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    Hair follicle bulge-derived stem cells promote tissue regeneration during skin expansion. Cheng Xiaoxi,Yu Zhou,Song Yajuan,Zhang Yu,Du Jing,Su Yingjun,Ma Xianjie Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Skin expansion is widely used in reconstructive surgery to obtain supplemental and optimal skin. Enhancing the regenerative capacity of expanded skin is therefore of great interest. Hair follicle bulge-derived stem cells (HFBSCs) located in hair follicle bulges are closely associated with skin; HFBSC transplantation could promote cutaneous wound repair. However, the effects of transplanted HFBSCs on skin regeneration during expansion remain unclear. The aim of the study was to reveal the potential effects of transplanted HFBSCs in the expanded skin and explore its mechanism. Our results showed higher skin area, tissue weight, epidermal thickness, dermal thickness, proliferating cell count, collagen content, microcirculatory blood flow, blood vessels, and lower retraction ratios were observed in HFBSC-injected rats compared to uninjected controls. Moreover, the transplanted HFBSCs directly contributed to tissue regeneration by differentiating into vascular endothelial cells, epidermal cells, and the outer root sheath cells of hair follicle. Higher expression of EGF, VEGF, bFGF, and TGF-β were observed in HFBSC-injected rats. Our research demonstrated the transplanted HFBSCs could promote skin regeneration by differentiating into various types of skin related cells and by up-regulating the expression of growth factors. Our results could form a basis for the development of novel strategies to enhance regeneration in expanded skin by using HFBSCs. 10.1016/j.biopha.2020.110805
    Bulge Region as a Putative Hair Follicle Stem Cells Niche: A Brief Review. Joulai Veijouye Sanaz,Yari Abazar,Heidari Fatemeh,Sajedi Nayereh,Ghoroghi Moghani Fatemeh,Nobakht Maliheh Iranian journal of public health BACKGROUND:Hair follicle stem cells exist in different sites. Most of the hair follicle stem cells are reside in niche called bulge. Bulge region is located between the opening of sebaceous gland and the attachment site of the arrector pili muscle. METHODS:Data were collected using databases and resources of PubMed, Web of Science, Science Direct, Scopus, MEDLINE and their references from the earliest available published to identify English observational studies on hair follicle bulge region. RESULTS:Bulge stem cells are pluripotent with high proliferative capacity. Specific markers allow the bulge cells to be isolated from mouse or human hair follicle. Stem cells isolated from bulge region are label retaining and slow cycling hence these cells are defined as label-retaining cells. Bulge cell populations, due to their plasticity nature are able to differentiate into distinct linage and could contribute in tissue regeneration. CONCLUSION:The current review discuss about bulge stem cells characteristics and biology including their cycle, location, plasticity, specific markers and regenerative nature. Also the differences between mouse and human hair follicles are investigated.
    LncRNA PlncRNA‑1 regulates proliferation and differentiation of hair follicle stem cells through TGF‑β1‑mediated Wnt/β‑catenin signal pathway. Si Yuan,Bai Jingzhu,Wu Jiang,Li Qun,Mo You,Fang Ruihua,Lai Wei Molecular medicine reports The present study demonstrated that hair follicle stem cells (HFSc) have multidirectional differentiation potential and participate in skin wound healing processes. Long non‑coding RNAs (lncRNAs) are defined as non‑protein coding transcripts longer than 200 nucleotides, which are important in the proliferation and differentiation of cells. The purpose of the present study was to investigate the role of PlncRNA‑1 in the proliferation and differentiation of HFSc. Results revealed that PlncRNA‑1, transforming growth factor (TGF)‑β1, Wnt and β‑catenin expression levels were significantly downregulated in HFSc. PlncRNA‑1 transfection promoted proliferation and differentiation of HFSc. TGF‑β1, Wnt and β‑catenin expression levels were upregulated in HFSc following transfection of PlncRNA‑1. Results demonstrated that TGF‑β1 inhibitor LY2109761 blocked proliferation and differentiation of HFSc promoted by PlncRNA‑1 transfection. In addition, TGF‑β1 inhibitor LY2109761 led to decreased Wnt and β‑catenin expression levels in HFSc. Furthermore, PlncRNA‑1 transfection stimulated the cell cycle of HFSc, whereas TGF‑β1 inhibitor LY2109761 inhibited the cell cycle of HFSc and decreased the acceleration of the cell cycle induced by PlncRNA‑1 transfection. In conclusion, these findings suggest that PlncRNA‑1 may promote proliferation and differentiation of HFSc through upregulation of TGF‑β1‑mediated Wnt/β‑catenin signaling pathway. 10.3892/mmr.2017.7944
    High Runx1 levels promote a reversible, more-differentiated cell state in hair-follicle stem cells during quiescence. Lee Song Eun,Sada Aiko,Zhang Meng,McDermitt David J,Lu Shu Yang,Kemphues Kenneth J,Tumbar Tudorita Cell reports Quiescent hair follicle (HF) bulge stem cells (SCs) differentiate to early progenitor (EP) hair germ (HG) cells, which divide to produce transit-amplifying matrix cells. EPs can revert to SCs upon injury, but whether this dedifferentiation occurs in normal HF homeostasis (hair cycle) and the mechanisms regulating both differentiation and dedifferentiation are unclear. Here, we use lineage tracing, gain of function, transcriptional profiling, and functional assays to examine the role of observed endogenous Runx1 level changes in the hair cycle. We find that forced Runx1 expression induces hair degeneration (catagen) and simultaneously promotes changes in the quiescent bulge SC transcriptome toward a cell state resembling the EP HG fate. This cell-state transition is functionally reversible. We propose that SC differentiation and dedifferentiation are likely to occur during normal HF degeneration and niche restructuring in response to changes in endogenous Runx1 levels associated with SC location with respect to the niche. 10.1016/j.celrep.2013.12.039
    Activated hair follicle stem cells and Wnt/β-catenin signaling involve in pathnogenesis of sebaceous neoplasms. Qiu Weiming,Lei Mingxing,Li Jin,Wang Ning,Lian Xiaohua International journal of medical sciences Sebaceous glands (SGs) undergo cyclic renewal independent of hair follicle stem cells (HFSCs) activation while HFSCs have the potential to differentiate into sebaceous gland cells, hair follicle and epidermal keratinocytes. Abnormalities of sebaceous gland progenitor cells contribute to the development of sebaceous neoplasms, but little is known about the role of HFSCs during sebaceous neoplasm development. Here, using dimethylbenzanthracene (DMBA) plus 12-o-tetradecanoyl phorbol-13-acetate (TPA) treatment developing sebaceous neoplasms (SNs) were identified with H&E and Oil red O staining. And then the molecular expression and activation of HFSCs and was characterized by immunostaining. Wnt10b/β-catenin signaling molecular which is important for activation of HFSCs were detected by immunostaining. We found hair follicle and epidermal cell markers were expressed in sebaceous neoplasms. Furthermore, SOX-9 and CD34-positive HFSCs were located in the basal layer of sebaceous lobules within the sebaceous neoplasms. Many appear to be in an active state. Finally, Wnt10b/β-catenin signaling was activated within the basal cells of sebaceous lobules in the sebaceous neoplasms. Collectively, our findings suggest that the abnormal activation of both HFSCs and Wnt10b/β-catenin signaling involves in the development of sebaceous neoplasms. 10.7150/ijms.8383
    Human hair follicle-derived mesenchymal stem cells: Isolation, expansion, and differentiation. Wang Bo,Liu Xiao-Mei,Liu Zi-Nan,Wang Yuan,Han Xing,Lian Ao-Bo,Mu Ying,Jin Ming-Hua,Liu Jin-Yu World journal of stem cells Hair follicles are easily accessible skin appendages that protect against cold and potential injuries. Hair follicles contain various pools of stem cells, such as epithelial, melanocyte, and mesenchymal stem cells (MSCs) that continuously self-renew, differentiate, regulate hair growth, and maintain skin homeostasis. Recently, MSCs derived from the dermal papilla or dermal sheath of the human hair follicle have received attention because of their accessibility and broad differentiation potential. In this review, we describe the applications of human hair follicle-derived MSCs (hHF-MSCs) in tissue engineering and regenerative medicine. We have described protocols for isolating hHF-MSCs from human hair follicles and their culture condition in detail. We also summarize strategies for maintaining hHF-MSCs in a highly proliferative but undifferentiated state after repeated passages, including supplementation of growth factors, 3D suspension culture technology, and 3D aggregates of MSCs. In addition, we report the potential of hHF-MSCs in obtaining induced smooth muscle cells and tissue-engineered blood vessels, regenerated hair follicles, induced red blood cells, and induced pluripotent stem cells. In summary, the abundance, convenient accessibility, and broad differentiation potential make hHF-MSCs an ideal seed cell source of regenerative medical and cell therapy. 10.4252/wjsc.v12.i6.462
    Induction of hair follicle dermal papilla cell properties in human induced pluripotent stem cell-derived multipotent LNGFR(+)THY-1(+) mesenchymal cells. Veraitch Ophelia,Mabuchi Yo,Matsuzaki Yumi,Sasaki Takashi,Okuno Hironobu,Tsukashima Aki,Amagai Masayuki,Okano Hideyuki,Ohyama Manabu Scientific reports The dermal papilla (DP) is a specialised mesenchymal component of the hair follicle (HF) that plays key roles in HF morphogenesis and regeneration. Current technical difficulties in preparing trichogenic human DP cells could be overcome by the use of highly proliferative and plastic human induced pluripotent stem cells (hiPSCs). In this study, hiPSCs were differentiated into induced mesenchymal cells (iMCs) with a bone marrow stromal cell phenotype. A highly proliferative and plastic LNGFR(+)THY-1(+) subset of iMCs was subsequently programmed using retinoic acid and DP cell activating culture medium to acquire DP properties. The resultant cells (induced DP-substituting cells [iDPSCs]) exhibited up-regulated DP markers, interacted with human keratinocytes to up-regulate HF related genes, and when co-grafted with human keratinocytes in vivo gave rise to fibre structures with a hair cuticle-like coat resembling the hair shaft, as confirmed by scanning electron microscope analysis. Furthermore, iDPSCs responded to the clinically used hair growth reagent, minoxidil sulfate, to up-regulate DP genes, further supporting that they were capable of, at least in part, reproducing DP properties. Thus, LNGFR(+)THY-1(+) iMCs may provide material for HF bioengineering and drug screening for hair diseases. 10.1038/srep42777
    Embryonic attenuated Wnt/β-catenin signaling defines niche location and long-term stem cell fate in hair follicle. Xu Zijian,Wang Wenjie,Jiang Kaiju,Yu Zhou,Huang Huanwei,Wang Fengchao,Zhou Bin,Chen Ting eLife Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/β-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/β-catenin signaling. Soon afterwards, a region with attenuated Wnt/β-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive long-term hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/β-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation. 10.7554/eLife.10567
    Exosomal Micro RNAs Derived from Dermal Papilla Cells Mediate Hair Follicle Stem Cell Proliferation and Differentiation. Yan Hailong,Gao Ye,Ding Qiang,Liu Jiao,Li Yan,Jin Miaohan,Xu Han,Ma Sen,Wang Xiaolong,Zeng Wenxian,Chen Yulin International journal of biological sciences Recent studies have demonstrated that dermal papilla cell-derived exosomes (DPC-Exos) promote the anagen stage of hair follicle (HF) growth and delay the catagen stage. However, the roles of DPC-Exos in regulating hair follicle stem cell (HFSC) quiescence and activation remain unknown. Here, we found that HFSC differentiation was induced by co-culture with DPCs, and that DPC-Exos attached to the surface of HFSCs. Using micro RNA (miRNA) high-throughput sequencing, we identified 111 miRNAs that were significantly differentially expressed between DPC-Exos and DPCs, and the predicted target genes of the top 34 differentially expressed miRNAs indicated that DPC-Exos regulate HFSCs proliferation and differentiation via genes involved in cellular signal transduction, fatty acid expression regulation, and cellular communication. The overexpression of miR-22-5p indicated that it negatively regulates HFSC proliferation and was revealed as the direct target gene of miR-22-5p. We therefore propose the miR-22-5p- axis as a novel pathway regulating HFSC proliferation. 10.7150/ijbs.33233
    Concise Review: Mechanisms of Quiescent Hair Follicle Stem Cell Regulation. Yi Rui Stem cells (Dayton, Ohio) Maintaining a pool of adult stem cells is essential for tissue homeostasis and wound repair. In mammalian tissues, notably hair follicles, blood, and muscle, stem cells acquire quiescence and infrequently divide for self-renewal. Mechanistic understanding of stem cell quiescence is critical for applying these multipotent cells in regenerative medicine and interrogating their roles in human diseases such as cancer. Quiescent and dividing epithelial stem cells located in hair follicle are conspicuously organized in a spatiotemporally specific manner, allowing them to be studied at a considerable depth. Recent advancements in mouse genetics, genomics, and imaging have revealed unprecedented insights into establishment, maintenance, and regulation of quiescent hair follicle stem cells. This concise review summarizes the progress with a focus on mechanisms mediated by signaling pathways and transcription factors and discusses their implications in the understanding of stem cell biology. Stem Cells 2017;35:2323-2330. 10.1002/stem.2696
    Postnatal neural crest stem cells from hair follicle interact with nerve tissue in vitro and in vivo. Kosykh Anastasiia,Beilin Arkadii,Sukhinich Kirill,Vorotelyak Ekaterina Tissue & cell Neural crest stem cells that located in the postnatal hair follicle (HF-NCSC) are considered a promising tool for treatment of nervous system diseases and injuries. It is well known that HF-NCSC can be used in the spinal cord and sciatic nerve reparation but their ability to restore brain structures is poorly studied. In this article we are investigating the interaction between HF-NCSC and a nerve tissue (embryonic and adult). We have found out that HF-NCSC isolated from adult mice grow and differentiate in accordance with the mouse embryo developmental stage when co-cultured with the embryonic nerve tissue. The HF-NCSC migration is slower in the late embryonic tissue co-culture system compared to the early one. This phenomenon is related to the motor function of the cells but not to their proliferation level. We have demonstrated that the embryonic nerve tissue maintains HF-NCSC an undifferentiated status, while an adult brain tissue inhibits the cell proliferation and activates the differentiation processes. Besides, HF-NCSC pre-differentiated into the neuronal direction shows a higher survival and migration rate after the transplantation into the adult brain tissue compared to the undifferentiated HF-NCSC. Thus, we have investigated the postnatal HF-NCSC response to the nerve tissue microenvironment to analyze their possible application to the brain repair processes. 10.1016/j.tice.2018.08.005
    Skin vasculature and hair follicle cross-talking associated with stem cell activation and tissue homeostasis. Li Kefei Nina,Jain Prachi,He Catherine Hua,Eun Flora Chae,Kang Sangjo,Tumbar Tudorita eLife Skin vasculature cross-talking with hair follicle stem cells (HFSCs) is poorly understood. Skin vasculature undergoes dramatic remodeling during adult mouse hair cycle. Specifically, a horizontal plexus under the secondary hair germ (HPuHG) transiently neighbors the HFSC activation zone during the quiescence phase (telogen). Increased density of HPuHG can be induced by reciprocal mutations in the epithelium () and endothelium () in adult mice, and is accompanied by prolonged HFSC quiescence and by delayed entry and progression into the hair growth phase (anagen). Suggestively, skin vasculature produces BMP4, a well-established HFSC quiescence-inducing factor, thus contributing to a proliferation-inhibitory environment near the HFSC. Conversely, the HFSC activator Runx1 regulates secreted proteins with previously demonstrated roles in vasculature remodeling. We suggest a working model in which coordinated remodeling and molecular cross-talking of the adult epithelial and endothelial skin compartments modulate timing of HFSC activation from quiescence for proper tissue homeostasis of adult skin. 10.7554/eLife.45977
    Interactions Between Epidermal Keratinocytes, Dendritic Epidermal T-Cells, and Hair Follicle Stem Cells. Badarinath Krithika,Dutta Abhik,Hegde Akshay,Pincha Neha,Gund Rupali,Jamora Colin Methods in molecular biology (Clifton, N.J.) The interplay of immune cells and stem cells in maintaining skin homeostasis and repair is an exciting new frontier in cutaneous biology. With the growing appreciation of the importance of this new crosstalk comes the requirement of methods to interrogate the molecular underpinnings of these leukocyte-stem cell interactions. Here we describe how a combination of FACS, cellular coculture assays, and conditioned media treatments can be utilized to advance our understanding of this emerging area of intercellular communication between immune cells and stem cells. 10.1007/7651_2018_155
    Treg-Cell Control of a CXCL5-IL-17 Inflammatory Axis Promotes Hair-Follicle-Stem-Cell Differentiation During Skin-Barrier Repair. Mathur Anubhav N,Zirak Bahar,Boothby Ian C,Tan Madge,Cohen Jarish N,Mauro Thea M,Mehta Pooja,Lowe Margaret M,Abbas Abul K,Ali Niwa,Rosenblum Michael D Immunity Restoration of barrier-tissue integrity after injury is dependent on the function of immune cells and stem cells (SCs) residing in the tissue. In response to skin injury, hair-follicle stem cells (HFSCs), normally poised for hair generation, are recruited to the site of injury and differentiate into cells that repair damaged epithelium. We used a SC fate-mapping approach to examine the contribution of regulatory T (Treg) cells to epidermal-barrier repair after injury. Depletion of Treg cells impaired skin-barrier regeneration and was associated with a Th17 inflammatory response and failed HFSC differentiation. In this setting, damaged epithelial cells preferentially expressed the neutrophil chemoattractant CXCL5, and blockade of CXCL5 or neutrophil depletion restored barrier function and SC differentiation after epidermal injury. Thus, Treg-cell regulation of localized inflammation enables HFSC differentiation and, thereby, skin-barrier regeneration, with implications for the maintenance and repair of other barrier tissues. 10.1016/j.immuni.2019.02.013
    The battle of the bulge: re-evaluating hair follicle stem cells in wound repair. Garcin Clare L,Ansell David M Experimental dermatology The hair follicle has an established role in wound re-epithelialisation, a phenomenon that has been appreciated since at least the first half of the last century. The bulge niche, one location of hair follicle epithelial stem cells has been of particular interest to researchers over recent years, with numerous studies showing its ability to directly contribute to epidermal repair. However, recent work has highlighted other progenitor regions of the hair follicle that appear to act as stem cells during epidermal repair. In addition, several studies within the last 12 months have questioned the importance of the bulge during re-epithelialisation, producing conflicting literature. Here we provide a new model to demonstrate how several important differences in experimental design between studies could account for these seemingly opposing findings, which may have implications for how future studies are conducted. 10.1111/exd.13184
    Functional Hair Follicle Regeneration by the Rearrangement of Stem Cells. Asakawa Kyosuke,Toyoshima Koh-Ei,Tsuji Takashi Methods in molecular biology (Clifton, N.J.) Hair follicles develop from the ectoderm in embryos and cyclically regenerate using proper spatiotemporal signaling molecules, which are conserved in organogenesis during adulthood. Previously, we demonstrated that bioengineered hair follicle germs could regenerate functional hair follicles via a three-dimensional cell manipulation technique, which we named the "organ germ method ." We could also regulate the type of hair follicle and pigmentation with correct structures by rearranging the source of the cells. In this article, we describe a detailed protocol for the regeneration of functional hair follicles and their stem cell niches by the rearrangement of embryonic or adult hair follicle-derived epithelial and mesenchymal cells. 10.1007/978-1-4939-6949-4_9
    Hair Follicle Bulge Stem Cells Appear Dispensable for the Acute Phase of Wound Re-epithelialization. Garcin Clare L,Ansell David M,Headon Denis J,Paus Ralf,Hardman Matthew J Stem cells (Dayton, Ohio) 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. 10.1002/stem.2289
    Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications. Vasyliev R G,Rodnichenko A E,Gubar O S,Zlatska A V,Gordiienko I M,Novikova S N,Zubov D O Experimental oncology AIM:The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. MATERIALS AND METHODS:Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. RESULTS:Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2+Sox10+Nestin+CD73CD90CD105CD140a+CD140b+CD146CD166+CD271+CD349+ CD34CD45CD56-HLA-DR, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. CONCLUSION:The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40-200·10 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice.Key Words: regenerative medicine, neural crest, hair follicle, neural crest-derived multipotent stem cells, directed differentiation, large-scale expansion.
    loss in Lgr5 hair follicle stem cells promotes SCC development. Chen Haiyan,Wang Xusheng,Chen Yu,Han Jimin,Kong Deqiang,Zhu Meizhong,Fu Xiaobing,Wu Yaojiong Theranostics Accumulating data support that tissue stem cells give rise to cancer cells. Hair follicle stem cells (HFSCs) undergo cyclic quiescence and activation and may sever as the origin of cutaneous squamous cell carcinoma (SCC). is a tumor suppressor gene that is frequently mutated in hereditary cancer syndromes such as Cowden disease, which is featured with papillomatosis in cutaneous tissues and hyperkeratosis in the acral region of the skin. Additionally, mice with keratinocyte-specific deficiency ( mice) show epidermal hyperplasia and spontaneous tumor formation. However, the impact of mutation in HFSCs, such as in Lgr5 HFSCs, on SCC formation is unclear. : We established experiments with wildtype and mice, and used DMBA/TPA two-stage skin carcinogenesis model to explore the effect of loss in Lgr5 HFSCs of 3 weeks old mice in skin carcinogenesis. experiments (cell culture and protein expression analysis) are employed to investigate molecular mechanisms involved. : loss in Lgr5 HFSCs promoted SCC formation, which was attenuated in mice. Notably, β-catenin loss in Lgr5 HFSCs decreased the formation of SCC. In addition, loss in cultured epidermal stem cells upregulated the levels of both phospho-Akt and β-catenin. : loss in Lgr5 cells induced Akt/β-catenin signaling, and SCCs can subsequently be raised as progeny from these primed Lgr5 stem cells. 10.7150/thno.35467
    Ex Vivo Imaging and Genetic Manipulation of Mouse Hair Follicle Bulge Stem Cells. Haensel Daniel,McNeil Melissa A,Dai Xing Methods in molecular biology (Clifton, N.J.) 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. 10.1007/7651_2018_136
    Highly Efficient Neural Differentiation of CD34-Positive Hair-Follicle-Associated Pluripotent Stem Cells Induced by Retinoic Acid and Serum-Free Medium. Sagha Mohsen,Najafzadeh Nowruz Methods in molecular biology (Clifton, N.J.) Neural differentiation of hair-follicle-associated pluripotent (HAP) stem cells residing in the bulge area is a promising autologous source for stem cell therapy. In the present chapter, we describe the identification and enrichment of CD34(+) HAP stem cells by magnetic-activated cell sorting (MACS), and induce them to differentiate into neuronal and glial cells using defined neural-induction media. The different neural cell populations arising during in vitro differentiation from HAP stem cells are characterized by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry assay. 10.1007/978-1-4939-3786-8_17
    Nestin-Expressing Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Promote Whisker Sensory-Nerve Growth in Long-Term 3D-Gelfoam® Histoculture. Mii Sumiyuki,Duong Jennifer,Tome Yasunori,Uchugonova Aisada,Liu Fang,Amoh Yasuyuki,Saito Norimitsu,Katsuoka Kensei,Hoffman Robert M Methods in molecular biology (Clifton, N.J.) Mouse whiskers containing hair-follicle-associated pluripotent (HAP) stem cells, from nestin-driven green fluorescent protein (ND-GFP) transgenic mice, were placed in 3D histoculture supported by Gelfoam(®). β-III tubulin-positive fibers, consisting of ND-GFP-expressing HAP stem cells, extended up to 500 mm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F-actin indicating they were growing axons. The growing whisker sensory nerve was highly enriched in ND-GFP HAP stem cells which appeared to play a major role in its elongation and interaction with other nerves placed in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results suggested that a major function of HAP stem cells in the hair follicle is for growth of the hair follicle sensory nerve. 10.1007/978-1-4939-3786-8_6
    Cutaneous applied nano-ZnO reduce the ability of hair follicle stem cells to differentiate. Ge Wei,Zhao Yong,Lai Fang-Nong,Liu Jing-Cai,Sun Yuan-Chao,Wang Jun-Jie,Cheng Shun-Feng,Zhang Xi-Feng,Sun Li-Lan,Li Lan,Dyce Paul W,Shen Wei Nanotoxicology The ability of metal oxide nanoparticles to penetrate the skin has aroused a great deal of interest during the past decade due to concerns over the safety of topically applied sunscreens that contain physical UV-resistant metal particles, such as nano-Zinc oxide (nZnO). Previous studies demonstrate that metal oxide nanoparticles accumulate in skin furrows and hair follicles following topical application while little is known about the consequence of these nanoparticles on skin homeostasis. The current investigation tested the effects of nZnO (0.5 mg/day mouse) on hair follicle physiology. Topical application of Vaseline containing nZnO, bulk ZnO (bZnO), or ionized Zn to newborn mice vibrissa pad over a period of 7 consecutive days revealed that nZnO accumulated within hair follicles, and this induced the apoptosis of hair follicle stem cells (HFSCs). In vitro studies also indicated that nZnO exposure caused obvious DNA damage and induced apoptosis in HFSCs. Furthermore, it was found that nZnO exposure perturbed genes associated with HFSC apoptosis, cell communication, and differentiation. HFSCs transplantation assay demonstrated that the potential of HFSCs to differentiate was reduced. This investigation indicates a potential risk of topically applied ZnO nanoparticles on skin homeostasis. 10.1080/17435390.2017.1310947
    Differential distribution of the epigenetic marker 5-hydroxymethylcytosine occurs in hair follicle stem cells during bulge activation. Leavitt Danielle,Wells Michael,Abarzua Phammela,Murphy George F,Lian Christine G Journal of cutaneous pathology BACKGROUND:Hair follicle (HF) cycling is dependent upon activation and differentiation of an epithelial subpopulation of cells with stem-like characteristics. These cells express cytokeratin 15 (CK15) and are sequestered within a specialized niche termed the follicular bulge. The pathways that mediate bulge activation are poorly understood, although growing evidence suggests a role for epigenetic events. METHODS:Here we investigated murine and human HFs to determine whether a recently described epigenetic hydroxymethylation marker, 5-hmC, known to mediate cell growth and differentiation, may play a role in bulge activation. RESULTS:We found the bulge region of murine HFs to show variable 5-hmC distribution within the nuclei of CK15-positive stem cells during early anagen, a pattern that was not associated with resting stem cells of telogen follicles, which did not express 5-hmC. Moreover, during phases of early anagen that were induced in an organ culture model, spatial alterations in bulge stem cell 5-hmC reactivity, as assessed by dual labeling, were noted. CONCLUSIONS:These preliminary findings suggest that 5-hmC may play a dynamic role in bulge activation during anagen growth, and provide a foundation for further experimental inquiry into epigenomic regulation of HF stem cells. 10.1111/cup.13434
    Beta-catenin can induce hair follicle stem cell differentiation into transit-amplifying cells through c-myc activation. Shen Qiong,Yu Weirong,Fang Yong,Yao Min,Yang Penggao Tissue & cell Hair follicle stem cells play important roles in maintaining homeostasis and skin tissue self-renewal. Transit-amplifying cells represent the transition of cells from hair follicle stem cells into differentiated epidermal cells. Thus far, the signaling pathway and the molecular biological mechanism that regulate the proliferation and differentiation of hair follicle stem cells remain unclear. In this paper, we studied the relationship between β-catenin and c-myc during the process of the differentiation of hair follicle stem cells into transit-amplifying cells. Based on our results, the expression of β-catenin can activate the nuclear gene c-myc and regulate the expression of transit-amplifying cell markers K15, K19, a6-integrin and β1-integrin, indicating that β-catenin is involved in the transformation process from hair follicle stem cells to transit-amplifying cells and suggesting that β-catenin plays an important biological role in the induction of this differentiation process. 10.1016/j.tice.2016.12.005
    BMP2-mediated PTEN enhancement promotes differentiation of hair follicle stem cells by inducing autophagy. Cai Bingjie,Zheng Yunpeng,Yan Jiadi,Wang Junmin,Liu Xiaojun,Yin Guangwen Experimental cell research The proliferation and differentiation of hair follicle stem cells (HFSCs) is regulated by several signaling pathways, including BMP and PTEN. Therefore, this study intended to clarify the potential effects of two such regulators, BMP2 and PTEN, on HFSC differentiation. HFSCs were subjected to BMP2, noggin (BMP2 ligand inhibitor), rapamycin (Rapa, autophagy inducer), 3-methyladenine (3-MA, autophagy inhibitor), or shRNA against PTEN. The differentiation of HFSCs was evaluated using oil red O staining and autophagy was assessed using the transmission electron microscope. Then expression of epidermal differentiation marker (K10 and involucrin), adipogenic markers (PPAR-γ2, aP2, perilipin2, and Adipoq), keratinocyte-specific marker (K15), proliferation-related markers (PCNA and Ki67) and autophagy-related factors (Atg5, Atg7, Atg12, Beclin-1 and LC3-II/LC3-I) was examined by RT-qPCR and Western blot analysis. Next, HFSCs were treated with 3-MA, or shRNA against Atg5 or Atg7 to verify the effect of autophagy on differentiation of BMP2-treated HFSCs. Finally, the effect of BMP2 on HFSC differentiation was verified by a mouse wound model. HFSCs overexpressing BMP2 exhibited elevated expression of epidermal differentiation marker, adipogenic markers and autophagy-related factors but inhibited expression of keratinocyte-specific marker and proliferation-related markers. Furthermore, we found that PTEN promoted the differentiation of BMP2-treated HFSCs by inducing autophagy. In vivo experiments further confirmed the roles of BMP2/PTEN on differentiation of HFSCs. Taken together, BMP2 up-regulated PTEN and consequently induced autophagy to facilitate HFSC differentiation. 10.1016/j.yexcr.2019.111647
    Foxi3 Deficiency Compromises Hair Follicle Stem Cell Specification and Activation. Shirokova Vera,Biggs Leah C,Jussila Maria,Ohyama Takahiro,Groves Andrew K,Mikkola Marja L Stem cells (Dayton, Ohio) The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ (HG). Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here, we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary HG marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary HG activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. Stem Cells 2016;34:1896-1908. 10.1002/stem.2363
    Cryopreservation of Hair-Follicle Associated Pluripotent (HAP) Stem Cells Maintains Differentiation and Hair-Growth Potential. Hoffman Robert M,Kajiura Satoshi,Cao Wenluo,Liu Fang,Amoh Yasuyuki Advances in experimental medicine and biology Hair follicles contain nestin-expressing pluripotent stem cells which originate above the bulge area of the follicle, below the sebaceous gland. We have termed these cells hair follicle-associated pluripotent (HAP) stem cells. We have established efficient cryopreservation methods of the hair follicle that maintain the pluripotency of HAP stem cells as well as hair growth. We cryopreserved the whole hair follicle by slow-rate cooling in TC-Protector medium or in DMSO-containing medium and storage in liquid nitrogen or at -80 °C. After thawing and culture of the cryopreserved whisker follicles, growing HAP stem cells formed hair spheres. The hair spheres contained cells that differentiated to neurons, glial cells, and other cell types. The hair spheres derived from slow-cooling cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. We have also previously demonstrated that cryopreserved mouse whisker hair follicles maintain their hair-growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. DMSO-cryopreserved hair follicles also maintained the HAP stem cells, evidenced by P75 expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair-shaft growth of cryopreserved hair follicles. HAP stem cells can be used for nerve and spinal-cord repair. This biobanking of hair follicles can allow each patient the potential for their own stem cell use for regenerative medicine or hair transplantation. 10.1007/978-3-319-45457-3_16
    Introduction to Hair-Follicle-Associated Pluripotent Stem Cells. Hoffman Robert M Methods in molecular biology (Clifton, N.J.) Nestin-expressing stem cells of the hair follicle, discovered by our laboratory, have been shown to be able to form outer-root sheaths of the follicle as well as neurons and many other non-follicle cell types. We have termed the nestin-expressing stem cells of the hair follicle as hair-follicle-associated pluripotent (HAP) stem cells. We have shown that the HAP stem cells from the hair follicle can effect the repair of peripheral nerve and spinal cord injury. The hair follicle stem cells differentiate into neuronal and glial cells after transplantation to the injured peripheral nerve and spinal cord, and enhance injury repair and locomotor recovery. When the excised hair follicle with its nerve stump was placed in Gelfoam(®) 3D histoculture, HAP stem cells grew and extended the hair follicle nerve which consisted of βIII-tubulin-positive fibers with F-actin expression at the tip. These findings indicate that βIII-tubulin-positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in HAP stem cells, which appeared to play a major role in its elongation and interaction with other nerves in 3D Gelfoam(®) histoculture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. These results suggest that a major function of the HAP stem cells in the hair follicle is for growth of the follicle sensory nerve. Recently, we have shown that HAP stem cells can differentiate into beating cardiac muscle cells. HAP stem cells have critical advantages for regenerative medicine over embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in that they are highly accessible from each patient, thereby eliminating immunological issues since they are autologous, require no genetic manipulation, are non-tumorigenic, and do not present ethical issues. 10.1007/978-1-4939-3786-8_1
    Deficiency of Crif1 in hair follicle stem cells retards hair growth cycle in adult mice. Shin Jung-Min,Ko Jung-Woo,Choi Chong-Won,Lee Young,Seo Young-Joon,Lee Jeung-Hoon,Kim Chang-Deok PloS one Hair growth is the cyclically regulated process that is characterized by growing phase (anagen), regression phase (catagen) and resting phase (telogen). Hair follicle stem cells (HFSCs) play pivotal role in the control of hair growth cycle. It has been notified that stem cells have the distinguished metabolic signature compared to differentiated cells, such as the preference to glycolysis rather than mitochondrial respiration. Crif1 is a mitochondrial protein that regulates the synthesis and insertion of oxidative phosphorylation (OXPHOS) polypeptides to inner membrane of mitochondria. Several studies demonstrate that tissue-specific knockout of Crif1 leads to mitochondrial dysfunction. In this study, we investigated the effect of mitochondrial dysfunction in terms of Crif1 deficiency on the hair growth cycle of adult mice. We created two kinds of inducible conditional knockout (icKO) mice. In epidermal specific icKO mice (Crif1 K14icKO), hair growth cycle was significantly retarded compared to wild type mice. Similarly, HFSC specific icKO mice (Crif1 K15icKO) showed significant retardation of hair growth cycle in depilation-induced anagen model. Interestingly, flow cytometry revealed that HFSC populations were maintained in Crif1 K15icKO mice. These results suggest that mitochondrial function in HFSCs is important for the progression of hair growth cycle, but not for maintenance of HFSCs. 10.1371/journal.pone.0232206
    Immunohistochemical study of hair follicle stem cells in regenerated hair follicles induced by Wnt10b. Zhang Yiming,Xing Yizhan,Guo Haiying,Ma Xiaogen,Li Yuhong International journal of medical sciences 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. 10.7150/ijms.16118
    Epidermal Stem Cells in Hair Follicle Cycling and Skin Regeneration: A View From the Perspective of Inflammation. Morgun Elena I,Vorotelyak Ekaterina A Frontiers in cell and developmental biology 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 T 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. 10.3389/fcell.2020.581697
    A crucial role of fibroblast growth factor 2 in the differentiation of hair follicle stem cells toward endothelial cells in a STAT5-dependent manner. Cai Bingjie,Zheng Yunpeng,Liu Xiaojun,Yan Jiadi,Wang Junmin,Yin Guangwen Differentiation; research in biological diversity Fibroblast growth factor (FGF2) is reported to affect the proliferation, differentiation, and survival abilities of stem cells. In this study, we hypothesize that FGF2 might promote the differentiation of hair follicle stem cell (HFSCs) into endothelial cells (ECs), in a manner dependent on STAT5 activation. We first treated human HFSCs with recombinant human FGF2 to determine the involvement of FGF2 in the differentiation of HFSCs. Then the expression of EC-specific markers including von Willebrand factor (vWF), VE-cadherin, CD31, FLT-1, KDR and Tie2 was evaluated using immunofluorescence and flow cytometry, while the expression of HFSC-specific markers such as K15, K19, Lgr5, Sox9 and Lhx2 was determined by flow cytometry. Next, in vitro tube formation was performed to confirm the function of FGF2, and low-density lipoprotein (LDL) uptake by ECs and HFSCs was studied by Dil-acetylated LDL assay. In addition, we transduced FGF2-treated HFSCs with constitutive-active or dominant-negative STAT5A adenovirus vectors. FGF2 up-regulated the expression of EC-specific markers, and promoted the differentiation of HFSCs into ECs, tube formation and LDL uptake. The phosphorylated STAT5 was translocated into the nucleus of HFSCs after FGF2 treatment, but this translocation was blocked by the dominant-negative STAT5A mutant. FGF2 increased the differentiation potential through the activation of STAT5 in vivo. Taken together, we find that FGF2 promotes the differentiation of HFSCs into ECs via activated STAT5, which gives a new perspective on the role of FGF2 in the development of ischemic vascular disease. 10.1016/j.diff.2019.10.004
    Identification and Characterization of Hair Follicle Stem Cells. Wang Xiaoyang,Xing Yizhan,Li Yuhong Methods in molecular biology (Clifton, N.J.) 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. 10.1007/978-1-4939-8697-2_5
    External light activates hair follicle stem cells through eyes via an ipRGC-SCN-sympathetic neural pathway. Fan Sabrina Mai-Yi,Chang Yi-Ting,Chen Chih-Lung,Wang Wei-Hung,Pan Ming-Kai,Chen Wen-Pin,Huang Wen-Yen,Xu Zijian,Huang Hai-En,Chen Ting,Plikus Maksim V,Chen Shih-Kuo,Lin Sung-Jan Proceedings of the National Academy of Sciences of the United States of America Changes in external light patterns can alter cell activities in peripheral tissues through slow entrainment of the central clock in suprachiasmatic nucleus (SCN). It remains unclear whether cells in otherwise photo-insensitive tissues can achieve rapid responses to changes in external light. Here we show that light stimulation of animals' eyes results in rapid activation of hair follicle stem cells with prominent hair regeneration. Mechanistically, light signals are interpreted by M1-type intrinsically photosensitive retinal ganglion cells (ipRGCs), which signal to the SCN via melanopsin. Subsequently, efferent sympathetic nerves are immediately activated. Increased norepinephrine release in skin promotes hedgehog signaling to activate hair follicle stem cells. Thus, external light can directly regulate tissue stem cells via an ipRGC-SCN autonomic nervous system circuit. Since activation of sympathetic nerves is not limited to skin, this circuit can also facilitate rapid adaptive responses to external light in other homeostatic tissues. 10.1073/pnas.1719548115
    Up-regulated lncRNA5322 elevates MAPK1 to enhance proliferation of hair follicle stem cells as a ceRNA of microRNA-19b-3p. Cai Bingjie,Wang Xinxin,Liu Hongtao,Ma Shanshan,Zhang Kun,Zhang Yanting,Li Qinghua,Wang Junmin,Yao Minghao,Guan Fangxia,Yin Guangwen Cell cycle (Georgetown, Tex.) Hair follicle stem cells (HFSCs), located in the bulge region of the follicle, maintain hair follicle growth and cycling. Long non-coding RNAs (lncRNAs), non-protein coding transcripts, are widely known to play critical roles in differentiation and proliferation of stem cells. Therefore, the current study aimed to explore the regulatory roles of lncRNA5322 in HFSCs proliferation and the underlying regulatory mechanisms. Initially, the expression patterns of lncRNA5322 and microRNA-19b-3p (miR-19b-3p) in HFSCs were detected. Subsequently, gain-and loss-of-functions analyses were conducted to explore the roles of lncRNA5322, miR-19b-3p and mitogen-activated protein kinase 1 (MAPK1) in cell proliferation, colony formation and apoptosis of HFSCs, with the expression of cyclin-dependent kinase (CDK)1 and CDK2 examined. Also, the interaction relationships among lncRNA5322, miR-19b-3p and MAPK1 were explored. Furthermore, a mouse model was established to detect the roles of lncRNA5322, miR-19b-3p, and MAPK1 in wound contraction and epidermal regeneration. Over-expressed lncRNA5322 was found to promote proliferation, colony formation ability but inhibit apoptosis of HFSCs, in addition to up-regulation of the expression of CDK1 and CDK2. LncRNA5322 was found to act as a ceRNA of miR-19b-3p which directly targeted MAPK1. Furthermore, up-regulation of lncRNA5322 enhanced wound contraction and epidermal regeneration by increasing the expression of MAPK1 through functioning as a ceRNA of miR-19b-3p. In summary, the results in this study suggested that lncRNA5322 serves as a ceRNA of miR-19b-3p to elevate the expression of MAPK1, ultimately promoting HFSCs proliferation, wound contraction and epidermal regeneration of mouse model. 10.1080/15384101.2019.1624111
    Hair Follicle-Associated Pluripotent(HAP) Stem Cells. Hoffman Robert M,Amoh Yasuyuki Progress in molecular biology and translational science The hair follicle has been known, since 1990, to contain stem cells located in the bulge area. In 2003, we reported a new type of stem cell in the hair follicle that expresses the brain stem-cell marker nestin. We have termed these cells as hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells can differentiate into neuronal and glial cells, beating cardiac-muscle cells, and other cell types in culture. HAP stem cells can be used for nerve and spinal-cord repair such that locomotor activity is recovered. A major function in situ of the HAP stem cells is for growth of the hair follicle sensory nerve. HAP stem cells have critical advantages over embryonic stem cells and induced pluripotent stem (IPS) cells for regenerative medicine in that they are highly accessible, require no genetic manipulation, are nontumorigenic, and do not present ethical issues. 10.1016/bs.pmbts.2018.09.001
    VEGF165 induces differentiation of hair follicle stem cells into endothelial cells and plays a role in in vivo angiogenesis. Quan Renfu,Du Weibin,Zheng Xuan,Xu Shichao,Li Qiang,Ji Xing,Wu Ximei,Shao Rongxue,Yang Disheng Journal of cellular and molecular medicine Within the vascular endothelial growth factor (VEGF) family of five subtypes, VEGF165 secreted by endothelial cells has been identified to be the most active and widely distributed factor that plays a vital role in courses of angiogenesis, vascularization and mesenchymal cell differentiation. Hair follicle stem cells (HFSCs) can be harvested from the bulge region of the outer root sheath of the hair follicle and are adult stem cells that have multi-directional differentiation potential. Although the research on differentiation of stem cells (such as fat stem cells and bone marrow mesenchymal stem cells) to the endothelial cells has been extensive, but the various mechanisms and functional forms are unclear. In particular, study on HFSCs' directional differentiation into vascular endothelial cells using VEGF165 has not been reported. In this study, VEGF165 was used as induction factor to induce the differentiation from HFSCs into vascular endothelial cells, and the results showed that Notch signalling pathway might affect the differentiation efficiency of vascular endothelial cells. In addition, the in vivo transplantation experiment provided that HFSCs could promote angiogenesis, and the main function is to accelerate host-derived neovascularization. Therefore, HFSCs could be considered as an ideal cell source for vascular tissue engineering and cell transplantation in the treatment of ischaemic diseases. 10.1111/jcmm.13089
    Hair-follicle-associated pluripotent stem cells derived from cryopreserved intact human hair follicles sustain multilineage differentiation potential. Obara Koya,Tohgi Natsuko,Mii Sumiyuki,Hamada Yuko,Arakawa Nobuko,Aki Ryoichi,Singh Shree Ram,Hoffman Robert M,Amoh Yasuyuki Scientific reports The bulge area of the hair follicle contains hair-follicle-associated pluripotent (HAP) stem cells. Here, we present effective cryopreservation procedures of the human hair follicle that preserve the differentiation potential of HAP stem cells. Whole hair follicles isolated from human scalp were cryopreserved by a slow-rate cooling medium and stored in liquid nitrogen. A careful thawing method was used to collect the upper parts of the human hair follicles which were cultured for four weeks in a Dulbecco's Modified Eagle's Medium with fetal bovine serum (FBS). Proliferating hair follicle cells were then shifted to DMEM/Ham's Nutrient Mixture F-12 medium without FBS and allowed to grow for one week. These proliferating cells were able to produce HAP stem cell colonies with multilineage differentiation capacity. They produced keratinocytes, smooth muscle cells, cardiac muscle cells, neurons and glial cells. Interestingly, these cryopreserved hair follicles produced pluripotent HAP stem cell colonies similar to fresh follicles. These findings suggest that the cryopreserved whole human hair follicle preserves the ability to produce HAP stem cells, which will enable any individual to preserve a bank of these stem cells for personalized regenerative medicine. 10.1038/s41598-019-45740-9
    Hair follicle epidermal stem cells define a niche for tactile sensation. Cheng Chun-Chun,Tsutsui Ko,Taguchi Toru,Sanzen Noriko,Nakagawa Asako,Kakiguchi Kisa,Yonemura Shigenobu,Tanegashima Chiharu,Keeley Sean D,Kiyonari Hiroshi,Furuta Yasuhide,Tomono Yasuko,Watt Fiona M,Fujiwara Hironobu eLife The heterogeneity and compartmentalization of stem cells is a common principle in many epithelia, and is known to function in epithelial maintenance, but its other physiological roles remain elusive. Here we show transcriptional and anatomical contributions of compartmentalized epidermal stem cells in tactile sensory unit formation in the mouse hair follicle. Epidermal stem cells in the follicle upper-bulge, where mechanosensory lanceolate complexes innervate, express a unique set of extracellular matrix (ECM) and neurogenesis-related genes. These epidermal stem cells deposit an ECM protein called EGFL6 into the collar matrix, a novel ECM that tightly ensheathes lanceolate complexes. EGFL6 is required for the proper patterning, touch responses, and αv integrin-enrichment of lanceolate complexes. By maintaining a quiescent original epidermal stem cell niche, the old bulge, epidermal stem cells provide anatomically stable follicle-lanceolate complex interfaces, irrespective of the stage of follicle regeneration cycle. Thus, compartmentalized epidermal stem cells provide a niche linking the hair follicle and the nervous system throughout the hair cycle. 10.7554/eLife.38883
    Pre-aggregation of scalp progenitor dermal and epidermal stem cells activates the WNT pathway and promotes hair follicle formation in in vitro and in vivo systems. Su Yiqun,Wen Jie,Zhu Junrong,Xie Zhiwei,Liu Chang,Ma Chuan,Zhang Qun,Xu Xin,Wu Xunwei Stem cell research & therapy BACKGROUND:Billions of dollars are invested annually by pharmaceutical companies in search of new options for treating hair loss conditions; nevertheless, the challenge remains. One major limitation to hair follicle research is the lack of effective and efficient drug screening systems using human cells. Organoids, three-dimensional in vitro structures derived from stem cells, provide new opportunities for studying organ development, tissue regeneration, and disease pathogenesis. The present study focuses on the formation of human hair follicle organoids. METHODS:Scalp-derived dermal progenitor cells mixed with foreskin-derived epidermal stem cells at a 2:1 ratio aggregated in suspension to form hair follicle-like organoids, which were confirmed by immunostaining of hair follicle markers and by molecular dye labeling assays to analyze dermal and epidermal cell organization in those organoids. The hair-forming potential of organoids was examined using an in vivo transplantation assay. RESULTS:Pre-aggregation of dermal and epidermal cells enhanced hair follicle formation in vivo. In vitro pre-aggregation initiated the interactions of epidermal and dermal progenitor cells resulting in activation of the WNT pathway and the formation of pear-shape structures, named type I aggregates. Cell-tracing analysis showed that the dermal and epidermal cells self-assembled into distinct epidermal and dermal compartments. Histologically, the type I aggregates expressed early hair follicle markers, suggesting the hair peg-like phase of hair follicle morphogenesis. The addition of recombinant WNT3a protein to the medium enhanced the formation of these aggregates, and the Wnt effect could be blocked by the WNT inhibitor, IWP2. CONCLUSIONS:In summary, our system supports the rapid formation of a large number of hair follicle organoids (type I aggregates). This system provides a platform for studying epithelial-mesenchymal interactions, for assessing inductive hair stem cells and for screening compounds that support hair follicle regeneration. 10.1186/s13287-019-1504-6
    Preliminary studies of hair follicle regeneration by injections of epidermal stem cells and dermal papilla cells into nude mice. Zhang Mingsheng,Ye Yan,Zhao Pin,Bai Liming,Li Xinping Cell and tissue banking The ultimate goal of organ regenerative therapy is to reproduce fully functional organs to replace which have been damaged as a result of diseases or injury. Although several studies claimed that using different types of cells in some animal models promote hair follicles regeneration, more researches can be done to develop a sufficient and efficient protocol to induce hair generation from different animal models. In this study, we investigated the therapeutic potentials for hair follicle formation by injecting a mixture of epidermal stem cells and dermal papilla cells. Those cells were isolated and culture-expanded. Then we randomly allocated 8 nude mice into two groups. The experiment group received an injection of a mixture that containing of epidermal stem cells and dermal papilla cells. The control group received injection of keratinocyte serum-free medium. The hair follicles regeneration was observed and the injection area was harvested for HE staining. 14 day later, the regenerated hair shafts were observed and HE staining indicated that the newly hair follicle formed the correct structures in experiment group. Furthermore, the mixture injection induced a regular and multilayered stratified epidermis and the epidermis contained of hair follicle-likes structures. Our data showed that injection of a mixture of epidermal stem cells and dermal papilla cells could induce hair follicles regeneration and well-ordered epidermis formation. This study emphasized that the rearrangement of the interactions during seed cells and the niches of the seed cells is essential and necessary for tissue-engineered construct success. 10.1007/s10561-020-09825-4
    Expression of anti-aging type-XVII collagen (COL17A1/BP180) in hair follicle-associated pluripotent (HAP) stem cells during differentiation. Shirai Kyoumi,Obara Koya,Tohgi Natsuko,Yamazaki Aiko,Aki Ryoichi,Hamada Yuko,Arakawa Nobuko,Singh Shree Ram,Hoffman Robert M,Amoh Yasuyuki Tissue & cell Hair-follicle-associated pluripotent (HAP) stem cells reside in the upper part of the bulge area of the the hair follicle. HAP stem cells are nestin-positive and keratin 15-negative and have the capacity to differentiate into various types of cells in vitro. HAP stem cells are also involved in nerve and spinal cord regeneration in mouse models. Recently, it was shown that the DNA-damage response in non-HAP hair follicle stem cells induces proteolysis of type-XVII collagen (COL17A1/BP180), which is involved in hair-follicle stem-cell maintenance. COL17A1 proteolysis stimulated hair-follicle stem-cell aging, characterized by the loss of stemness signatures and hair-follicle miniaturization associated with androgenic alopecia. In the present study, we demonstrate that HAP stem cells co-express nestin and COL17A1 in vitro and in vivo. The expression of HAP stem cell markers (nestin and SSEA1) increased after HAP stem-cell colonies were formed, then decreased after differentiation to epidermal keratinocytes. In contrast COL17A1 increased after differentiation to epidermal keratinocytes. These results suggest that COL17A1 is important in differentiation of HAP stem cells. 10.1016/j.tice.2019.06.001