Hair Follicle Bulge Stem Cells Research Articles

Piperine reduces hair oiliness by inhibiting adipogenesis of hair stem cells

Piperine, an alkaloid compound in black pepper (Piper nigrum), has beneficial bioactivities. Specifically, piperine inhibits adipogenesis in 3T3-L1 cells by suppressing the transcriptional activity of PPARγ. Control of hair oiliness, which is related to adipogenic regulation, is important to prevent hair loss. Excessive sebum from the sebaceous gland (SG) can cause acne, folliculitis, or irritated skin by clogging pores. To investigate the in vivo function of piperine in SG, we used mice fed a high-fat diet (HFD). The HFD increased the size and Oil Red O (ORO) staining intensity of SG, which were significantly reduced by piperine. The HFD also upregulated the expression of sebocyte-associated genes, including PPARγ target genes, an effect reversed by piperine. In CD34/CD49f double-positive hair follicle bulge stem cells isolated from mouse vibrissae, piperine inhibited cellular adipogenesis, likely via transcriptional repression of related genes. Furthermore, piperine reduced the thickness of subcutaneous fat. In human dermal papilla cells, piperine inhibited cellular adipogenesis, as shown by the reduction in ORO staining and the downregulation of PPARγ target genes. In conclusion, piperine can be used to reduce hair greasiness by suppressing adipogenesis in hair stem cells.

MicroRNA-148a Controls Epidermal and Hair Follicle Stem/Progenitor Cells by Modulating the Activities of ROCK1 and ELF5

Skin and hair development is regulated by complex programs of gene activation and silencing and microRNA-dependent modulation of gene expression to maintain normal skin and hair follicle development, homeostasis, and cycling. In this study, we show that miR-148a, through its gene targets, plays an important role in regulating skin homeostasis and hair follicle cycling. RNA and protein analysis of miR-148a and its gene targets were analyzed using a combination of invitro and invivo experiments. We show that the expression of miR-148a markedly increases during telogen (bulge and hair germ stem cell compartments). Administration of antisense miR-148a inhibitor into mouse skin during the telogen phases of the postnatal hair cycle results in accelerated anagen development and altered stem cell activity in the skin. We also show that miR-148a can regulate colony-forming abilities of hair follicle bulge stem cells as well as control keratinocyte proliferation/differentiation processes. RNA and protein analysis revealed that miR-148a may control these processes by regulating the expression of Rock1 and Elf5 invitro and invivo. These data provide an important foundation for further analyses of miR-148a as a crucial regulator of these genes target in the skin and hair follicles and its importance in maintaining stem/progenitor cell functions during normal tissue homeostasis and regeneration.

Metformin Promotes Mechanical Stretch-Induced Skin Regeneration by Improving the Proliferative Activity of Skin-Derived Stem Cells.

BackgroundSkin expansion by mechanical stretch is an essential and widely used treatment for tissue defects in plastic and reconstructive surgery; however, the regenerative capacity of mechanically stretched skin limits clinical treatment results. Here, we propose a strategy to enhance the regenerative ability of mechanically stretched skin by topical application of metformin.MethodsWe established a mechanically stretched scalp model in male rats (n = 20), followed by their random division into two groups: metformin-treated (n = 10) and control (n = 10) groups. We measured skin thickness, collagen volume fraction, cell proliferation, and angiogenesis to analyze the effects of topical metformin on mechanically stretched skin, and immunofluorescence staining was performed to determine the contents of epidermal stem cells and hair follicle bulge stem cells in mechanically stretched skin. Western blot was performed to detect the protein expression of skin-derived stem cell markers.ResultsCompared with the control group, metformin treatment was beneficial to mechanical stretch-induced skin regeneration by increasing the thicknesses of epidermis (57.27 ± 10.24 vs. 31.07 ± 9.06 μm, p < 0.01) and dermis (620.2 ± 86.17 vs. 402.1 ± 22.46 μm, p < 0.01), number of blood vessels (38.30 ± 6.90 vs. 17.00 ± 3.10, p < 0.01), dermal collagen volume fraction (60.48 ± 4.47% vs. 41.28 ± 4.14%, p < 0.01), and number of PCNA+, Aurora B+, and pH3+ cells. Additionally, we observed significant elevations in the number of proliferating hair follicle bulge stem cells [cytokeratin (CK)15+/proliferating cell nuclear antigen (PCNA)+] (193.40 ± 35.31 vs. 98.25 ± 23.47, p < 0.01) and epidermal stem cells (CK14+/PCNA+) (83.00 ± 2.38 vs. 36.38 ± 8.96, p < 0.01) in the metformin-treated group, and western blot results confirmed significant increases in CK14 and CK15 expression following metformin treatment.ConclusionTopical application of metformin enhanced the regenerative capacity of mechanically stretched skin, with the underlying mechanism possibly attributed to improvements in the proliferative activity of skin-derived stem cells.

The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function

It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.

The Developmental & Molecular Requirements for Ensuring that Human Pluripotent Stem Cell-Derived Hair Follicle Bulge Stem Cells Have Acquired Competence for Hair Follicle Generation Following Transplantation

When using human induced pluripotent stem cells (hiPSCs) to achieve hair follicle (HF) replacement, we found it best to emulate the earliest fundamental developmental processes of gastrulation, ectodermal lineage commitment, and dermogenesis. Viewing hiPSCs as a model of the epiblast, we exploited insights from mapping the dynamic up- and down-regulation of the developmental molecules that determine HF lineage in order to ascertain the precise differentiation stage and molecular requirements for grafting HF-generating progenitors. To yield an integrin-dependent lineage like the HF in vivo, we show that hiPSC derivatives should co-express, just prior to transplantation, the following combination of markers: integrins α6 and β1 and the glycoprotein CD200 on their surface; and, intracellularly, the epithelial marker keratin 18 and the hair follicle bulge stem cell (HFBSC)-defining molecules transcription factor P63 and the keratins 15 and 19. If the degree of trichogenic responsiveness indicated by the presence of these molecules is not achieved (they peak on Days 11-18 of the protocol), HF generation is not possible. Conversely, if differentiation of the cells is allowed to proceed beyond the transient intermediate progenitor state represented by the HFBSC, and instead cascades to their becoming keratin 14+ keratin 5+ CD200– keratinocytes (Day 25), HF generation is equally impossible. We make the developmental case for transplanting at Day 16-18 of differentiation—the point at which the hiPSCs have lost pluripotency, have attained optimal expression of HFBSC markers, have not yet experienced downregulation of key integrins and surface glycoproteins, have not yet started expressing keratinocyte-associated molecules, and have sufficient proliferative capacity to allow a well-populated graft. This panel of markers may be used for isolating (by cytometry) HF-generating derivatives away from cell types unsuited for this therapy as well as for identifying trichogenic drugs.

Fisetin Promotes Hair Growth by Augmenting TERT Expression.

Although thinning hair and alopecia are not recognized as severe diseases, hair loss has implications for mental health and quality of life; therefore, a large number of studies have been carried out to develop novel hair growth agents. In the present study, we aimed to examine the potential of telomerase reverse transcriptase (TERT), because TERT overexpression in skin activates resting hair follicle bulge stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To this end, we screened polyphenols that activate TERT expression in keratinocytes, and identified resveratrol and fisetin as strong hTERT-augmenting compounds. These polyphenols also regulated the gene expression of cytokines such as IGF-1 and KGF, which activate the β-catenin pathway, and TGF-β1, which plays an important role in maintaining the niche of hair follicle stem cells, thus are thought to play roles in promoting hair growth. We additionally showed that these polyphenols, especially fisetin, promoted hair growth from the shaved dorsal skin of mice, which suggests that these polyphenols activate the transition from telogen to anagen phase. Histological studies indicated that the dorsal skin of mice treated with these polyphenols contained numerous hair follicles and was thickened compared with that in control mice. Furthermore, on the dorsal skin of mice treated with resveratrol and fisetin, a number of proliferating cells (Ki67+ cells) were observed around the hair papilla. These results suggest that resveratrol and fisetin induce a shift from telogen to anagen in the hair follicle by inducing proliferation of hair follicle bulge stem cells, thus promoting hair growth.

Hair follicle stem cells promote cutaneous wound healing through the SDF-1α/CXCR4 axis: an animal model.

An appropriate source of adult stem cells for therapeutic use is stem cells deriving from the hair follicle bulge. Following injury, ischaemic tissues produce a variety of cytokines and growth factors that are essential for tissue repair. This study sought to investigate the temporal effects of hair follicle bulge stem cells (HFSCs) on cutaneous wound healing in rats using the SDF-1α/CXCR4 axis. HFSCs obtained from rat vibrissa, labeled with DiI and then special markers, were detected using flow cytometry. The animals were divided into five groups: control (non-treated, n=18), sham (PBS, n=18), AMD (treated with AMD3100, n=18), HFSC + AMD (treated with HFSCs + AMD3100, n=18) and HFSC (treated with HFSCs, n=18). A full-thickness excisional wound model was created and DiI-labeled HFSCs were injected around the wound bed. Wound healing was recorded with digital photographs. The animals were sacrificed 3, 7 and 14 days after the surgery and were used for histological (H&E, Masson's trichrome staining) and molecular (ELISA and q-PCR) assays. The flow cytometry results demonstrated that HFSCs were CD34-positive, nestin-positive, but Kr15-negative. The morphological analysis of the HFSC-treated wounds showed accelerated wound closure. The histological analysis of the photomicrographs exhibited more re-epithelialisation and dermal structural regeneration in the HFSC-treated wounds compared with the control group. In the HFSC + AMD group, the histological parameters improved on the same days, but showed a significant decrease compared with the HFSC group in all the days assayed. In the AMD group, there was a significant reduction in the noted parameters. qRT-PCR and ELISA showed a high expression level of SDF-1α, CXCR4 and VEGFR-2 in the HFSC-treated wounded skin tissue, but the expression of CXCR4 and VEGFR-2 showed a significant reduction in the HFSC + AMD group compared with the HFSC group. Based on the findings of this study, HFSC transplantation affects wound closure parameters and the expression of SDF-1α and CXCR4. As the SDF-1α expression level increases in the injured area, the HFSCs contribute to wound repair through the SDF-1α/CXCR4 axis. This result is extremely valuable because it raises the possibility of wounds healed by isolating autologous HFSCs from the patient.

Deriving Keratinocyte Progenitor Cells and Keratinocytes from Human-Induced Pluripotent Stem Cells.

Skin or hair loss (alopecia) may occur due to a wide variety of causes ranging from trauma to pathological processes including acquired or congenital causes. It would be ideal to replace them with immunologically compatible cells to avoid potentially exacerbating the condition. Deriving the replacement cells from human-induced pluripotent stem cells (hiPSCs) allows for sufficient scale up and using hiPSCs as the choice of human pluripotent stem cells (hPSC) will ensure immunocompatibility. Here we offer a protocol for differentiating hiPSCs into keratinocyte progenitor cells (KPC) and keratinocytes employing all-trans retinoic acid (ATRA) and L-ascorbic acid, (L-AA), bone morphogenic protein-4 (BMP4), and epidermal growth factor (EGF). We observed that the hiPSC-derived KPCs express the same panel of markers as primary hair follicle bulge stem cells (HFBSCs), including CD200, integrin α-6 (ITGA6), integrin β-1 (ITGB1), the transcription factor P63, keratin 15 (KRT15), and keratin 19 (KRT19). If permitted to differentiate further, the hiPSC-derived KPC lose CD200 expression and rather come to express keratin 14 (KRT14) indicating emergence of more mature terminally-differentiated keratinocytes. The HFBSCs are transplantable for hair follicle (HF) restoration, and the keratinocytes may be transplantable for therapy for large burns or ulcers. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Reprogramming of normal human skin fibroblasts into normal hiPSCs using episomal DNA cocktail Basic Protocol 2: Differentiation of hiPSCs into KPCs and keratinocytes Alternate Protocol 2: EBS formation protocol using AggreWell™ plates (Antonchuk, 2013) Support Protocol 1: Passage hiPSC-KPC Support Protocol 2: Immunocytochemistry (ICC) Support Protocol 3: Immunofluorescence staining of cells for flow cytometry (FC).

Calcitonin gene-related peptide receptor Calcrl is enriched in hair follicles stem cells and differentially expressed in interfollicular epidermis in murine skin.

Calcitonin gene-related peptide (CGRP) and adrenomedullin are structurally similar neuropeptides acting as potent vasodilators of blood pressure and mediator of inflammation in skin. Revealing the expression pattern of their common receptor-Calcitonin gene-related peptide receptor (Calcrl) in their targeted cells is important to explain the functions of CGRP and adrenomedullin in skin. Our immunostaining results showed that Calcrl is enriched in hair follicles bulge stem cells and differentially expressed in basal stem cells of interfollicular epidermis. In addition, Calcrl expression in interfollicular epidermis is dependent on presence of nerve fibers. Long-term ablation of the murine cutaneous nerve leads to loss of Calcrl expression in interfollicular epidermis but not in hair follicle bulge stem cells. Our results demonstrate a tight interaction between neuronal components and epidermis, and indicates potential roles of Calcrl in epidermal stem cells.

Dicer- and Bulge Stem Cell-Dependent MicroRNAs During Induced Anagen Hair Follicle Development.

MicroRNAs (miRNAs) are a major class of conserved non-coding RNAs that have a wide range of functions during development and disease. Biogenesis of canonical miRNAs depend on the cytoplasmic processing of pre-miRNAs to mature miRNAs by the Dicer endoribonuclease. Once mature miRNAs are generated, the miRNA-induced silencing complex (miRISC), or miRISC, incorporates one strand of miRNAs as a template for recognizing complementary target messenger RNAs (mRNAs) to dictate post-transcriptional gene expression. Besides regulating miRNA biogenesis, Dicer is also part of miRISC to assist in activation of the complex. Dicer associates with other regulatory miRISC co-factors such as trans-activation responsive RNA-binding protein 2 (Tarbp2) to regulate miRNA-based RNA interference. Although the functional role of miRNAs within epidermal keratinocytes has been extensively studied within embryonic mouse skin, its contribution to the normal function of hair follicle bulge stem cells (BSCs) during post-natal hair follicle development is unclear. With this question in mind, we sought to ascertain whether Dicer-Tarpb2 plays a functional role within BSCs during induced anagen development by utilizing conditional knockout mouse models. Our findings suggest that Dicer, but not Tarbp2, functions within BSCs to regulate induced anagen (growth phase) development of post-natal hair follicles. These findings strengthen our understanding of miRNA-dependency within hair follicle cells during induced anagen development.

Keratinocytes-hair follicle bulge stem cells-fibroblasts co-cultures on a tri-layer skin equivalent derived from gelatin/PEG methacrylate nanofibers

Generation of full thickness skin equivalent models is of increasing interest in tissue engineering because of the limitations inherent to current models. In recent years, considerable interest has been given to electrospun hybrid nanofibers prepared using natural and synthetic combinations of polymers. By blending two polymers, gelatin and PEG methacrylate we created a novel functional hydrogel—named GelMet. By adjusting the concentration of GelMet between 14 and 20wt%, three types of electrospun membranes were fabricated. Keratinocytes, hair follicle bulge stem cells (HFBSCs) and fibroblasts were successfully isolated and cultured in 14 wt%, 17 wt% and 20 wt% GelMet scaffolds respectively and generated a tri-layered electrospun construct. Characterization of GelMet electrospun membranes were compared with those of the pure gelatin nanofibers. Due to plasticity, by incorporating HFBSCs, it is expected to increase the cell content of skin substitute without the need to incorporate several different cell populations. The fiber diameter and pore size of the scaffold for each layer were fabricated in such a way to mimic the structural gradation of collagen matrix across the native skin. Good mechanical properties and dimensional stability of GelMet scaffold, combined with the ability to support cell growth in vitro, suggest its tremendous potential application in skin tissue engineering.

Beta-caryophyllene enhances wound healing through multiple routes.

Beta-caryophyllene is an odoriferous bicyclic sesquiterpene found in various herbs and spices. Recently, it was found that beta-caryophyllene is a ligand of the cannabinoid receptor 2 (CB2). Activation of CB2 will decrease pain, a major signal for inflammatory responses. We hypothesized that beta-caryophyllene can affect wound healing by decreasing inflammation. Here we show that cutaneous wounds of mice treated with beta-caryophyllene had enhanced re-epithelialization. The treated tissue showed increased cell proliferation and cells treated with beta-caryophyllene showed enhanced cell migration, suggesting that the higher re-epithelialization is due to enhanced cell proliferation and cell migration. The treated tissues also had up-regulated gene expression for hair follicle bulge stem cells. Olfactory receptors were not involved in the enhanced wound healing. Transient Receptor Potential channel genes were up-regulated in the injured skin exposed to beta-caryophyllene. Interestingly, there were sex differences in the impact of beta- caryophyllene as only the injured skin of female mice had enhanced re-epithelialization after exposure to beta-caryophyllene. Our study suggests that chemical compounds included in essential oils have the capability to improve wound healing, an effect generated by synergetic impacts of multiple pathways.

154 Dissecting the keratinocyte lineage of basal cell carcinoma using single cell RNA sequencing

It remains unclear whether basal cell carcinomas (BCCs) originate from the stem cells in the hair follicle bulge or inter-follicular epithelial (IFE) regions. To explore the lineage derivation of BCC tumors, we applied single-cell RNA sequencing to 56219 cells isolated from 4 BCCs and 2 normal skin samples, profiling tumor keratinocyte, immune, stromal, melanocyte and endothelial cell subpopulations. Pseudotime trajectories of keratinocyte and fibroblast clusters displayed a common origin of normal cells, then a bifurcation into two branches in BCCs. In particular, tumor keratinocyte clusters characterized by high levels of EPCAM also expressed high levels of human bulge markers (KRT15, LGR5, DKK3) and low levels of human IFE markers (CD34, KIT). In contrast, keratinocyte clusters with low EPCAM expression had low levels of human bulge markers and high levels of mouse IFE markers (GRHL3, OVOL1). Single-cell analyses also defined distinct tumor keratinocyte-stromal interaction patterns. Our findings support the hair follicle bulge stem cells as the lineage of origin for BCC and elucidate the tumor and microenvironment pathways that evolve during BCC development.

Oral Mucosal Stem Cells, Human Immature Dental Pulp Stem Cells and Hair Follicle Bulge Stem Cells as Adult Stem Cells Able to Correct Limbal Stem Cell Deficiency.

Adult stem cells are somatic stem cells distributed all over the body. They represent a promising future for regenerative medicine because of their multiple advantages as they are widely available, accessible, easily stored and manipulated to a wide range of cells and with minimal invasive extraction. This review describes three examples of adult stem cells: oral mucosal epithelial stem cells, human immature dental pulp stem cells and hair follicle bulge stem cells that show an ability to correct limbal stem cell deficiency, their isolation and cultivation methods, feeder layers, carriers, markers expressed, successfulness to regenerate the ocular surface and mimic the corneal function in LSCD. Although hair follicle bulge stem cells and human immature dental pulp stem cells are a promising adult stem cell source to correct limbal stem cell deficiency, but the published research evaluating this ability is limited. Therefore, this article emphasize further research in this area.

Ex Vivo Imaging and Genetic Manipulation of Mouse Hair Follicle Bulge Stem Cells.

Stem cells that reside in the bulge of adult mouse hair follicles are a leading model of tissue stem cell research. Ex vivo culturing, molecular and cell biological characterizations, as well as genetic manipulation of fluorescence-activated cell sorting-isolated bulge stem cells offer a useful experimental pipeline to complement in vivo studies. Here we describe detailed methods for culturing, immunostaining, live cell imaging, and adenoviral infection of bulge stem cells for downstream applications such as in vitro clonal and in vivo patch assays.

Repigmentation of Human Vitiligo Skin by NBUVB Is Controlled by Transcription of GLI1 and Activation of the β-Catenin Pathway in the Hair Follicle Bulge Stem Cells

Vitiligo repigmentation is a complex process in which the melanocyte-depleted interfollicular epidermis is repopulated by melanocyte precursors from hair follicle bulge that proliferate, migrate, and differentiate into mature melanocytes on their way to the epidermis. The strongest stimulus for vitiligo repigmentation is narrow-band UVB (NBUVB), but how the hair follicle melanocyte precursors are activated by UV light has not been extensively studied. To better understand this process, we developed an application that combined laser capture microdissection and subsequent whole transcriptome RNA sequencing of hair follicle bulge melanocyte precursors and compared their gene signatures to that of regenerated mature epidermal melanocytes from NBUVB-treated vitiligo skin. Using this strategy, we found up-regulation of TNC, GJB6, and THBS1 in the hair follicle bulge melanocytes and of TYR in the epidermal melanocytes of the NBUVB-treated vitiligo skin. We validated these results by quantitative real-time-PCR using NBUVB-treated vitiligo skin and untreated normal skin. We also identified that GLI1, a candidate stem cell-associated gene, is significantly up-regulated in the melanocytes captured from NBUVB-treated vitiligo bulge compared with untreated vitiligo bulge. These signals are potential key players in the activation of bulge melanocyte precursors during vitiligo repigmentation.

Essential Role of Polarity Protein Par3 for Epidermal Homeostasis through Regulation of Barrier Function, Keratinocyte Differentiation, and Stem Cell Maintenance

Partitioning-defective (Par) proteins contribute to multiprotein complexes that drive cell polarity and fate in invertebrates. Of these, the ternary Par3-atypical protein kinase C-Par6 polarity complex mediates asymmetry in various systems, whereas Par3 and aPKC/Par6 can also act independently. aPKC-λ has recently been implicated in epidermal differentiation and stem cell fate; however, whether Par3 contributes to the homeostasis of adult stratified epithelia is currently unknown. Here, we provide functional evidence that epidermal Par3 loss disturbed the inside-out skin barrier, coinciding with altered expression and localization of principle tight junction components, and that epidermal differentiation and thickness were increased. Moreover, Par3 inactivation caused an initial expansion and later decline of hair follicle bulge stem cells, accompanied by an enrichment of committed progenitors, formation of hypertrophic sebaceous glands, and increased epidermal differentiation, suggesting aberrant cell fate decisions. Importantly, and opposite to aPKCλ deletion, Par3 loss did not enhance perpendicular cell divisions. Instead, in Par3-deficient hair follicles, spindles were shifted toward planar orientation, indicating that abnormal differentiation after Par3 inactivation is unlikely to be attributed to increased perpendicular spindle orientation. Collectively, mammalian Par3 controls the epidermal barrier, differentiation, and stem cell maintenance in the pilosebaceous unit, which are all essential for the homeostasis of an important barrier-forming epithelium.

Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/β-catenin signaling

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.

Hair Follicle Bulge Stem Cells Appear Dispensable for the Acute Phase of Wound Re-epithelialization.

The cutaneous healing response has evolved to occur rapidly, in order to minimize infection and to re‐establish epithelial homeostasis. Rapid healing is achieved through complex coordination of multiple cell types, which importantly includes specific cell populations within the hair follicle (HF). Under physiological conditions, the epithelial compartments of HF and interfollicular epidermis remain discrete, with K15+ve bulge stem cells contributing progeny for HF reconstruction during the hair cycle and as a basis for hair shaft production during anagen. Only upon wounding do HF cells migrate from the follicle to contribute to the neo‐epidermis. However, the identity of the first‐responding cells, and in particular whether this process involves a direct contribution of K15+ve bulge cells to the early stage of epidermal wound repair remains unclear. Here we demonstrate that epidermal injury in murine skin does not induce bulge activation during early epidermal wound repair. Specifically, bulge cells of uninjured HFs neither proliferate nor appear to migrate out of the bulge niche upon epidermal wounding. In support of these observations, Diphtheria toxin‐mediated partial ablation of K15+ve bulge cells fails to delay wound healing. Our data suggest that bulge cells only respond to epidermal wounding during later stages of repair. We discuss that this response may have evolved as a protective safeguarding mechanism against bulge stem cell exhaust and tumorigenesis. Stem Cells 2016;34:1377–1385

Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis.

Immunosuppressive therapies using calcineurin inhibitors, such as cyclosporine A, are associated with a higher incidence of squamous cell carcinoma formation in mice and humans. Calcineurin is believed to suppress tumorigenesis in part through Nfatc1, a transcription factor expressed primarily in hair follicle bulge stem cells in mice. However, mice overexpressing a constitutively active Nfatc1 isoform in the skin epithelium developed increased spontaneous skin squamous cell carcinomas. Because follicular stem cells can contribute to skin tumorigenesis, whether the endogenous expression of Nfatc1 inhibits or enhances skin tumorigenesis is unclear. Here we show that loss of the endogenous expression of Nfatc1 suppresses the rate of DMBA/TPA-induced skin tumorigenesis. Inducible deletion of Nfatc1 in follicular stem cells before tumor initiation significantly reduces the rate of tumorigenesis and the contribution of follicular stem cells to skin tumors. We find that skin tumors from mice lacking Nfatc1 display reduced Hras codon 61 mutations. Furthermore, Nfatc1 enhances the expression of genes involved in DMBA metabolism and increases DMBA-induced DNA damage in keratinocytes. Together these data implicate Nfatc1 in the regulation of skin stem cell-initiated tumorigenesis via the regulation of DMBA metabolism.