Bulge Region Of Hair Follicle Research Articles

High expression of NF-κB inducing kinase in the bulge region of hair follicle induces tumor

The bulge region, a reservoir of multipotent stem cells, is possibly responsible for tumorigenesis. NF-κB-inducing kinase (NIK) is a kinase involved in the activation of the noncanonical NF-κB pathway and exhibits positive staining in tumor cells. However, whether high expression of NIK can result in tumorigenesis has not been reported in published papers. By establishing Nik-coe (Nik-stopF/F crossed with Chat-cre) and Nik-soe (Nik-stopF/F crossed with Sox9-cre) mice, we found that overexpression of Nik in the bulge region of hair follicles induced hair follicle loss and tumorigenesis. Furthermore, RNA sequencing, proteomic and phosphopeptide analyses revealed that multiple cancer pathways are involved in tumor formation. Taken together, these findings indicate that constitutive activation of Nik in the bulge region induces tumorigenesis.

Optogenetically Controlled TrkA Activity Improves the Regenerative Capacity of Hair-Follicle-Derived Stem Cells to Differentiate into Neurons and Glia.

Hair-follicle-derived stem cells (HSCs) originating from the bulge region of the mouse vibrissa hair follicle are able to differentiate into neuronal and glial lineage cells. The tropomyosin receptor kinase A (TrkA) receptor that is expressed on these cells plays key roles in mediating the survival and differentiation of neural progenitors as well as in the regulation of the growth and regeneration of different neural systems. In this study, the OptoTrkA system is introduced, which is able to stimulate TrkA activity via blue-light illumination in HSCs. This allows to determine whether TrkA signaling is capable of influencing the proliferation, migration, and neural differentiation of these somatic stem cells. It is found that OptoTrkA is able to activate downstream molecules such as ERK and AKT with blue-light illumination, and subsequently able to terminate this kinase activity in the dark. HSCs with OptoTrkA activity show an increased ability for proliferation and migration and also exhibited accelerated neuronal and glial cell differentiation. These findings suggest that the precise control of TrkA activity using optogenetic tools is a viable strategy for the regeneration of neurons from HSCs, and also provides a novel insight into the clinical application of optogenetic tools in cell-transplantation therapy.

Immunohistochemistry of Canine Hair Follicle Stem Cells (cHFSCs) by using CK15 and CK19

Adult body harbors powerful reservoir of stem cells that maintains homeostasis by tissue regeneration and in response to disease and injury. Hair follicle is a dynamic mini organ supporting important biological functions of the body in maintaining homeostasis and skin tissue self-renewal. This study was carried out with the objective of finding the adult stem cells in canine hair follicular tissue. To conduct this study, adult canine skin samples (n=12) irrespective of breed and sex were collected. To characterize the hair follicle stem cells, paraffin sections of canine hair follicles were immunostained with positive hair follicle stem cell markers like Anti-cytokeratin 15 (CK15) and Anti-cytokeratin 19 (CK19) and FITC conjugated and HRP conjugated secondary antibodies were used. Immunoreactivities for CK15 and CK19 were observed in the bulge/isthmus region of hair follicles in between the infundibulum and suprabulbar regions and occupied most part of the peripheral layer of outer root sheath cell. Immunophenotyping of canine Hair Follicle Stem Cells (cHFSCs) in the bulge region of hair follicle helps in confirmation of in vitro culture of cHFSCs from the bulge region which will be further used for translational research.

Ultrastructural and immunohistochemical characteristics of telocytes in human scalp tissue

This study was designed to characterize the location, morphology and ultrastructure of telocytes (TCs) in human scalp tissue. After obtaining approval for this study and informed consent from the patient, a scalp specimen was obtained. The distribution and morphology of TCs in human scalp tissue was assessed by immunohistochemical staining of CD34 and CD117/c-KIT, and the ultrastructure of TCs was investigated using transmission electron microscopy (TEM). Immunohistochemical staining of CD34 revealed that TCs were located in the connective tissue of human scalp, and were concentrated around hair follicles (HFs), blood vessels, sweat glands, sebaceous glands and adipose lobules. Immunohistochemical staining of CD117 revealed that TCs were mainly located in the dermis of human scalp, surrounding the HFs and sweat glands. Under TEM, TCs were seen and confirmed by their special morphological features. These cells were spindle-shaped, had small cell bodies and long thin processes, and surrounded stem cell clusters in the bulge region of HFs. These results demonstrate that TCs in human scalp were positive for CD34 and CD117, and their strategic positioning surrounding stem cells suggests their possible involvement in local regeneration, remodeling and homeostasis of the skin.

Autologous smashed follicular dermal graft with epidermal cell suspension in chronic nonhealing leg ulcers

Wound healing is a multifaceted process. Restoration of the epithelium may occur either by migration of activated epithelial keratinocytes from the adjoining epithelium or by centrifugal migration of putative stem cells found in the bulge region of the hair follicle. Dermal collagen and glycosaminoglycans also play a key role in laying down the scaffold for vascular in-growth and population of the matrix with host fibroblasts. Despite the availability of a wide range of treatment modalities, chronic nonhealing leg ulcers remain a therapeutic challenge and cause significant patient morbidity. Hence, there is a further need to develop novel techniques which would be effective, safe, easily available, and affordable for the patient.

Simulation of UV power absorbed by follicular stem cells during sun exposure and possible implications for melanoma development.

The incidence of melanoma in children is extremely rare. However, there is convincing evidence supporting a higher risk of developing melanoma in individuals who have experienced excessive sun exposure during their childhood. A possible explanation for this phenomenon is that prior to puberty, the melanocyte stem cells in the bulge region of hair follicles are much shallower in the dermis, resulting in their increased vulnerability to sun exposure. To validate this hypothesis, a Monte Carlo simulation of light transport is applied to quantify the dose of solar UV power absorbed in the stem-cell layers at different depths in both child and adult skins. The simulated results suggest that the stem cells in vellus hair follicles would absorb over 250times higher UV photons than those in the terminal hair follicles. Due to the thinner epidermis in child skin, the stem cells in vellus hair follicles absorb about 1.9 and 3.2times greater ultraviolet A and ultraviolet B, respectively, than those in adult skin. These findings provide a possible explanation to why children are particularly vulnerable to sun exposure.

Hair follicle stem cells isolated from newborn Yangtze River Delta White Goats

Adult stem cells are self-renewing populations that originate from embryonic progenitor cells during organogenesis and retain multipotency to support tissue and organ regeneration throughout the lifetime of an organism. The hair follicle (HF) is a small organ that is ideal for studying the biology and regulation of adult stem cells. A distinct, permanent pool of adult stem cells is located in the HF bulge region. Most methods used to isolate hair follicle stem cells (HFSCs) begin with mouse or human follicles. Here, we describe two methods of isolating HFSCs from newborn Yangtze River Delta White Goats. A suitable method was found. The cell viability and expression of HFSC marker proteins differed in the two methods. CD49f-positive (integrin alpha 6) HFSCs were sorted by fluorescence activated flow cytometry. Sorted HFSCs can be used in various in vivo grafting models and are useful as an in vitro model to study multipotency, quiescence and activation.

IL-17-positive mast cell infiltration in the lesional skin of lichen planopilaris: Possible role of mast cells in inducing inflammation and dermal fibrosis in cicatricial alopecia.

Lichen planopilaris (LPP) is a primary cicatricial alopecia characterized by the infiltration of lymphocytes in the upper portion of hair follicles. Inflammation around the bulge region of hair follicles induces destruction of hair follicle stem cells and tissue fibrosis, resulting in permanent hair loss. Treatment is still challenging, and the precise pathophysiology of this disorder is unknown. To clarify the pathogenesis of LPP, we performed histological and immunohistochemical analysis on specimens obtained from LPP patients. Formalin-fixed and paraffin-embedded samples were evaluated by staining with haematoxylin and eosin (HE), toluidine blue stain, immunohistochemistry and immunofluorescence. The immunohistochemical analysis demonstrated that CD4-positive T cells preferentially infiltrated into the follicular infundibulum in the LPP lesions. Toluidine blue stain detected a large number of mast cells in the inflammatory lesions of LPP. Interestingly, immunohistochemical analysis demonstrated that the mast cells harboured IL-17A- and IL-23-producing activity and expressed the IL-23 receptor. The number of IL-17A-positive mast cells was significantly higher in the LPP lesions than in normal scalp. Moreover, the IL-17 receptor was expressed exclusively in the follicular epithelial cells in the LPP lesions. These results suggested that mast cells infiltrating hair follicles might play a role in the pathogenesis of LPP via the IL-23/IL-17 axis.

CXCL12 regulates differentiation of human immature melanocyte precursors as well as their migration.

Melanocyte stem cells (McSCs) are localized in the bulge region of hair follicles and supply melanocytes, which determine hair color by synthesizing melanin. Ectopic differentiation of McSCs, which are usually undifferentiated in the bulge region, causes depletion of McSCs and results in hair graying. Therefore, to prevent hair graying, it is essential to maintain McSCs in the bulge region, but the mechanism of McSC maintenance remains unclear. To address this issue, we investigated the role of CXCL12, a chemokine which was previously suggested to induce migration of melanocyte lineage cells, as a niche component of McSCs. Immunohistological analysis revealed that CXCL12 was highly expressed in the bulge region of human hair follicles. CXCL12 mRNA expression level was significantly lower in white hairs plucked from human scalps than in black hairs. CXCL12 attracted the migration of early-passage normal human epidermal melanocytes (eNHEMs), an in vitro model of McSCs, which had characteristics of immature melanocyte precursors. We also found that CXCL12 suppressed their differentiation. These results suggest that CXCL12 regulates differentiation of McSCs as well as their proper localization, and maintaining McSCs by regulating CXCL12 expression level in the bulge region may be a key to preventing hair graying.

The role of Sox9 in maintaining the characteristics and pluripotency of Arbas Cashmere goat hair follicle stem cells.

In our previous work, we isolated Arbas Cashmere goat hair follicle stem cells (gHFSCs) and explored the pluripotency. In this study, we investigated the expression and putative role of Sox9 in the gHFSCs. Immunofluorescence staining showed that Sox9 is predominantly expressed in the bulge region of the Arbas Cashmere goat hair follicle, and also positively expressed in both nucleus and cytoplasm of the gHFSCs. When the cells were transfected using Sox9-shRNA, cell growth slowed down and the expression of related genes decreased significantly, cell cycle was abnormal, while the expression of terminal differentiation marker loricrin was markedly increased; cells lost the typical morphology of HFSCs; the mRNA and protein expression of gHFSCs markers and stem cell pluripotency associated factors were all significantly decreased; the expression of Wnt signaling pathway genes LEF1, TCF1,c-Myc were significantly changed. These results suggested that Sox9 plays important role in gHFSCs characteristics and pluripotency maintenance.

Identification and Characterization of Hair Follicle Stem Cells.

Hair follicle stem cells (HFSCs) are epithelial cells that inhabit in the bulge region of hair follicles. They govern development of hair follicle as well as periodically regeneration of hair follicle. Under special condition, they also play roles in homeostasis of skin and other skin appendages. To characterize HFSCs in vitro, HFSCs must be isolated and cultured. In this chapter, we introduce a mechanical method to isolate HFSCs from mouse vibrissa hair follicle, and a modified method to culture isolated HFSCs. We also describe methods to characterize HFSCs, including clone formation assay and chamber graft assay.

The Tgf--/Bmp Co-Receptor Endoglin Is a Key Component of the Molecular Oscillator That Regulates the Hair Follicle Cycle

The hair follicle is a biological oscillator that sequentially alternates phases of growth, regression and rest. The cyclic activity of this oscillator is driven by a stem cell population regulated by nurse cells and located in the hair follicle bulge region. As a whole, this morpho‐functional structure constitutes a suitable model to study the dynamics of the stem cell niches that actually maintain homeostatic and regeneration processes during development and in adult tissues. In the hair follicle, the activation of the stem cell niche and subsequent entry into the growing phase is mainly regulated by Wnt/β‐catenin signalling, while regression and resting phases are mainly regulated by Tgf‐β/Bmp/Smad activity. A major question still unresolved is the nature of the molecular switch that dictates the transition between both signalling pathways during the hair follicle growth cycle. Here we have focused on the role of Endoglin (Eng), a key co‐receptor for members of the Tgf‐β/Bmp family of growth factors. Using a haploinsufficient mouse model we have demonstrated that Eng is required for the stimulation of the hair follicle stem cell niche and to maintain a correct follicle cycling pattern. We further report that β‐catenin binds to the Eng promoter depending on Bmp signalling. Moreover, β‐catenin interacts with Smad4 in a Bmp/Eng dependent context and both proteins act synergistically to activate Eng promoter transcription. These observations point to the existence of a growth/rest switching mechanism in the hair follicle that is based on an Eng‐dependent feedback crosstalk between Wnt/β‐ catenin and Bmp/Smad signals.

In vitro identification of a stem cell population from canine hair follicle bulge region.

Skin is an extensive and easily accessible organ possessing various cell types that are constantly renewed. Previous studies have suggested the presence of a stem cell niche at the bulge region of the hair follicle, which contains cells positive for CD200 and CD34. Thus, this study sought to identify these cell populations in canine skin cells using the following methods 1- collecting samples of adult and fetal skin and isolating and culturing these cells using a method of simple enzymatic digestion and 2- testing the cell cultures for CD200 and CD34 in vitro and comparing them with skin tissue samples (in situ). Immunofluorescence results were negative for both CD200 and CD34 in frozen and paraffin embedded tissue, whereas the analysis showed that cultured cells positive for CD34, CD200 and double positive cells could be visualized in different percentages. Additionally, the pluripotency marker OCT4 was positive in the isolated cells. Analysis of CD34, CD200 and OCT4 by RT-qPCR showed that there is expression in fetal and adult cells, although no difference was observed between groups. Our results suggest that bulge stem cells from both fetuses and adult dogs were reported with the use of CD34 and CD200 markers in this study, and further techniques for cell isolation and in vitro cultivation are needed in order to obtain enriched populations of skin stem cells in dogs.

Hypoxia and hyperoxia differentially control proliferation of rat neural crest stem cells via distinct regulatory pathways of the HIF1α-CXCR4 and TP53-TPM1 proteins.

Neural crest stem cells (NCSCs) are a population of adult multipotent stem cells. We are interested in studying whether oxygen tensions affect the capability of NCSCs to self-renew and repair damaged tissues. NCSCs extracted from the hair follicle bulge region of the rat whisker pad were cultured in vitro under different oxygen tensions. We found significantly increased and decreased rates of cell proliferation in rat NCSCs (rNCSCs) cultured, respectively, at 0.5% and 80% oxygen levels. At 0.5% oxygen, the expression of both hypoxia-inducible factor (HIF) 1α and CXCR4 was greatly enhanced in the rNCSC nuclei and was suppressed by incubation with the CXCR4-specific antagonist AMD3100. In addition, the rate of cell apoptosis in the rNCSCs cultured at 80% oxygen was dramatically increased, associated with increased nuclear expression of TP53, decreased cytoplasmic expression of TPM1 (tropomyosin-1), and increased nuclear-to-cytoplasmic translocation of S100A2. Incubation of rNCSCs with the antioxidant N-acetylcysteine (NAC) overcame the inhibitory effect of 80% oxygen on proliferation and survival of rNCSCs. Our results show for the first time that extreme oxygen tensions directly control NCSC proliferation differentially via distinct regulatory pathways of proteins, with hypoxia via the HIF1α-CXCR4 pathway and hyperoxia via the TP53-TPM1 pathway. Developmental Dynamics 246:162-185, 2017. © 2016 Wiley Periodicals, Inc.

Simulated Microgravity Disrupts Cytoskeleton Organization and Increases Apoptosis of Rat Neural Crest Stem Cells Via Upregulating CXCR4 Expression and RhoA-ROCK1-p38 MAPK-p53 Signaling.

Neural crest stem cells (NCSCs) are a population of multipotent stem cells that are distributed broadly in many tissues and organs and are capable of differentiating into a variety of cell types that are dispersed throughout three germ layers. We are interested in studying the effects of simulated microgravity on the survival and self-renewal of NCSCs. NCSCs extracted from the hair follicle bulge region of the rat whisker pad were cultured in vitro, respectively, in a 2D adherent environment and a 3D suspension environment using the rotatory cell culture system (RCCS) to simulate microgravity. We found that rat NCSCs (rNCSCs) cultured in the RCCS for 24 h showed disrupted organization of filamentous actin, increased globular actin level, formation of plasma membrane blebbing and neurite-like artifact, as well as decreased levels of cortactin and vimentin. Interestingly, ∼70% of RCCS-cultured rNCSCs co-expressed cleaved (active) caspase-3 and neuronal markers microtubule-associated protein 2 (MAP2) and Tuj1 instead of NCSC markers, suggesting stress-induced formation of neurite-like artifact in rNCSCs. In addition, rNCSCs showed increased C-X-C chemokine receptor 4 (CXCR4) expression, RhoA GTPase activation, Rho-associated kinase 1 (ROCK1) and p38 mitogen-activated protein kinase (MAPK) phosphorylation, and p53 expression in the nucleus. Incubation of rNCSCs with the Gα protein inhibitor pertussis toxin or CXCR4 siRNA during RCCS-culturing prevented cytoskeleton disorganization and plasma membrane blebbing, and it suppressed apoptosis of rNCSCs. Taken together, we demonstrate for the first time that simulated microgravity disrupts cytoskeleton organization and increases apoptosis of rNCSCs via upregulating CXCR4 expression and the RhoA-ROCK1-p38 MAPK-p53 signaling pathway.

396 Induced expression of gasdermin A3 in the skin caused keratinocyte necrosis and anagen delay in the hair cycle

The epidermis forms a critical physical and immune barrier. Dysregulation of keratinocyte to cell death upon external stimuli could compromise the epidermal barrier immunity and cause skin inflammation. Gasdermin A was involved in gastric epithelial apoptosis; its function in the skin was hinted by the skin inflammation and hair loss phenotype in mice with dominant mutations in gasdermin A3 (Gsdma3). The mode of GSDMA3’s action and pathogenesis are in dispute. We and other group have demonstrated that N-terminal portion of Gsdma3 (N-Gsdma3) contains the functional domain, which is auto-regulated by its C-terminal domain. However, overexpression of N-Gsdma3 has been shown to induce autophagy or pyroptosis in some cell culture models. To study the biological function of GSDMA3 in vivo, we generated a doxycycline-inducible double transgenic (DTg) mouse model to express GSDMA3 in the epidermis using K14-rtTA mouse. We found that GSDMA3 overexpression caused epidermal hyperplasia and inflammatory infiltrations, which were associated with increased TNF-α and IL-1α expression. In addition, hair plucking was employed to induce hair cycle reentry at telogen phase. We found a significant delay in anagen initiation in DTg mice but their catagen phase seemed normal. We also observed increased macrophages around and in the bulge region of DTg hair follicles compared with control hair follicles, suggesting that bulge immune privilege was compromised. Regarding the underlying pathogenesis mechanism, we found increased TUNEL-positive staining and spontaneous necrotic-like keratinocytes in DTg mice. In primary keratinocytes isolated from the DTg mice, induced expression of Gsdma3 caused increased mitochondria ROS production, leakage of intracellular components, and spontaneous necrosis. Our data indicated that an innate immune-mediated mechanism could underlie the Gsdma3-mediated skin inflammation phenotype.

Expression characteristics in regenerating hair follicles induced by Wnt10b

Objective To investigate expression characteristics in regenerating hair follicles induced by Wnt10b, and to explore mechanisms underlying Wnt10b - induced regeneration of hair follicles. Methods Both adenovirus containing the Wnt10b gene (AdWnt10b) and that containing the green fluorescent protein-encoding gene (AdGFP) were amplified in HEK-293 cells and purified by caesium chloride density gradient centrifugation. A total of 36 C57BL/6J mice were randomly and equally divided into the AdWnt10b group and AdGFP group to be intracutaneously injected with AdWnt10b and AdGFP on the back respectively. Three mice were sacrificed on day 2.5, 5, 7, 9, 14 and 28 after the injection separately, and skin samples were resected from the injected sites subsequently. Hematoxylin and eosin (HE) staining and immunohistochemical staining were performed to observe hair follicle structure, analyze expression characteristics of the Wnt signaling pathway, and to estimate proliferative activity of regenerating hair follicles induced by Wnt10b. Results As HE staining showed, new hair follicles appeared as early as day 5 after the injection, then grew normally, and entered into catagen phase on day 28 in the AdWnt10b group. Immunohistochemical staining showed that AE15 expression was observed in new hair follicles as early as day 5 after the injection in the AdWnt10b group, then increased along with the growth of hair follicles, but decreased on day 28. On day 5 after AdWnt10b injection, both β-catenin and Lef1 expressions were seen in the cell nucleus. Lef1 was expressed specifically in hair germs and hair matrix, and its expression began to decrease on day 28. In addition, Ki67 expression was observed in the epidermis and outer root sheath of hair follicles as early as day 2.5 after the injection, in the bulge region of hair follicles on day 2.5, 7, 9 and 14, and in hair matrix cells as early as day 7. Conclusion Wnt10b could induce regeneration of hair follicles with normal structure, likely by activating the canonical Wnt signaling pathway in hair follicle stem cells and their daughter cells. Key words: Hair follicle; Regeneration; Stem cells; Wnt signaling pathway; Ki-67 antigen; beta Catenin

TR3 is preferentially expressed by bulge epithelial stem cells in human hair follicles

TR3 is an orphan member of the steroid/thyroid/retinoid nuclear receptor superfamily of transcription factors and it plays a pivotal role in regulating cell growth and apoptosis. The expression and function of TR3 in skin have not been well investigated. Using a cDNA expression assay, we discover that TR3 is significantly enriched in human telogen bulge compared with anagen bulb. Immunohistochemical staining confirms that TR3 is highly expressed in the bulge region of human hair follicles and it colocalizes with cytokeratin 15 (K15), an epithelial stem cell marker. To study the function of TR3 in the effect of androgens in keratinocytes, we treat HaCaT keratinocytes and primary human keratinocytes with dihydrotestosterone (DHT) and testosterone (T). The treated keratinocytes show a dose-dependent growth reduction to DHT and T. DHT increases the expression of TR3 in keratinocytes, associated with a concomitant increase of BAD and decrease of Bcl-2 expression. Knockdown TR3 expression by siRNA blocks the inhibitory effect of DHT on keratinocyte proliferation. Our results demonstrate that TR3 is localized to the stem cell compartment in the human hair follicles. Androgen increases TR3 expression in cultured keratinocytes. Our data suggest that TR3 mediates at least part of the inhibitory effect of androgens on keratinocytes.

Epidermal keratinocytes initiate wound healing and pro-inflammatory immune responses following percutaneous schistosome infection

Keratinocytes constitute the majority of cells in the skin’s epidermis, the first line of defence against percutaneous pathogens. Schistosome larvae (cercariae) actively penetrate the epidermis to establish infection, however the response of keratinocytes to invading cercariae has not been investigated. Here we address the hypothesis that cercariae activate epidermal keratinocytes to promote the development of a pro-inflammatory immune response in the skin. C57BL/6 mice were exposed to Schistosoma mansoni cercariae via each pinna and non-haematopoietic cells isolated from epidermal tissue were characterised for the presence of different keratinocyte sub-sets at 6, 24 and 96h p.i. We identified an expansion of epidermal keratinocyte precursors (CD45−, CD326−, CD34+) within 24h of infection relative to naïve animals. Following infection, cells within the precursor population displayed a more differentiated phenotype (α6integrin−) than in uninfected skin. Parallel immunohistochemical analysis of pinnae cryosections showed that this expansion corresponded to an increase in the intensity of CD34 staining, specifically in the basal bulge region of hair follicles of infected mice, and a higher frequency of keratinocyte Ki67+ nuclei in both the hair follicle and interfollicular epidermis. Expression of pro-inflammatory cytokine and stress-associated keratin 6b genes was also transiently upregulated in the epidermal tissue of infected mice. In vitro exposure of keratinocyte precursors isolated from neonatal mouse skin to excretory/secretory antigens released by penetrating cercariae elicited IL-1α and IL-1β production, supporting a role for keratinocyte precursors in initiating cutaneous inflammatory immune responses. Together, these observations indicate that S.mansoni cercariae and their excretory/secretory products act directly upon epidermal keratinocytes, which respond by initiating barrier repair and pro-inflammatory mechanisms similar to those observed in epidermal wound healing.

Treatment of Alopecia Areata in Mice by Stimulating the Hair Follicles Using Parathyroid Hormone Agonists Linked to a Collagen Binding Domain

Abstract Alopecia Areata is a patchy hair loss from autoimmune-mediated destruction of hair follicles, for which there is no adequate therapy. PTH-CBD is a fusion protein of parathyroid hormone and a bacterial collagen-binding domain, providing targeted delivery of a hair cycle stimulator to skin and promoting hair growth. Objectives: We tested the effects of PTH-CBD on hair growth in an established animal model for alopecia areata, the C3H/HeJ engrafted mouse. Methods: C3H/HeJ engrafted mice (Jackson Laboratories, Bar Harbor, ME) were treated subcutaneously for 8 weeks with either vehicle or different doses of PTH-CBD (320 mcg/kg x1, 1000 mcg/kg x1 or 1000 mcg/kg/wk). Results: Vehicle animals showed progressive hair loss, as expected. In PTH-CBD treated animals, grey scale quantification showed a greater proportion of PTH-CBD treated animals without significant hair loss at the end of the 2 month period (18/22 PTH-CBD vs. 4/11 vehicle), with no observed differences between the different PTH-CBD treatment regimens. Histological examination revealed no change in CD8+ cells, but there was a marked increases in the number of anagen VI hair follicles in PTH-CBD treated animals. There were also increased levels of betacatenin, a known initiator of the hair cycle, observed around the bulge region of hair follicles. Conclusions: C3H/HeJ engrafted animals treated with PTH-CBD showed rapid and persistent improvements in hair growth in the majority of tested animals. There were marked increases in anagen hair follicles despite an ongoing immune reaction. Increased beta catenin levels suggest that PTH-CBD stimulates hair growth by activating the Wnt pathway.