinned animals such as humans. Future Directions: While early findings in mice are promising, comparison across model systems is needed to resolve conflicting mechanisms and to identify conserved master regulators of skin regeneration. There also remains a dire need for studies on mechanomodulation of wounds in large, tight-skinned animals, such as red Duroc pigs, which better approximate human wound healing.

Symmetry breaking of tissue mechanics in wound induced hair follicle regeneration of laboratory and spiny mice

Tissue regeneration is a process that recapitulates and restores organ structure and function. Although previous studies have demonstrated wound-induced hair neogenesis (WIHN) in laboratory mice (Mus), the regeneration is limited to the center of the wound unlike those observed in African spiny (Acomys) mice. Tissue mechanics have been implicated as an integral part of tissue morphogenesis. Here, we use the WIHN model to investigate the mechanical and molecular responses of laboratory and African spiny mice, and report these models demonstrate opposing trends in spatiotemporal morphogenetic field formation with association to wound stiffness landscapes. Transcriptome analysis and K14-Cre-Twist1 transgenic mice show the Twist1 pathway acts as a mediator for both epidermal-dermal interactions and a competence factor for periodic patterning, differing from those used in development. We propose a Turing model based on tissue stiffness that supports a two-scale tissue mechanics process: (1) establishing a morphogenetic field within the wound bed (mm scale) and (2) symmetry breaking of the epidermis and forming periodically arranged hair primordia within the morphogenetic field (μm scale). Thus, we delineate distinct chemo-mechanical events in building a Turing morphogenesis-competent field during WIHN of laboratory and African spiny mice and identify its evo-devo advantages with perspectives for regenerative medicine.

620 RNase L is a regeneration repressor gene

Mammalian injury responses are characterized by fibrosis and scarring rather than functional regeneration. Limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we performed comprehensive transcript screening in human subjects after laser rejuvenation treatment and cross-referenced these transcripts to those found in mice with enhanced Wound Induced Hair Neogenesis (WIHN), a rare example of mammalian organogenesis. We find the anti-viral endoribonuclease RNase L to be a powerful suppressor of regeneration. Rnasel-/- mice exhibit remarkable regenerative capacity, with elevated WIHN (n=10, p<0.0001) through enhanced IL-36α (n=3, p<0.01). Consistent with the known role of RNase L to stimulate caspase-1, we find that pharmacologic inhibition of caspases promotes regeneration (n=3 versus 4, p<0.001) in a novel IL-36-dependent manner (n=4, n.s.= not significant). Additionally, these responses are not limited to skin but extend to other organs, such as the colon (n=4, p<0.05), suggesting that suppression of regeneration is a fundamental characteristic of epithelial wound healing. Taken together, this work suggests that RNaseL functions as a regeneration repressor gene in a functional tradeoff that prioritizes host antiviral abilities and is a target to enhance healing in multiple epithelial organs, perhaps even during viral infection.

Diverse cellular players orchestrate regeneration after wounding.

Fibrosis is one of the largest sources of human morbidity. The skin is a complex organ where interplay between diverse cell types and signalling pathways is essential both in homeostasis and wound repair, which can result in fibrosis or regeneration. This makes skin a useful model to study fibrosis and regeneration. While fibrosis often occurs postinjury, both clinical and laboratory observations suggest skin regeneration, complete with reconstituted cell diversity and de novo hair follicles, is possible. Extensive research performed in pursuit of skin regeneration has elucidated the key players, both cellular and molecular. Interestingly, some cells known for their homeostatic function are not implicated in regeneration or wound-induced hair neogenesis (WIHN), suggesting regeneration harnesses separate functional pathways from embryogenesis or other non-homeostatic mechanisms. For example, classic bulge cells, noted for their role in normally cycling hair follicles, do not finally contribute to long-lived cells in the regenerated tissue. During healing, multiple populations of cells, among them specific epithelial lineages, mesenchymal cells, and immune cells promote regenerative outcomes in the wounded skin. Ultimately, targeting specific populations of cells will be essential in manipulating a postwound environment to favour regeneration in lieu of fibrosis.

Wound Induced Hair Neogenesis - A Novel Paradigm for Studying Regeneration and Aging.

Hair follicles are the signature dermal appendage of mammals. They can be thought of as mini-organs with defined polarity, distinct constituent cell types, dedicated neurovascular supply, and specific stem cell compartments. Strikingly, some mammals show a capacity for adult hair follicle regeneration in a phenomenon known as wound-induced hair neogenesis (WIHN). In WIHN functional hair follicles reemerge during healing of large cutaneous wounds, and they can be counted to provide an index of regeneration. While age-related decline in hair follicle number and cycling are widely appreciated in normal physiology, it is less clear whether hair follicle regeneration also diminishes with age. WIHN provides an extraordinary quantitative system to address questions of mammalian regeneration and aging. Here we review cellular and molecular underpinnings of WIHN, explore known age-related changes to these elements, and present unanswered questions for future exploration.

The Viral Defense Gene RNaseL Acts as a Regeneration Repressor

Mammalian injury responses are characterized by fibrosis and scarring rather than functional regeneration. Limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we performed comprehensive transcript screening in human subjects after laser rejuvenation treatment. We searched for overlapping transcripts also associated with Wound Induced Hair Neogenesis (WIHN), a rare example of regeneration in mice. We found the anti-viral endoribonuclease RNase L to be a powerful suppressor of regeneration. Rnasel-/- mice exhibit remarkable regenerative capacity and accelerated wound healing following injury, through the production of IL-36α. Consistent with the known role of RNase L to stimulate caspase-1 signaling, we find that pharmacologic inhibition of caspases promotes regeneration in a novel IL-36-dependent manner. We also find a gene set of inner root sheath keratins, such as KRT71, that is associated with poor wound healing in mouse and man, and decreases with caspase inhibition. Finally, these responses are not limited to skin, but occur following intestinal injury as well, suggesting that suppression of regeneration is a general characteristic of epithelial wound healing. In all, this work suggests that RNaseL functions as a regeneration repressor gene in a functional tradeoff that prioritizes host antiviral abilities and is a target to enhance healing in multiple epithelial organs, perhaps even during viral infection.

794 RNase L is a regeneration repressor gene

When compared to animals across other phyla, mammals have restricted regeneration and more fibrosis. This limited regenerative capacity may reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover the programs governing regeneration in mammals, we investigated Wound Induced Hair Neogenesis (WIHN), a rare example of regeneration in adult mammals. Through comprehensive screening of transcripts associated with WIHN—as well as after rejuvenation lasers in human subjects--, we found that the endoribonuclease RNase L associates with regeneration/rejuvenation, but actually functions as a powerful suppressor of regeneration.

Hair Regeneration under Stress

The phenomenon of wound-induced hair neogenesis in adult mice and rabbits offers a tantalizing window into the mechanisms of regeneration. By comparing wounds in mice and several rat strains, Guerrero-Juarez etal. attempted to identify factors that may contribute to the failure of wound-induced hair neogenesis to occur in the rat. In addition to biochemical, cellular, and molecular variation, worthwhile comparisons could include the magnitude, distribution, and source of tensional forces within the wound environment.