Wound-induced Hair Follicle Neogenesis Research Articles

Wound-Induced Hair Follicle Neogenesis as a Promising Approach for Hair Regeneration.

The mammalian skin contains hair follicles, which are epidermal appendages that undergo periodic cycles and exhibit mini-organ features, such as discrete stem cell compartments and different cellular components. Wound-induced hair follicle neogenesis (WIHN) is the remarkable ability to regenerate hair follicles after large-scale wounding and occurs in several adult mammals. WIHN is comparable to embryonic hair follicle development in its processes. Researchers are beginning to identify the stem cells that, in response to wounding, develop into neogenic hair follicles, as well as to understand the functions of immune cells, mesenchymal cells, and several signaling pathways that are essential for this process. WIHN represents a promising therapeutic approach to the reprogramming of cellular states for promoting hair follicle regeneration and preventing scar formation. In the scope of this review, we investigate the contribution of several cell types and molecular mechanisms to WIHN.

EGFR marks a subpopulation of dermal mesenchymal cells highly expressing IGF1 which enhances hair follicle regeneration.

The skin harbours transcriptionally and functionally heterogeneous mesenchymal cells that participate in various physiological activities by secreting biochemical cues. In this study, we aimed to identify a new subpopulation of dermal mesenchymal cells that enhance hair follicle regeneration through a paracrine mechanism. Integrated single-cell RNA sequencing (scRNA-seq) data analysis revealed epidermal growth factor receptor (EGFR) as a marker of distinct fibroblast subpopulation in the neonatal murine dermis. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) were used to validate the existence of the cell population in Krt14-rtTA-H2BGFP mouse. The difference of gene expression between separated cell subpopulation was examined by real-time PCR. Potential effect of the designated factor on hair follicle regeneration was observed after the application on excisional wounds in Krt14-rtTA-H2BGFP mouse. Immunofluorescence staining demonstrated the existence of dermal EGFR+ cells in neonatal and adult mouse dermis. The EGFR+ mesenchymal population, sorted by FACS, displayed a higher expression level of Igf1 (insulin-like growth factor 1). Co-localisation of IGF1 with EGFR in the mouse dermis and upregulated numbers of hair follicles in healed wounds following the application of exogenous IGF1 illustrated the contribution of EGFR+ cells in promoting wound-induced hair follicle neogenesis. Our results indicate that EGFR identifies a subpopulation of dermal fibroblasts that contribute to IGF1 promotion of hair follicle neogenesis. It broadens the understanding of heterogeneity and the mesenchymal cell function in skin and may facilitate the potential translational application of these cells.

Commensal microbiome promotes hair follicle regeneration by inducing keratinocyte HIF-1α signaling and glutamine metabolism.

Tissue injury induces metabolic changes in stem cells, which likely modulate regeneration. Using a model of organ regeneration called wound-induced hair follicle neogenesis (WIHN), we identified skin-resident bacteria as key modulators of keratinocyte metabolism, demonstrating a positive correlation between bacterial load, glutamine metabolism, and regeneration. Specifically, through comprehensive multiomic analysis and single-cell RNA sequencing in murine skin, we show that bacterially induced hypoxia drives increased glutamine metabolism in keratinocytes with attendant enhancement of skin and hair follicle regeneration. In human skin wounds, topical broad-spectrum antibiotics inhibit glutamine production and are partially responsible for reduced healing. These findings reveal a conserved and coherent physiologic context in which bacterially induced metabolic changes improve the tolerance of stem cells to damage and enhance regenerative capacity. This unexpected proregenerative modulation of metabolism by the skin microbiome in both mice and humans suggests important methods for enhancing regeneration after injury.

Pyruvate Kinase M2 Promotes Hair Regeneration by Connecting Metabolic and Wnt/β-Catenin Signaling.

Hair follicle stem cells (HFSCs) utilize glycolytic metabolism during their activation and anagen induction. However, the role of pyruvate kinase M2 (PKM2), which catalyzes the final step of glycolysis, in hair regeneration has not been elucidated. In this study, we investigated the expression pattern and activity of PKM2 during the depilation-induced anagen progression in mice. We found that TEPP-46, a selective activator of PKM2, enhanced hair re-growth and proliferation of HFSCs. PKM2 expression was increased via up-regulation of Wnt/β-catenin signaling, which is involved in hair regeneration. Moreover, a combined treatment with KY19382, a small molecule that activates Wnt/β-catenin signaling, and TEPP-46 significantly enhanced hair re-growth and wound-induced hair follicle neogenesis (WIHN). These results indicate that simultaneous activation of the PKM2 and Wnt/β-catenin signaling could be a potential strategy for treating alopecia patients.

Wnt/β-catenin signaling activator restores hair regeneration suppressed by diabetes mellitus

Diabetes mellitus is one of the most prevalent diseases in modern society. Many complicationssuch as hepatic cirrhosis, neuropathy, cardiac infarction, and so on are associated with diabetes. Although a relationship between diabetes and hair loss has been recently reported, the treatment of diabetic hair loss by Wnt/β-catenin activators has not been achieved yet. In this study, we found that the depilation-induced anagen phase was delayed in both db/db mice and high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic mice. In diabetic mice, both hair regrowth and wound-induced hair follicle neogenesis (WIHN) were reduced because of suppression of Wnt/β-catenin signaling and decreased proliferation of hair follicle cells. We identified that KY19382, a small molecule that activates Wnt/β-catenin signaling, restored the capabilities of regrowth and WIHN in diabetic mice. The Wnt/β-catenin signaling activator also increased the length of the human hair follicle which was decreased under high glucose culture conditions. Overall, the diabetic condition reduced both hair regrowth and regeneration with suppression of the Wnt/β-catenin signaling pathway. Consequently, the usage of Wnt/β-catenin signaling activators could be a potential strategy to treat diabetes-induced alopecia patients. [BMB Reports 2022; 55(11): 559-564].

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.

755 Repurposing of DPP4 inhibition to improve hair follicle activation and regeneration

Injury to the skin results in establishment of fibrosis, which inversely correlates with capacity for regeneration of appendages such as hair follicles (HF). Large wounds on the back of mice can regenerate HF in a central area with reduced expression of fibrosis markers (wound induced hair follicle neogenesis, WIHN), which is facilitated by similar pathways of anagen activation (Wnt, AKT). Identification of modulators of anagen entry and WIHN might aid pinpointing targets for allowing regeneration of skin appendages after trauma or inducing hair growth in individuals affected for example by alopecia.

762 Commensal microbiome promotes hair follicle regeneration by inducing keratinocyte HIF-1α signaling and glutamine metabolism

Tissue injury induces metabolic changes in stem cells, which likely modulate regeneration. Using a model of organ regeneration called Wound-Induced Hair Follicle Neogenesis (WIHN), we identify skin-resident bacteria as key modulators of keratinocyte metabolism, demonstrating a positive correlation between bacterial load, glutamine metabolism, and regeneration. Specifically, through comprehensive multi-omic analysis and single-cell RNA sequencing in murine skin, we show that bacterially-induced hypoxia drives increased glutamine metabolism in keratinocytes with attendant enhancement of skin and hair follicle regeneration. In human skin wounds, topical broad-spectrum antibiotics inhibit glutamine production and are partially responsible for reduced healing. These findings reveal a conserved mechanism by which bacterially-induced metabolic changes improve the tolerance of stem cells to damage and enhance regenerative capacity. This surprising, pro-regenerative function of the skin microbiome affords novel approaches to wound healing, and suggests a refinement of the role of cutaneous antimicrobials.

Cell Population Dynamics in Wound-Induced Hair Follicle Neogenesis Model.

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

Neutrophil extracellular traps impair regeneration.

Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound‐induced hair follicle neogenesis (WIHN) as a model of non‐fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound‐associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single‐cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3−/− mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro‐regenerative cue, their presence and NETs hinder regeneration.

362 Not just a cell marker: DPP4 modulates regeneration of hair follicles in large wounds

Dipeptidyl peptidase IV (DPP4) is a serine peptidase which cleaves the last 2 amino acids at N-terminus of substrates with penultimate proline or Alanine. The majority of mammalian skin fibroblast express this molecule on their surface and pharmacological inhibition of DPP4 in splinted wounds results in decreased deposition of scar tissue. Other cell types of the skin have been reported to express this molecule and it is hypothesized to participate in many physiological and pathological processes. So far the expression pattern, regulation and exact functions of DPP4 in the skin during homeostasis and wound healing remain unknown. We investigate DPP4 in the context of healing of large wounds, which are able to regenerate hair follicles. In comparison, small wounds do not show this wound induced hair follicle neogenesis (WIHN). For WHIN, Wnt-Signals from the dermis and later from the epidermis need to be produced in a window of time of approximately 9 to 15 days post wounding (pw). Our studies show a downregulation of DPP4/Dpp4 upon wounding at the beginning of the inflammatory phase and at the beginning of activationd of WIHN (pw9). Interestingly the center of wounds show reduced numbers of DPP4 positive cells compared to the wound margins, where normally no WIHN takes place. Detailed Investigation of cutaneous cell populations show a differential Regulation of DPP4 in dermal, epithelial and immune cells. Inhibition of DPP4 around pw9 interestingly increases the number of WIHN hair follicles and the Expression of Wnt Targets and ligands in the Skin. Together our observations support the notion of DPP4 being a inhibitory factor for hair follicle regeneration

KY19382, a novel activator of Wnt/β-catenin signalling, promotes hair regrowth and hair follicle neogenesis.

Background and PurposeThe promotion of hair regeneration and growth heavily depends on the activation of Wnt/β‐catenin signalling in the hair follicle, including dermal papilla (DP). KY19382, one of the newly synthesized analogues of indirubin‐3′‐monoxime (I3O), was identified as a Wnt/β‐catenin signalling activator via inhibition of the interaction between CXXC‐type zinc finger protein 5 (CXXC5) and dishevelled (Dvl). Given the close relationship between the Wnt/β‐catenin signalling and hair regeneration, we investigated the effect of KY19382 on hair regrowth and hair follicle neogenesis.Experimental ApproachIn vitro hair induction effects of KY19382 were performed in human DP cells. The hair elongation effects of KY19382 were confirmed through the human hair follicle and vibrissa culture system. In vivo hair regeneration abilities of KY19382 were identified in three models: hair regrowth, wound‐induced hair follicle neogenesis (WIHN) and hair patch assays using C57BL/6 mice. The hair regeneration abilities were analysed by immunoblotting, alkaline phosphatase (ALP) and immunohistochemical staining.Key ResultsKY19382 activated Wnt/β‐catenin signalling and elevated expression of ALP and the proliferation marker PCNA in DP cells. KY19382 also increased hair length in ex vivo‐cultured mouse vibrissa and human hair follicles and induced hair regrowth in mice. Moreover, KY19382 significantly promoted the generation of de novo hair follicles as shown by WIHN and hair patch assays.Conclusion and ImplicationsThese results indicate that KY19382 is a potential therapeutic drug that exhibits effective hair regeneration ability via activation of the Wnt/β‐catenin signalling for alopecia treatments.

613 Bacteria induce skin regeneration via IL-1β signaling

Environmental factors that enhance regeneration are largely unknown. We hypothesized that skin bacteria modulate regeneration. Here, we assessed low, medium, and high levels of bacterial burden in wound healing and Wound Induced Hair follicle Neogenesis (WIHN), a rare adult organogenesis model. WIHN levels and stem cell markers indeed correlated with bacterial counts, being lowest in germ free (GF) (fold= -17.9, n=13, p=1.9X10-6), intermediate in conventional specific pathogen free (SPF), and highest even in mice infected with pathogenic Staphylococcus aureus (fold= 3.3, n=12, p=7.5X10-5). We identified IL-1β and keratinocyte-dependent IL-1R-MyD88 signaling as necessary and sufficient for bacteria to promote regeneration. Finally, in a small clinical trial, we found that a topical broad-spectrum antibiotic slowed skin wound healing. These results demonstrate a novel role for IL-1β to control morphogenesis and counter conventional notions that infection inhibits regeneration with a need for full sterility of small wounds.

Bacteria induce skin regeneration via IL-1β signaling

Environmental factors that enhance regeneration are largely unknown. The immune system and microbiome are attributed roles in repairing and regenerating structure but their precise interplay is unclear. Here, we assessed the function of skin bacteria in wound healing and wound-induced hair follicle neogenesis (WIHN), a rare adult organogenesis model. WIHN levels and stem cell markers correlate with bacterial counts, being lowest in germ-free (GF), intermediate in conventional specific pathogen-free (SPF), and highest in wild-type mice, even those infected with pathogenic Staphylococcus aureus. Reducing skin microbiota via cage changes or topical antibiotics decreased WIHN. Inflammatory cytokine IL-1β and keratinocyte-dependent IL-1R-MyD88 signaling are necessary and sufficient for bacteria to promote regeneration. Finally, in a small trial, a topical broad-spectrum antibiotic also slowed skin wound healing in adult volunteers. These results demonstrate a role for IL-1β to control morphogenesis and support the need to reconsider routine applications of topical prophylactic antibiotics.

Single-cell transcriptomic analysis of small and large wounds reveals the distinct spatial organization of regenerative fibroblasts.

Wound‐induced hair follicle neogenesis (WIHN) has been an important model to study hair follicle regeneration during wound repair. However, the cellular and molecular components of the dermis that make large wounds more regenerative are not fully understood. Here, we compare and contrast recently published scRNA‐seq data of small scarring wounds to wounds that regenerate in hope to elucidate the role of fibroblasts lineages in WIHN. Our analysis revealed an over‐representation of the newly identified upper wound fibroblasts in regenerative wound conditions, which express the retinoic acid binding protein Crabp1. This regenerative cell type shares a similar gene signature to the murine papillary fibroblast lineage, which are necessary to support hair follicle morphogenesis and homeostasis. RNA velocity analysis comparing scarring and regenerating wounds revealed the divergent trajectories towards upper and lower wound fibroblasts and that the upper populations were closely associated with the specialized dermal papilla. We also provide analyses and explanation reconciling the inconsistency between the histological lineage tracing and the scRNA‐seq data from recent reports investigating large wounds. Finally, we performed a computational test to map the spatial location of upper wound fibroblasts in large wounds which revealed that upper peripheral fibroblasts might harbour equivalent regenerative competence as those in the centre. Overall, our scRNA‐seq reanalysis combining multiple samples suggests that upper wound fibroblasts are required for hair follicle regeneration and that papillary fibroblasts may migrate from the wound periphery to the centre during wound re‐epithelialization. Moreover, data from this publication are made available on our searchable web resource: https://skinregeneration.org/.

Epidermal Stem Cells in Hair Follicle Cycling and Skin Regeneration: A View From the Perspective of Inflammation.

There are many studies devoted to the role of hair follicle stem cells in wound healing as well as in follicle self-restoration. At the same time, the influence of the inflammatory cells on the hair follicle cycling in both injured and intact skin is well established. Immune cells of all wound healing stages, including macrophages, γδT cells, and Tregs, may activate epidermal stem cells to provide re-epithelization and wound-induced hair follicle neogenesis. In addition to the ability of epidermal cells to maintain epidermal morphogenesis through differentiation program, they can undergo de-differentiation and acquire stem features under the influence of inflammatory milieu. Simultaneously, a stem cell compartment may undergo re-programming to adopt another fate. The proportion of skin resident immune cells and wound-attracted inflammatory cells (e.g., neutrophils and macrophages) in wound-induced hair follicle anagen and plucking-induced anagen is still under discussion to date. Experimental data suggesting the role of reactive oxygen species and prostaglandins, which are uncharacteristic of the intact skin, in the hair follicle cycling indicates the role of neutrophils in injury-induced conditions. In this review, we discuss some of the hair follicles stem cell activities, such as wound-induced hair follicle neogenesis, hair follicle cycling, and re-epithelization, through the prism of inflammation. The plasticity of epidermal stem cells under the influence of inflammatory microenvironment is considered. The relationship between inflammation, scarring, and follicle neogenesis as an indicator of complete wound healing is also highlighted. Taking into consideration the available data, we also conclude that there may exist a presumptive interlink between the stem cell activation, inflammation and the components of programmed cell death pathways.

Bacteria Induce Skin Regeneration Via Il-1β Signaling

Environmental factors that enhance regeneration are largely unknown. We hypothesized that skin bacteria modulate regeneration. Here, we assessed low, medium, and high levels of bacterial burden in wound healing and Wound Induced Hair follicle Neogenesis (WIHN), a rare adult organogenesis model (Ito et al., 2007; Plikus et al., 2017). WIHN levels and stem cell markers indeed correlated with bacterial counts, being lowest in germ free (GF), intermediate in conventional specific pathogen free (SPF), and highest even in mice infected with pathogenic Staphylococcus aureus. We identified IL-1β and keratinocyte-dependent IL-1R-MyD88 signaling as necessary and sufficient for bacteria to promote regeneration. Finally, in a small clinical trial, we found that a topical broad-spectrum antibiotic slowed skin wound healing. These results demonstrate a novel role for IL-1β to control morphogenesis and counter conventional notions that infection inhibits regeneration with a need for full sterility of small wounds.

IL-36α Promoted Wound Induced Hair Follicle Neogenesis via Hair Follicle Stem/Progenitor Cell Proliferation.

BackgroundWound-induced hair follicle neogenesis (WIHN) is a phenomenon of hair neogenesis that occurs at the center of a scar when the wound area is sufficiently large. Neogenic hair follicles are separated from the pre-existing follicles at the wound edge by a hairless circular region. This WIHN study provides a unique model for developing treatments for hair loss and deciphering the mechanisms underlying organogenesis in adult mammals.MethodsThe skin of a mouse was wounded by excising a 1.5 × 1.5 cm2 square of full-thickness dorsal skin. iTRAQ technology was used to screen proteins differentially expressed between the inner and outer scar areas in a mouse model of WIHN, on post-wounding day 15, to identify the regulators of WIHN. Owing to the overexpression of interleukin-36α (IL-36α) in the de novo hair follicle growth area, the regulating effect of IL-36α overexpression in WIHN was investigated. Hair follicle stem/progenitor cells were counted by flow cytometry while the expression of hair follicle stem/progenitor cell markers (Lgr5, Lgr6, Lrig1, K15, and CD34) and that of Wnt/β-catenin and IL-6/STAT3 pathway intermediaries was detected by qPCR and western blotting.ResultsWe found that wounding induced IL-36α expression. Incorporation of recombinant murine IL-36α (mrIL-36α) into murine skin wounds resulted in a greater number of regenerated hair follicles (p < 0.005) and a faster healing rate. The expression of hair follicle stem/progenitor cell markers was upregulated in the mrIL-36α-injected site (p < 0.05). Additionally, mrIL-36α upregulated the IL-6/STAT3 pathway intermediaries.ConclusionIL-36α is upregulated in de novo hair follicle growth areas and can promote wound epithelialization and WIHN.

Wound-induced hair follicle neogenesis: a new perspective on hair follicles regeneration in adult mammals

To explore the research progress of the cell sources and related signaling pathways of the wound-induced hair follicle neogenesis (WIHN) in recent years. The literature related to WIHN in recent years was reviewed, and the cell sources and molecular mechanism were summarized and discussed. Current research shows that WIHN is a rare regeneration phenomenon in the skin of adult mammals, with multiple cell origins, both hair follicle stem cells and epithelial stem cells around the wound. Its molecular mechanism is complicated, which is regulated by many signaling pathways. Besides, the process is closely related to the immune response, the immunocytes and their related cytokines provide suitable conditions for this process. There are still many unsolved problems on the cellular origins and molecular mechanisms of the WIHN. Further study on the mechanisms will enhance the understanding of adult mammals' hair follicle regeneration and may provide new strategy for functional healing of the human skin.

Noncoding dsRNA induces retinoic acid synthesis to stimulate hair follicle regeneration via TLR3

How developmental programs reactivate in regeneration is a fundamental question in biology. We addressed this question through the study of Wound Induced Hair follicle Neogenesis (WIHN), an adult organogenesis model where stem cells regenerate de novo hair follicles following deep wounding. The exact mechanism is uncertain. Here we show that self-noncoding dsRNA activates the anti-viral receptor toll like receptor 3 (TLR3) to induce intrinsic retinoic acid (RA) synthesis in a pattern that predicts new hair follicle formation after wounding in mice. Additionally, in humans, rejuvenation lasers induce gene expression signatures for dsRNA and RA, with measurable increases in intrinsic RA synthesis. These results demonstrate a potent stimulus for RA synthesis by non-coding dsRNA, relevant to their broad functions in development and immunity.