Plasticity of hair follicle dermal cells in wound healing and induction

The capacity of adult hair follicle dermal cells to participate in new follicle induction and regeneration, and to elicit responses from diverse epithelial partners, demonstrates a level of developmental promiscuity and influence far exceeding that of interfollicular fibroblasts. We have recently suggested that adult follicle dermal cells have extensive stem or progenitor cell activities, including an important role in skin dermal wound healing. Given that up to now tissue engineered skin equivalents have several deficiencies, including the absence of hair follicles, we investigated the capacity of follicle dermal cells to be incorporated into skin wounds; to form hair follicles in wound environments; and to create a hair follicle-derived skin equivalent. In our study, we implanted rat follicle dermal cells labelled with a vital dye into ear and body skin wounds. We found that they were incorporated into the new dermis in a manner similar to skin fibroblasts, but that lower follicle dermal sheath also assimilated into hair follicles. Using different combinations of follicle dermal cells and outer root sheath epithelial cells in punch biopsy wounds, we showed that new hair follicles were formed only with the inclusion of intact dermal papillae. Finally by combining follicle dermal sheath and outer root sheath cells in organotypic chambers, we created a skin equivalent with characteristic dermal and epidermal architecture and a normal basement membrane - the first skin to be produced entirely from hair follicle cells. These data support the hypothesis that follicle dermal cells may be important in wound healing and demonstrate their potential usefulness in human skin equivalents and skin substitutes. While we have made progress towards producing skin equivalents that contain follicles, we suggest that the failure of cultured dermal papilla cells to induce follicle formation in wounds illustrates the complex role the follicle dermis may play in skin. We believe that it demonstrates a genuine dichotomy of activity for follicle cells within skin.     

Role of bulge cells in wound healing: possible implications for hidradenitis suppurativa

The hair follicle bulge harbors a cluster of quiescent epithelial stem cells, which generate the new lower hair follicle during follicle cycling. The role of bulge cells for maintenance of the hair follicle and epidermis during normal homeostasis and after wounding remains controversial. To address these questions, we targeted the suicide gene, thymidine kinase (TK), or CrePR recombinase to hair follicle bulge cells using a K15 promoter. Administration of ganciclovir to K15‐TK mice caused their death due to gastrointestinal injury. We then treated immunodeficient mice carrying K15‐TK skin grafts with ganciclovir. This resulted in injury of bulge cells within several days. Loss of hair and permanent injury to hair follicles ensued. Dermal scarring was also present. The epidermis without follicles survived for several months. To further assess the contribution of bulge cells to epidermis, we used double transgenic K15‐crePR1, R26R mice. After 5 days of treatment with RU‐486 (time 0), bulge cells and a small fraction of epidermal cells expressed LacZ under control of the ROSA promoter. To determine whether bulge cells moved into the epidermis under normal homeostatic conditions, we counted labeled epidermal basal cells at times 0, 30 days, and 180 days. We found that the percentage of labeled epidermal basal cells (approximately 1%) remained constant, indicating an absence of cell movement from the bulge to the epidermis. After punch wounding, bulge progeny were detected in a radial pattern in the re‐epithelialized area. Radial streaks of bulge cell progeny emanated from the hair follicles at the wound edge. Bulge cell progeny proliferated and expressed normal epidermal differentiation markers such as keratin 10 and loricrin. Overall, these results indicate that follicular stem cells in the bulge are necessary for hair follicle survival and that these cells functionally contribute to epidermal regeneration in response to wounding; however, the epidermis self‐renews autonomously of the bulge under normal conditions. The role of bulge cells in hidradenitis suppurativa (HS) is not known; however, pathological studies suggest aberrant proliferation of the hair follicle may accompany HS and inappropriate stem cell activation and differentiation could be a component of this disorder. The K15‐crePR1 transgenic mouse will serve as a powerful tool for evaluating the role of hair follicle stem cells in mouse models of HS. Studying hair follicle stem cells with recently defined markers for these cells will be useful for evaluating stem cell behavior in HS lesions.     

Epithelial stem cells: a folliculocentric view

Putative epithelial stem cells were identified in the hair follicle bulge as quiescent "label retaining cells". The study of these cells was hindered until the identification of bulge cell molecular markers, such as CD34 expression and K15 promoter activity. This allowed for the isolation and characterization of bulge cells from mouse follicles. Bulge cells possess stem cell characteristics, including multipotency, high proliferative potential, and their cardinal feature of quiescence. Lineage analysis demonstrated that all epithelial layers within the adult follicle and hair originated from bulge cells. Bulge cells only contribute to the epidermis during wound healing, but after isolation, when combined with neonatal dermal cells, they regenerate new hair follicles, epidermis, and sebaceous glands. Bulge cells maintain their stem cell characteristics after propagation in vitro, thus ultimately they may be useful for tissue engineering applications. Understanding the signals important for directing movement and differentiation of bulge cells into different lineages will be important for developing treatments based on stem cells as well as clarifying their role in skin disease.     

Is the Hair Follicle Necessary for Normal Wound Healing?

The hair follicle contributes cells to the interfollicular epidermis after wounding, but the functional role of these cells has not been resolved. To address this question, Langton et al. (this issue, 2008) take advantage of the Edaradd mutant mouse, which lacks hair follicles on its tail. They discover an initial sluggish response of the hairless tail epidermis to wounding that is rapidly compensated for by recruitment of epidermal cells from outside the normally responsive area. This suggests that the hair follicle is important but not necessary for normal wound healing.     

Epigenetic control of skin and hair regeneration after wounding

Skin wound healing is a complex regenerative phenomenon that can result in hair follicle neogenesis. Skin regeneration requires significant contribution from the immune system and involves substantial remodelling of both epidermal and dermal compartments. In this viewpoint, we consider epigenetic regulation of reepithelialization, dermal restructuring and hair neogenesis. Because little is known about the epigenetic control of these events, we have drawn upon recent epigenetic mapping and functional studies of homeostatic skin maintenance, epithelial–mesenchymal transition in cancer, and new works on regenerative dermal cell lineages and the epigenetic events that may shape their conversion into myofibroblasts. Finally, we speculate on how these various healing components might converge for wound‐induced hair follicle neogenesis.     

中波紫外线诱导小鼠皮肤光损伤中miRNA141表达的研究

【摘要】 目的 探讨小室移植法重建小鼠毛囊,观察细胞成分对毛囊再生的影响。 方法 取0 ～ 2 d的C57BL/6乳鼠背部皮肤,胰酶消化后分离表真皮,再分离出毛囊上皮细胞。实验分表皮细胞混合毛囊胚芽组、真皮细胞组、表皮细胞混合毛囊胚芽 + 真皮细胞组、毛囊上皮细胞 + 真皮细胞组。用小室移植法接种于裸鼠背部,并于移植后1、2、4、8周观察变化,HE染色观察毛囊组织学形态。 结果 小鼠毛囊细胞移植1周时,背部小室开始脱落,伤口结痂;2周时除表皮细胞组外,另3组均长出了短小的毛发,镜下可见毛囊样结构;4周及8周时除表皮细胞组外,均长出了正常的毛发,表皮细胞与真皮细胞混合组及毛囊上皮细胞与真皮细胞混合组毛发生长情况良好,优于单独的真皮细胞组。 结论 毛囊细胞小室移植后可形成新的毛囊,上皮细胞及真皮细胞在毛囊重建中具有重要的作用。 【关键词】 毛囊; 毛囊重建; 鼠科     

Reflections on how wound healing‐promoting effects of the hair follicle can be translated into clinical practice

Clinicians have long reported that hair‐bearing areas tend to heal more rapidly than those lacking hair follicles. In the past decade, numerous scientific studies have corroborated clinical evidence, showing a direct nexus between the human hair follicle and the wound healing process. The migration of epithelial follicular stem cells to the skin surface to help in the wound re‐epithelialization and the effect of the hair cycle on the wound healing rate underline the influence of the hair follicle in the healing process. In clinical practice, non‐healing wounds are pathologies of high prevalence with significant associated burden costs for the healthcare system. As the population ages, the prevalence of this pathology is expected to increase in future years. The recent advances in understanding the biology of hair follicle stem cells have created the challenges of using this newly acquired knowledge in practical therapeutic applications. Chronic leg ulcers are an example of the targeted pathologies that urgently need better therapies. In this essay, our aim is to raise interest in this question, reviewing what is known in relation to the connections between hair follicles and wound healing, and elaborating on future directions that the field might take, including implications for clinical practice.     

Keratinocyte stem cell assays: an evolving science

Although the existence of epithelial stem cells in the skin has been known for some decades from cell kinetic studies performed in vivo, attempts to prospectively isolate these cells for further biological characterization have been made possible relatively recently facilitated by the availability of antibodies that detect cell surface markers on epidermal cells. Elegant gene marking studies in vivo have provided confirmation of the patterns of epithelial tissue replacement predicted by classical cell turnover studies. But, the identification of candidate epidermal stem cells ex vivo remains an area of great controversy, requiring the re-evaluation of current experimental approaches that rely of necessity on predicted epidermal stem cell behavior in culture. Here we review the diverse experimental approaches utilized to identify keratinocyte stem cells and their underlying assumptions. We conclude that hair follicles and interfollicular epidermis each have their own self-renewing stem cell populations, contributing to distinct regions of the epithelium during homeostasis, although this is perturbed during wound healing. The need for the development of more rigorous assays for stem cell activity is highlighted given our recent observations using current assays and the discovery of new surface markers that identify putative epidermal stem cells.     

Molecular mechanisms regulating hair follicle development

Clinical conditions causing hair loss, such as androgenetic alopecia, alopecia areata, and scarring alopecia, can be psychologically devastating to individuals and are the target of a multimillion dollar pharmaceutical industry. The importance of the hair follicle in skin biology, however, does not rest solely with its ability to produce hair. Hair follicles are self-renewing and contain reservoirs of multipotent stem cells that are capable of regenerating the epidermis and are thought to be utilized in wound healing. Hair follicles are also the sites of origin of many neoplasias, including some basal cell carcinomas and pilomatricoma. These diseases result from inappropriate activation of signaling pathways that regulate hair follicle morphogenesis. Identification of the signaling molecules and pathways operating in developing and postnatal, cycling, hair follicles is therefore vital to our understanding of pathogenic states in the skin and may ultimately permit the development of novel therapies for skin tumors as well as for hair loss disease. The purpose of this review is to summarize recent progress in our understanding of the molecular mechanisms regulating hair follicle formation, and to discuss ways in which this information may eventually be utilized in the clinic.     

Betacellulin regulates hair follicle development and hair cycle induction and enhances angiogenesis in wounded skin

Betacellulin (BTC) belongs to the EGF family, whose members play important roles in skin morphogenesis, homeostasis, and repair. However, the role of BTC in skin biology is still unknown. We employed transgenic mice overexpressing BTC ubiquitously to study its role in skin physiology. Immunohistochemistry revealed increased levels of BTC especially in the hair follicles and in the epidermis of transgenic animals. Expression of key markers of epithelial differentiation was unaltered, but keratinocyte proliferation was significantly increased. At post-natal day 1 (P1), transgenic mice displayed a significant retardation of hair follicle morphogenesis. At P17, when most follicles in control mice had initiated hair follicle cycling and had already entered into their first late catagen or telogen phase, all follicles of transgenic mice were still at the mid- to late catagen phases, indicating retarded initiation of hair follicle cycling. Healing of full-thickness excisional wounds and bursting strength of incisional wounds were similar in control and transgenic mice. However, an increase in the area covered by blood vessels at the wound site was detected in transgenic animals. These results provide evidence for a role of BTC in the regulation of epidermal homeostasis, hair follicle morphogenesis and cycling, and wound angiogenesis.     

Myo/Nog cells in normal, wounded and tumor-bearing skin

Murine and human skin were examined for the presence of Myo/Nog cells that were originally discovered in the chick embryo by their expression of MyoD mRNA, noggin and the G8 antigen. Myo/Nog cells are the primary source of noggin in telogen hair follicles. They are scarce within the interfollicular dermis and absent in the epidermis. Within 24 h following epidermal abrasion, Myo/Nog cells increase in number in the follicles and appear in the wound. Myo/Nog cells are also recruited to the stroma of tumors formed from v‐Ras‐transformed keratinocytes (Ker/Ras). Human squamous cell carcinomas and malignant melanomas contain significantly more Myo/Nog cells than basal cell carcinomas. Myo/Nog cells are distinct from macrophages, granulocytes and cells expressing alpha smooth muscle actin in the tumor stroma. Myo/Nog cells may be modulators of skin homoeostasis and wound healing, and potential diagnostic and therapeutic targets in skin cancer.     

Epidermal and hair follicle progenitor cells express melanoma-associated chondroitin sulfate proteoglycan core protein

Basal keratinocytes in the epidermis and hair follicle are biologically heterogeneous but must include a stable subpopulation of epidermal stem cells. In animal models these can be identified by their retention of radioactive label due to their slow cycle (label-retaining cells) but human studies largely depend on in vitro characterization of colony forming efficiency and clonogenicity. Differential integrin expression has been used to detect cells of increased proliferative potential but further stem cell markers are urgently required for in vivo and in vitro characterization. Using LHM2, a monoclonal antibody reacting with a high molecular weight melanoma-associated proteoglycan core protein, a subset of basal keratinocytes in both the interfollicular epidermis and the hair follicle has been identified. Coexpression of melanoma-associated chondroitin sulfate proteoglycan with keratins 15 and 19 as well as beta 1 and alpha 6 integrins has been examined in adult and fetal human skin from hair bearing, nonhair bearing, and palmoplantar regions. Although melanoma-associated chondroitin sulfate proteoglycan coexpression with a subset of beta 1 integrin bright basal keratinocytes within the epidermis suggests that melanoma-associated chondroitin sulfate proteoglycan colocalizes with epidermal stem cells, melanoma-associated chondroitin sulfate proteoglycan expression within the hair follicle was more complex and multiple subpopulations of basal outer root sheath keratinocytes are described. These data suggest that epithelial compartmentalization of the outer root sheath is more complex than interfollicular epidermis and further supports the hypothesis that more than one hair follicle stem cell compartment may exist.     

Sheep vibrissa dermal papillae induce hair follicle formation in heterotypic skin equivalents

Cultured skin equivalents were constructed by combining keratinocytes, outer root sheath cells or isolated epidermis, in vitro, with a matrix composed of collagen and cultured fibroblasts. When equivalents were grafted on to host animals, the epidermis thickened considerably, and tongues of cells penetrated the dermis, giving the dermal/epidermal junction a deeply sculptured profile. No cutaneous appendages were found in these grafts. We explored the possibility of inducing hair follicles by incorporating ovine hair follicle dermal papillae into constructs composed of an isolated epidermal sheet and a contracted dermal equivalent. In vitro, no morphogenetic changes associated with follicle formation were observed in the recombinants, but when grafted on to nude mice, follicle-like structures were identified. The follicles were large, and had developed adjacent to the epidermis, indicating that the matrix environment of the induced follicles may not have been compatible with the downgrowth of the epidermal plugs normally observed during follicle formation in living skin. Nevertheless, in histological sections, the induced structures displayed many of the morphological characteristics of follicles in vivo, including the production of keratinized hairs. These results indicate that skin equivalents provide a useful model for the study of the chemical and structural features of matrices that facilitate hair follicle development.     

Photoepilation: a potential threat to wound healing in a mouse

Background Theoretically, the bulge area which is known to be a reservoir of epidermal stem cells should be destroyed to achieve permanent photoepilation. We wished to determine whether wound healing capability is perturbed after photoepilation. Methods Twenty C57/BL6 mice were used. After wax epilation to synchronize the hair cycle, one‐half of the backs of mice were photoepilated in the early anagen stage. After the two hair cycles of the mice to confirm the hair removal effect, 30% trichloroacetic acid was applied to the both halves of the backs of the mice. A skin biopsy was performed on both sides before and just after the injury, and 2, 6, 9, and 14 days thereafter. The specimens were evaluated histologically after staining with hematoxylin and eosin, Masson trichrome, and Verhoeff‐van Gieson. Results No differences in wound healing times were evident upon gross observation by the naked eye. However, the photoepilated hairless skin was observed to have a thicker epidermis and dermis than normal hairy skin by histological evaluation. The cellularity of the healed wound was much denser in the photoepilated. Collagen production of the neodermis in the normal hairy skin was first observed around the lower part of hair follicle, while it started from the upper papillary dermis in photoepilated skin. Conclusion Photoepilation may disturb the normal wound healing process, especially dermal wound healing, and increases the risk of producing hypertropic scar or keloid.     

Two‐photon microscopy for intracutaneous imaging of stem cell activity in mice

The adult skin is a typical example of a highly regenerative tissue. Terminally differentiated keratinocytes are shed from the external layers of the epidermis or extruded from the skin as part of the growing hair shaft on a daily basis. These are effectively replenished through the activity of skin‐resident stem cells. Precise regulation of stem cell activity is critical for normal skin homoeostasis or wound healing and irregular stem cell proliferation or differentiation can lead to skin disease. The scarcity and dynamic nature of stem cells presents a major challenge for elucidating their mechanism of action. To address this, we have recently established a system for visualizing stem cell activity, in real time or long term, in the intact skin of live mice using two‐photon microscopy. The purpose of this review was to provide essential information to researchers who wish to incorporate two‐photon microscopy and live imaging into their experimental toolbox for studying aspects of skin and stem biology in the mouse model. We discuss fundamental principles of the method, instrumentation and basic experimental approaches to interrogate stem cell activity in the interfollicular epidermis and hair follicle.     

治疗性皮肤创伤诱导白癜风皮损复色的机制与临床研究进展

【摘要】 人们很早就注意到用皮肤磨削术、中医梅花针或皮肤微针,甚至剥脱性点阵CO2激光等对常规中波紫外线光疗抵抗的白癜风皮损可有效诱导复色。这些治疗除了因创伤增加药物透皮吸收外,还启动了创伤修复以及对毛囊或表皮黑素细胞的活化。伤口周围基底层角质形成细胞、血管内皮细胞和成纤维细胞分泌趋化因子12以招募毛囊隆突区或皮损周围表达趋化因子受体CXCR4/CXCR7的黑素细胞或黑素干细胞向白斑区移行。本文综述治疗性皮肤创伤诱导白癜风皮损复色的研究进展。     

Expression of the hair stem cell-specific marker nestin in epidermal and follicular tumors

Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells give rise to the outer-root sheath. Nestin-expressing hair follicle stem cells that are negative for the keratinocyte marker keratin 15 (K15) can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Recent studies suggest that the epithelial stem cells are important in tumorigenesis. In this study, we immunohistochemically examined the expression of three hair follicle stem cell and progenitor cell markers, nestin, K15, and CD34, in normal human epidermis and hair follicles and in epidermal and follicular tumors, trichilemmoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). In normal human skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34. The hair follicle cells below the sebaceous glands were also positive for nestin and K15 and negative for CD34. The outer-root sheath cells under this area could be divided into three parts: an upper part of the outer-root sheath cells that was partially positive for nestin and positive for K15 and negative for CD34; a middle part that was CD34-positive and K15-negative; and a lower part that was positive for K15 and negative for CD34. In the tumor tissues, nestin immunoreactivity was observed in trichilemmoma but not in BCC. Also, immunoreactivity for K15 was strong in BCC and weak in trichilemmoma, and SCC was negative for nestin and partially positive for K15. No CD34 immunoreactivity was observed in any of the cases. These results suggested that trichilemmoma originates in the nestin-positive/K15-positive/CD34-negative outer-root sheath cells below sebaceous glands, BCC tumor cells from the more mature nestin-negative/K15-positive/CD34-negative outer-root sheath cells, and SCC from the nestin-negative/K15-positive/CD34-negative keratinocytes of the basal cell layer in the epidermis.     

CRH peptides modulate proliferation, melanogenesis and dendri-city in human follicular melanocytes

Recent findings have established that cutaneous nerves modulate both skin homeostasis and various skin diseases, by influencing cell growth and differentiation, inflammation and wound healing. In order to study the influence of epidermis, hair follicles and capillaries on sensory neurons, and vice-versa, we developed a tissue-engineered model of innervated endothelialized reconstructed skin (MIERS). Mouse dorsal root ganglia neurons were seeded on a collagen sponge populated with human fibroblasts and human endothelial cells. Keratinocytes or mice newborn immature hair follicle buds were then seeded on the opposite side of the MIERS to study their influence on sensory nerves growth, and vice versa. A vigorous neurite elongation was detected inside the reconstructed dermis after 14 and 31 days of neurons culture. The presence of endothelial cells induced a significant increase of the neurite elongation after 14 days of culture. The addition of human keratinocytes totally avoided the twofold decrease in the amount of neurites observed between 14 and 31 days in controls. We have successfully developed the MIERS that allowed us to study the effects of epidermis and capillaries on nerve growth. This model will be a useful tool to study the modulation of sensory nerves on wound healing, angiogenesis, hair growth and neurogenic inflammation in the skin.     

毛囊间充质干细胞的研究进展

 毛囊间充质干细胞（HFMSCs）是一类具有自我再生能力、高度增殖潜能、可多向分化且来源丰富的慢周期标记滞留细胞,可促进表皮、毛囊和皮脂腺再生。得益于其来源丰富、易获得、可于体外扩增、无需基因操作、不会形成肿瘤和无伦理限制等特点,毛囊间充质干细胞在再生医学中具有重要优势。Wnt、Shh、Notch和BMP等信号通路之间的协同和拮抗作用在干细胞稳态调节、表皮发育和毛囊再生过程中发挥至关重要的作用,这些途径的失调可能导致毛发脱落或者肿瘤的发生。本文综述毛囊间充质干细胞的分类及其特异性生物标记物、毛囊间充质干细胞的活化以及影响毛发再生的生物分子途径,为毛发疾病的治疗提供新的线索和靶点。     

Expression of cyclooxygenase isozymes during morphogenesis and cycling of pelage hair follicles in mouse skin: precocious onset of the first catagen phase and alopecia upon cyclooxygenase-2 overexpression

Cyclooxygenase (COX)-1 and -2 catalyze the key reaction in prostaglandin biosynthesis. Whereas COX-1 is found in most tissues, COX-2, with a few exceptions, is not expressed in normal tissues but becomes transiently induced in the course of inflammatory reactions. In many neoplastic epithelia, COX-2 is constitutively overexpressed. Here we show that COX isozymes are spatiotemporally expressed during morphogenesis of dorsal skin epithelium of NMRI mice. COX-1 and COX-2 mRNA and protein were detected in embryonic and postnatal epidermal tissue by RT-PCR, northern blot, and immunoblot analysis indicating that both isoforms may contribute to prostaglandin production. Being barely detectable in interfollicular epidermis and resting hair follicles of adult mice, COX-2 protein appeared in embryonic skin first in epidermal precursor cells and later on in the basal cells and the peridermal layer of the stratified epidermis. In the course of pelage hair follicle morphogenesis, COX-2 remained expressed in the basal interfollicular compartment and, in addition, became apparent in elongated hair germs and hair pegs and later on in the outer root sheath cells of the distal and proximal hair follicles as well as in basal sebaceous gland cells. During the subsequent synchronous phases of hair cycling, COX-2 expression declined in catagen, was barely detectable in telogen, and was reinduced in the basal outer root sheath and basal sebaceous gland cells of anagen hair follicles. COX-1 immunosignals were detected predominantly in the interfollicular spinous and granular layers of the developing, neonatal, and adult epidermis but not in follicular epithelial cells of developing or cycling hair follicles. Dendritic cells in the interfollicular epidermis and distal hair follicles were also COX-1-positive. Transgenic overexpression of COX-2 under the control of a keratin 5 promoter in basal cells of the interfollicular and follicular epidermis induced a precocious entry into the first catagen stage of postnatal hair follicle cycling and a subsequent disturbance of hair follicle phasing. Furthermore, transgenic mice developed an alopecia. Inhibition of transgenic COX-2 activity by feeding the specific COX-2 inhibitor valdecoxib suppressed the development of alopecia, indicating that COX-2-mediated prostaglandin synthesis is involved in hair follicle biology.