Modelling the hair follicle dermal papilla using spheroid cell cultures

Please cite this paper as: Modelling the hair follicle dermal papilla using spheroid cell cultures. Experimental Dermatology 2010; 19: 546–548. Abstract: Human dermal papilla (DP) cells grown in two‐dimensional (2D) culture have been studied extensively. However, key differences exist between DP cell activities in vivo and in vitro. Using a suspension method of cell culture to maintain DP cells, we created three‐dimensional (3D) dermal spheres morphologically akin to intact (anagen) DPs. Analysis of these spheres using immunocytochemistry demonstrates that they have expression profiles different from papilla cells cultured in 2D but with many similarities to intact DPs. This method of DP cell culture may provide us with a tool to elucidate our understanding of signalling within the DP as it relates to induction, maintenance or even inhibition of hair growth.     

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.     

PDGF signalling in the dermis and in dermal condensates is dispensable for hair follicle induction and formation

Embryonic hair follicle (HF) induction and formation is dependent on signalling crosstalk between the dermis and specialized dermal condensates on the mesenchymal side and epidermal cells and incipient placodes on the epithelial side, but the precise nature and succession of signals remain unclear. Platelet‐derived growth factor (PDGF) signalling is involved in the development of several organs and the maintenance of adult tissues, including HF regeneration in the hair cycle. As both PDGF receptors, PDGFRα and PDGFRβ, are expressed in embryonic dermis and dermal condensates, we explored in this study the role of PDGF signalling in HF induction and formation in the developing skin mesenchyme. We conditionally ablated both PDGF receptors with Tbx18^Cre in early dermal condensates before follicle formation, and with Prx1‐Cre broadly in the ventral dermis prior to HF induction. In both PDGFR double mutants, HF induction and formation ensued normally, and the pattern of HF formation and HF numbers were unaffected. These data demonstrate that mesenchymal PDGF signalling, either in the specialized niche or broadly in the dermis, is dispensable for HF induction and formation.     

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.     

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。     

At the dawn of hair research – testing the limits of hair follicle regeneration

In the late 1960s, tissue recombination studies by Roy Oliver on the model of rat vibrissae provided invaluable information about the morphogenetic properties of hair follicles. Now more than ever, the field is hopeful that a clinically reproducible procedure for cell‐based hair regeneration is achievable. Highly inductive mesenchymal cells are thought to be the key ingredient necessary to achieve robust hair regeneration, and efforts are underway to develop protocols to improve the naturally low inductive properties of human dermal papilla cells. In this respect, the original studies by Oliver provide essential rodent research benchmarks, which current‐day human studies should aim to reach.     

Dickkopf‐1 is involved in dexamethasone‐mediated hair follicle regression

The stress‐related neurohormones including glucocorticoids (GCs) are secreted by hair follicles (HFs), and GCs suppress murine hair growth in vivo. In this study, we found that dexamethasone (Dex), a synthetic GC, increased the expression of dickkopf‐1 (DKK1), a known catagen inducer, in dermal papilla (DP) cells, but not in follicular keratinocytes. The neutralizing DKK1 antibody significantly attenuated the Dex‐induced inhibition of human hair shaft elongation. In addition, the neutralizing Dkk1 antibody delayed Dex‐induced catagen in mice. Collectively, our data strongly suggest that stress‐related neurohormones cause DP cells to secrete DKK1, thereby leading to stress‐associated disturbances in hair growth.     

Cxcr4 is transiently expressed in both epithelial and mesenchymal compartments of nascent hair follicles but is not required for follicle formation

Hair follicle (HF) morphogenesis relies on the coordinated exchange of signals between mesenchymal and epithelial compartments of embryonic skin. Chemokine receptor Cxcr4 expression was recently identified in dermal condensates (DCs) of nascent HFs, but its role in promoting HF morphogenesis remains unknown. Our analyses confirmed Cxcr4 expression in condensate cells, and additionally revealed transient Cxcr4 expression in incipient epithelial hair placodes. Placodal Cxcr4 appeared prior to detection in DCs, representing a switch of expression between epithelial and mesenchymal compartments. To explore the functional role of this receptor in both compartments for early HF formation, we conditionally ablated Cxcr4 with condensate‐targeting Tbx18^cre knock‐in and epidermis‐targeting Krt14‐cre transgenic mice. Conditional knockouts for both crosses were viable throughout embryogenesis and into adulthood. Morphological and biochemical marker analyses revealed comparable numbers of HFs forming in knockout embryos compared to wild‐type littermate controls in both cases, suggesting that neither dermal nor epithelial Cxcr4 expression is required for early HF morphogenesis. We conclude that Cxcr4 expression and chemokine signaling through this receptor in embryonic mouse skin is dispensable for HF formation.     

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.     

某些中药煎剂对阿霉素抑制的猪毛囊真皮鞘细胞增殖的影响

目的:探讨何首乌、女贞子等中药煎剂对阿霉素抑制的猪毛囊真皮鞘细胞增殖的影响。方法:用MTT法通过比色分析测定吸光度值(OD),检测不同剂量(0、5、10、20)mg/L阿霉素作用24h和10mg/L阿霉素作用不同时间(4、8、12、24、48h)对猪毛囊真皮鞘细胞增殖的影响;用流式细胞仪检测不同剂量的阿霉素作用24h后猪毛囊真皮鞘细胞的凋亡率,从而找出用于以下研究中阿霉素的最适浓度。用MTT法检测不同剂量中药煎剂和10mg/L阿霉素同时给药共同作用24h以及先给予不同剂量中药煎剂作用24h,然后给予10mg/L阿霉素作用24h两种条件下对猪毛囊真皮鞘细胞增殖的影响。结果:阿霉素抑制体外培养的猪毛囊真皮鞘细胞增殖,其抑制效应随阿霉素浓度的增大和作用时间的延长而增强;细胞凋亡率以阿霉素浓度为10mg/L时最高。中药煎剂和阿霉素10mg/L同时给药条件下,前者不能有效升高OD值(P>0.05);如果先给予中药煎剂处理,后给10mg/L阿霉素,则中等剂量的中药煎剂即能有效升高OD值(P<0.001)。结论:阿霉素抑制体外培养的猪毛囊真皮鞘细胞增殖,先给予适当剂量的中药煎剂处理细胞,能在一定程度上减轻阿霉素对细胞的损伤。     

毛乳头细胞对毛囊前体黑素细胞作用的评价

目的：确定毛乳头细胞（DPC）对毛囊前体黑素细胞（MP）的激活和趋化作用。方法：采用三维分离式共培养体系,将MP与毛囊外毛根鞘角质形成细胞（KC）以1：10比例于6孔培养板中接触式共培养,DPC以1×10^6个／孔分离培养于Transwell inserts。检测DPC对MP的趋化作用。MP内黑素小体、酪氨酸酶（TYR）、酪氨酸酶相关蛋白1（TRP-1）和酪氨酸酶相关蛋白2（TRP-2）的表达。结果：MP与KC共培养后MP内出现Ⅲ期黑素小体,当三种细胞共培养后MP内主要为Ⅲ和Ⅳ期黑素小体。DPC可促进MP中TYR和TRP-1的表达,但对TRP-2的表达无明显影响,对MP具有明显趋化作用。结论：构建了MP、KC和DPC的三维培养体系,发现DPC对MP具有明显激活和趋化移行作用。     

Effect of Plantago asiatica L. extract on the anagen phase in human hair follicle dermal papilla cells

Background

The hair growth cycle consists of the anagen, catagen, and telogen phases, and hair follicle dermal papilla (HDP) cells of human hair play a role in the initiation and maintenance of the anagen phase. Reduction in HDP cells contributes to hair loss; however, the limited treatment options are associated with negative side effects. Therefore, a naturally derived substance with hair loss-preventing properties is needed.

Aim

We investigated the hair growth-stimulating activities of Plantago asiatica L. extract (PAE) and its molecular mechanism in HDP cells.

Methods

Cell proliferation was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution. Relative mRNA and protein expression levels of hair growth factors were determined using quantitative real-time polymerase chain reaction and western blotting, respectively. Additionally, a tube formation assay was performed in human umbilical vein endothelial cells (HUVEC).

Results

Plantago asiatica L. extract significantly increased the cell proliferation and expression of hair growth factors, including keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2) and MYC, in HDP cells. Moreover, PAE led to the accumulation of β-catenin by promoting the phosphorylation of glycogen synthase kinase-3 beta (GSK-3β) at Ser9 and cAMP response element-binding protein (CREB) at Ser133 via phosphorylation of extracellular signal-regulated kinase (ERK) (Thr202/Tyr204). PAE also increased tube formation in HUVECs, which promoted angiogenesis for the anagen phase.

Conclusions

Plantago asiatica L. extract amplified tube formation and production of growth factors (KGF, VEGF) via the activation of GSK-3β/β-catenin and mitogen-activated protein kinase (MAPK)/CREB signaling pathways, demonstrating its potential to safely promote hair growth by inducing the anagen phase.     

Squamous cell carcinoma induced by ultraviolet radiation originates from cells of the hair follicle in mice

Short-wave ultraviolet radiation (UVB) is the most carcinogenic part of the ultraviolet spectrum. The target cells of skin cancer are believed to be the bulge stem cells and/or their offspring, the transit-amplifying cells that reside in the epidermis. However, the amount of UVB penetrating epidermis and reaching the bulge cells is very low, which questions if these cells suffer sufficient DNA damage to transform into cancer stem cells. We performed this study to determine whether UV-induced squamous cell carcinoma (SCC) originates from the epidermis or the hair follicles in mice. Hairless mice had their epidermis removed at different levels using CO(2) laser ablation. Simulated solar irradiations were administered either preoperatively (in total 7 weeks) or pre- and postoperatively (in total 30 weeks). Control groups were untreated or treated only with solar-simulated radiation or with laser. Blinded clinical assessments of skin tumors were carried out weekly during 12 months observation. Only mice irradiated with solar-simulated radiation both pre- and postoperatively developed tumors. Median time to first, second and third tumor ranged from 19 to 20.5 weeks and was not significantly different between the non-laser and laser-treated groups (P > 0.05). The tumor response was thus similar in UV-exposed mice whether they had their epidermis removed or not. No tumors appeared in control groups. Hence, UV-induced SCC of mice originates from cells of the hair follicle, presumably the bulge stem cells, indicating that ultraviolet radiation penetrates epidermis sufficiently to cause irreversible DNA damage in cells located beneath the epidermis.