Reversibly immortalization establishes a hair follicle stem cell line with hair follicle reconstruction ability.

Hair follicle stem cells (HFSCs) play critical roles in the periodic regeneration of hair follicles. HFSCs are also a good model for stem cell biology research. However, no stable mouse HFSC cell line has been reported, which restricts the research and application of HFSCs. We isolated HFSCs from mouse hair follicles and immortalized them by inducing a reversible SV40 large T antigen. Through monoclonal screening, we identified a reversibly immortalized cell line, immortalized HFSC (iHFSC2). RNA sequencing, fluorescence-activated cell sorting, western blotting and immunofluorescence experiments revealed that the expression patterns of iHFSC2 and HFSC were similar at the protein and mRNA levels. After that, iHFSC2s were passaged and morphologically monitored for up to 40 times to detect their long-term culture potential. The long-term cultured iHFSC2 could regenerate hair follicles with complete hair follicle structure and HFSCs in the bulge area. This work successfully established an HFSC cell line with the ability of hair follicle reconstruction.

Correction to: The balance of Bmp6 and Wnt10b regulates the telogen-anagen transition of hair follicles.

Following publication of the original article [1], the authors reported that they would like to correct the second last sentence of "Authors' information" section as PW is an undergraduate, but was incorrectly described as a Ph.D. in the sentence. The sentence should read "PW is an undergraduate. YZ, YX, WX, HG, FD and YL are Ph.D.". The authors sincerely apologize for having this unintentional error in the article, and apologize for any inconvenience caused.

The balance of Bmp6 and Wnt10b regulates the telogen-anagen transition of hair follicles.

BACKGROUND: The periodic growth of hair follicles is regulated by the balance of activators and inhibitors. The BMP signaling pathway plays an important role during hair follicle regeneration, but the exact BMP protein that controls this process has not been revealed. METHODS: The expression of BMP6 was determined via in situ hybridization and immunofluorescence. The in vivo effect of BMP6 overexpression was studied by using a previously established adenovirus injection model. The hair follicle regeneration was assessed by gross observation, H&E staining and 5-bromo-2-deoxyuridine (BrdU) tracing. The expression patterns of BMP6 signaling and Wnt10b signaling in both AdBMP6-treated and AdWnt10b-treated skins were determined by in situ hybridization and immunofluorescence. RESULTS: BMP6 was expressed differently in the stages of hair follicle cycle. The telogen-anagen transition of hair follicles was inhibited by adenovirus-mediated overexpression of BMP6. In the in vivo model, the BMP6 signaling was inhibited by Wnt10b and the Wnt10b signaling was inhibited by BMP6. The activation of hair follicle stem cells (HFSCs) was also competitively regulated by Wnt10b and BMP6. CONCLUSIONS: Combined with previously reported data of Wnt10b, our findings indicate that BMP6 and Wnt10b are major inhibitors and activators respectively and their balance regulates the telogen-anagen transition of hair follicles. To the best of our knowledge, our data provide previously unreported insights into the regulation of hair follicle cycling and provide new clues for the diagnosis and therapies of hair loss.

Gsdma3 is required for mammary gland development in mice.

AE (alopecia and excoriation) is a mouse mutant phenotype that harbours a mutation in Gsdma3. Gsdma3 has been reported to regulate the development of skin and hair follicles. However, its role in the mammary glands has not been reported. In this study, we found that descendants bred from an AE mother died within 12 days after birth. Then, we found that the expression of Gsdma3 varied among the developmental stages of mammary glands. Subsequently, we systematically studied the phenotype of the mammary glands of AE and wild-type mice, revealing that the mammary glands were smaller in AE mice. The mammary glands of AE mice exhibited shorter ductal extension and less bifurcation. Immunohistochemistry staining indicated that the mammary glands of AE mice displayed more proliferating cells during puberty while secreting less ß-casein during pregnancy and lactation. The lymph nodes in the mammary glands of the AE mice were larger and showed some pigmentation, suggesting that the immune reaction in the mammary glands was up-regulated. Under a transmission electron microscope, residual bodies were observed in the lymph nodes in the mammary glands of AE mice. Thus, we report a new function of Gsdma3 in regulating the development of mammary glands, and we demonstrate that the Gsdma3 gene may act as a suppressor of the immune reaction.

Immunohistochemical study of hair follicle stem cells in regenerated hair follicles induced by Wnt10b.

The regulation of the periodic regeneration of hair follicles is complicated. Although Wnt10b has been reported to induce hair follicle regeneration, the characteristics of induced hair follicles, especially the target cells of Wnt10b, have not yet been clearly elucidated. Thus, we systematically evaluated the expression and proliferation patterns of Wnt10b-induced hair follicles. We found that Wnt10b promoted the proliferation of hair follicle stem cells from 24 hours after AdWnt10b injection. Seventy-two hours after AdWnt10b injection, cells outside of bulge area began to proliferate. When the induced hair follicle entered full anagen, although the hair follicle stem cells were normal, canonical Wnt signaling was maintained in the hair precortex cells. Our results reveal that the target cells that overexpressed Wnt10b included hair follicle stem cells, hair precortex cells, and matrix cells.

Wnt5a Suppresses ß-catenin Signaling during Hair Follicle Regeneration.

Hair follicles display periodic growth. Wnt signaling is a critical regulator for hair follicle regeneration. Previously, we reported that Wnt5a inhibits the telogen-to-anagen transition of hair follicles, but the mechanism by which this process occurs has not yet been reported. Here, we determined the expression patterns of Wnt signaling pathway molecules by quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry and found that ß-catenin signaling was suppressed by Wnt5a. We then compared the phenotypes and expression patterns following ß-catenin knockdown and Wnt5a overexpression during hair follicle regeneration induced by hair depilation and observed similar patterns. In addition, we performed a rescue experiment in the JB6 cell line and found that the inhibitory effect of Wnt5a on cell proliferation could be rescued by the addition of Wnt3a. Our data reveal that Wnt5a suppresses the activation of ß-catenin signaling during hair follicle regeneration.

Recent progress in hair follicle stem cell markers and their regulatory roles.

Hair follicle stem cells (HFSCs) in the bulge are a multipotent adult stem cell population. They can periodically give rise to new HFs and even regenerate the epidermis and sebaceous glands during wound healing. An increasing number of biomarkers have been used to isolate, label, and trace HFSCs in recent years. Considering more detailed data from single-cell transcriptomics technology, we mainly focus on the important HFSC molecular markers and their regulatory roles in this review.

Wnt10b promotes differentiation of mouse hair follicle melanocytes.

Previous research has revealed that Wnt10b activates canonical Wnt signaling, which is integral to melanocyte differentiation in hair follicles (HFs). However, the function of Wnt10b in HF melanocytes remains poorly understood. We determined using Dct-LacZ transgenic mice that Wnt10b is mainly expressed near and within melanocytes of the hair bulbs during the anagen stage of the hair cycle. We also found that Wnt10b promotes an increase in melanocyte maturation and pigmentation in the hair bulbs of the mouse HF. To further explore the potential functions of Wnt10b in mouse HF melanocytes, we infected iMC23 cells with Ad-Wnt10b to overexpress Wnt10b. We demonstrated that Wnt10b promotes the differentiation of melanocytes by activating canonical Wnt signaling in melanocytes.

Adenovirus-mediated Wnt5a expression inhibits the telogen-to-anagen transition of hair follicles in mice.

The canonical Wnt/ß-catenin pathway plays an important role in hair cycle induction. Wnt5a is a non-canonical Wnt family member that generally antagonizes canonical Wnt signaling in other systems. In hair follicles, Wnt5a and canonical Wnt are both expressed in cells in the telogen stage. Wnt5a has been shown to be critical for controlling hair cell fate. However, the role that Wnt5a plays in the transition from the telogen to anagen stage is unknown. In this study, using whole-mount in situ hybridization, we show that Wnt5a is produced by several other cell types, excluding dermal papilla cells, throughout the hair cycle. For example, Wnt5a is expressed in bulge and secondary hair germ cells in the telogen stage. Our studies focused on the depilated 8-week-old mouse as a synchronized model of hair growth. Interestingly, overexpression of adenovirus Wnt5a in the dorsal skin of mice led to the elongation of the telogen stage and inhibition of the initiation of the anagen stage. However, following an extended period of time, four pelage hair types grew from hairless skin that was induced by Wnt5a, and the structure of these new hair shafts was normal. Using microarray analysis and quantitative arrays, we showed that the expression of ß-catenin and some target genes of canonical Wnt signaling decreased after Wnt5a treatment. These data demonstrate that Wnt5a may inhibit the telogen stage to maintain a quiescent state of the hair follicle.

Wnt4 increases the thickness of the epidermis in burn wounds by activating canonical Wnt signalling and decreasing the cell junctions between epidermal cells.

Background: Burn wound healing is a complex process and the role of Wnt ligands varies in this process. Whether and how Wnt4 functions in burn wound healing is not well understood. In this study, we aim to reveal the effects and potential mechanisms of Wnt4 in burn wound healing. Methods: First, the expression of Wnt4 during burn wound healing was determined by immunofluorescence, Western blotting and qPCR. Then, Wnt4 was overexpressed in burn wounds. The healing rate and healing quality were analysed by gross photography and haematoxyline and eosin staining. Collagen secretion was observed by Masson staining. Vessel formation and fibroblast distribution were observed by immunostaining. Next, Wnt4 was knocked down in HaCaT cells. The migration of HaCaT cells was analysed by scratch healing and transwell assays. Next, the expression of ß-catenin was detected by Western blotting and immunofluorescence. The binding of Frizzled2 and Wnt4 was detected by coimmunoprecipitation and immunofluorescence. Finally, the molecular changes induced by Wnt4 were analysed by RNA sequencing, immunofluorescence, Western blotting and qPCR in HaCaT cells and burn wound healing tissues. Results: The expression of Wnt4 was enhanced in burn wound skin. Overexpression of Wnt4 in burn wound skin increased the thickness of epidermis. Collagen secretion, vessel formation and fibroblast distribution were not significantly impacted by Wnt4 overexpression. When Wnt4 was knocked down in HaCaT cells, the ratio of proliferating cells decreased, the ratio of apoptotic cells increased and the ratio of the healing area in the scratch healing assay to the number of migrated cells in the transwell assay decreased. The nuclear translocation of ß-catenin decreased in shRNA of Wnt4 mediated by lentivirus-treated HaCaT cells and increased in Wnt4-overexpressing epidermal cells. RNA-sequencing analysis revealed that cell junction-related signalling pathways were significantly impacted by Wnt4 knockdown. The expression of the cell junction proteins was decreased by the overexpression of Wnt4. Conclusions: Wnt4 promoted the migration of epidermal cells. Overexpression of Wnt4 increased the thickness of the burn wound. A potential mechanism for this effect is that Wnt4 binds with Frizzled2 and increases the nuclear translocation of ß-catenin, thus activating the canonical Wnt signalling pathway and decreasing the cell junction between epidermal cells.

Wnt3a promotes melanin synthesis of mouse hair follicle melanocytes.

Although the importance of Wnt3a in melanocyte development has been well recognized, the effect of Wnt3a in normal HF melanocytes has not been clearly elucidated yet. Thus, we sought to examine the presence and location of Wnt3a in HF during hair cycle. By using melanocyte-targeted Dct-LacZ transgenic mice, we found that Wnt3a signaling is activated in mouse HF melanocytes during anagen of hair cycle. To further explore the potential functions of Wnt3a in mouse melanocytes, we infected melan-a cells with AdWnt3a to serve as the production source of Wnt3a protein. We demonstrated that Wnt3a promoted melanogenesis through upregulation of MITF and its downstream genes, tyrosinase and TRP1, in melanocytes. In vivo, AdWnt3a rescued the effects of AdsimMITF on HF melanocytes and promoted melanin synthesis. Our results suggest that Wnt3a plays an important role in mouse HF melanocytes homeostasis.

Secreted Frizzled-related protein 4 inhibits the regeneration of hair follicles.

Secreted Frizzled-related Protein 4 (sFRP4) belongs to Wnt inhibitors. Previously, we reported that sFRP4 inhibited the differentiation of melanocyte. Here, by using of immunostaining, we showed that sFRP4 is expressed in both human and mouse hair follicles, especially in the outer root sheath and inner root sheath. To reveal the role of sFRP4 in hair follicle growth and hair cycle, we induced synchronized hair cycle in the dorsal skin of mice by depilation, and injected sFRP4 intradermally into the skin. By hematoxylin and eosin staining, we found that the regeneration of hair follicles was inhibited by sFRP4. However, the structure of hair follicles remained complete. Compared with phosphate buffer saline-treated hair follicles, the sFRP4-treated hair follicles still had the same expression pattern of keratins. Our findings reveal that sFRP4 inhibits but not blocks the regeneration of hair follicles, and supply a potential therapeutic application to treat hair follicle regeneration disorders.

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.

Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy.

Dermal papilla (DP) plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

Paracrine Secreted Frizzled-Related Protein 4 Inhibits Melanocytes Differentiation in Hair Follicle.

Wnt signaling plays crucial role in regulating melanocyte stem cells/melanocyte differentiation in the hair follicle. However, how the Wnt signaling is balanced to be overactivated to control follicular melanocytes behavior remains unknown. Here, by using immunofluorescence staining, we showed that secreted frizzled-related protein 4 (sFRP4) is preferentially expressed in the skin epidermal cells rather than in melanocytes. By overexpression of sFRP4 in skin cells in vivo and in vitro, we found that sFRP4 attenuates activation of Wnt signaling, resulting in decrease of melanocytes differentiation in the regenerating hair follicle. Our findings unveiled a new regulator that involves modulating melanocytes differentiation through a paracrine mechanism in hair follicle, supplying a hope for potential therapeutic application to treat skin pigmentation disorders.

Wnt/ß-catenin signaling promotes aging-associated hair graying in mice.

Canities is an obvious sign of aging in mouse and human, shown as hair graying. Melanocytes in the hair follicle show cyclic activity with hair cycling, which transitions from anagen, catagen to telogen. How the hairs turn gray during aging is not completely uncovered. Here, by using immunostaining and LacZ staining in Dct-LacZ mice, we show that ß-catenin is expressed in melanocytes during hair cycling. RT-PCR, western blot and immunostaining show that ß-catenin expression is significantly increased in both anagen and telogen skin of aged mice, when compared to the anagen and telogen skin of young mice, respectively. Overexpression of Wnt10b not only accelerates hair follicle to enter anagen phase, but also promotes melanocytes differentiation in young adult mice (2-month old), with increased ß-catenin expression in melanocytes at the secondary hair germ and matrix region of regenerated hair follicles. Overexpression of Wnt10b also promotes melanocyte progenitor cells differentiation in vitro. Our data suggest that increased Wnt signaling promotes excessive differentiation of melanocytes, leading to exhaustion of melanocyte stem cells and eventually canities in aged mice.

Gsdma3 regulates hair follicle differentiation via Wnt5a-mediated non-canonical Wnt signaling pathway.

Hair follicle is a mini-organ that consists of complex but well-organized structures, which are differentiated from hair follicle progenitor or stem cells. How non-canonical Wnt signaling pathway is involved in regulating hair follicle differentiation remains elusive. Here we showed that Wnt5a regulates hair follicle differentiation through an epithelial-mesenchymal interaction mechanism in mice. We first observed that Wnt5a is expressed in the epithelial and dermal papilla cells during hair follicle development and growth. For the upstream of Wnt5a, RT-PCR and immunohistochemistry staining showed that Wnt5a expression is significantly decreased in the Gsdma3-mutant mice in vivo. Overexpression of Gsdma3 results in a significantly increased expression of Wnt5a in the cultured epidermal cells in vitro. We also checked the downstream factors of Wnt5a by adenovirus-mediated overexpression of Wnt5a to the dermal papilla cells isolated from the mouse whisker. We found that overexpression of Wnt5a suppresses canonical Wnt signaling pathway effectors such as ß-catenin and Lef1. In addition, genes involved in maintaining cell quiescent state are also significantly decreased in their expression to the DP cells which were treated by Wnt5a. Our study indicates that Wnt5a mediates epithelia-expressed Gsdma3 to influence DP cell behaviors, which in turn regulate hair follicle epithelia differentiation in mice.

Wnt/ß-catenin signaling pathway activates melanocyte stem cells in vitro and in vivo.

BACKGROUND: Melanocyte stem cells (McSCs) are the origin of melanocytes that are periodically refreshed in skin and hair follicle. Previously, we reported that Wnt3a could promote melanogenesis, but the mechanism of McSCs activation remains unclear. OBJECTIVE: We aimed to illustrate the roles of Wnt/ß-catenin signaling pathway during McSC activation. METHODS: Adenovirus-mediated overexpression of Wnt3a and Wnt10b were used. In vitro experiments were performed on the immortalized melanocyte progenitor cell line iMC23, wheres in vivo experiments were performed in Dct-LacZ mice. Immunofluorescence and western blot were used to determine the protein expression. RESULTS: Wnt3a promotes the differentiation and melanogenesis of iMC23, by activating Wnt/ß-catenin signaling pathway. Wnt3a induces hair follicle regeneration and McSC activation. Detailed analysis indicats that Wnt3a activated Wnt/ß-catenin signaling pathway, thus promoting the differentiation of McSCs during this process. Wnt10b, another canonical Wnt signaling ligand, induces hair follicle regeneration and McSC activation as well. CONCLUSION: Wnt/ß-catenin signaling pathway activates McSCs both in vitro and in vivo.

Adenovirus-mediated Wnt10b overexpression induces hair follicle regeneration.

Hair follicles periodically undergo regeneration. The balance between activators and inhibitors may determine the time required for telogen hair follicles to reenter anagen. We previously reported that Wnt10b (wingless-type mouse mammary tumor virus integration site family member 10b) could promote the growth of hair follicles in vitro. To unveil the roles of Wnt10b in hair follicle regeneration, we established an in vivo mouse model using intradermal injection. On the basis of this model, we found that Wnt10b could induce the biological switch of hair follicles from telogen to anagen when overexpressed in the skin. The induced hair follicles expressed structure markers and could cycle normally into catagen. Conversely, anagen onset was abrogated by the knockdown of Wnt10b with small interfering RNA (siRNA). The Wnt10b aberrant expression data suggest that it is one of the activators of hair follicle regeneration. The ß-catenin protein is translocated to the nucleus in Wnt10b-induced hair follicles. The biological effects of Wnt10b were abrogated when ß-catenin expression was downregulated with siRNA. These data revealed that Wnt10b might induce hair follicle regeneration in vivo via the enhanced activation of the canonical Wnt signaling pathway. To our knowledge, our data provide previously unreported insights into the regulation of hair follicle cycling and provide potential therapeutic targets for hair follicle-related diseases.

Wnt3a inhibits proliferation but promotes melanogenesis of melan-a cells.

Melanocytes are pigment-producing cells responsible for coloration of skin and hair. Although the importance of Wnt3a in melanocyte development has been well recognized, the role of Wnt3a in mature melanocytes has not been elucidated. This study was conducted to further explore the effects of Wnt3a on melanocyte proliferation and melanogenesis, and to elucidate the possible mechanisms involved. We infected melan-a cells with AdWnt3a to serve as the production source of the Wnt3a protein. MTT assay, 5-bromodeoxyuridine incorporation assay and flow cytometric analysis showed that Wnt3a inhibited the proliferation of melan-a cells and this was associated with decrease of cells in the S phase and increase of cells in the G(1) phase. Melanin content and tyrosinase activity assay revealed that Wnt3a significantly promoted melanogenesis of melan-a cells. Furthermore, western blot analysis showed that Wnt3a upregulated the expression of microphthalmia-associated transcription factor and its downstream target genes, tyrosinase and tyrosinase-related protein 1 in melan-a cells. Collectively, our results suggest that Wnt3a plays an important role in melanocyte homeostasis.