Niche crosstalk: intercellular signals at the hair follicle.

A recent series of papers, including Festa et al. (2011) in this issue, has revealed unexpected interdependent relationships among cell populations residing in and around the hair follicle. These interactions between different lineages of stem cells are crucial for hair follicle growth and cycling and point to a complex crosstalk in stem cell niches.

Dominant effect of gap junction communication in wound-induced calcium-wave, NFAT activation and wound closure in keratinocytes.

Wounding induces a calcium wave and disrupts the calcium gradient across the epidermis but mechanisms mediating calcium and downstream signalling, and longer-term wound healing responses are incompletely understood. As expected, live-cell confocal imaging of Fluo-4-loaded normal human keratinocytes showed an immediate increase in [Ca2+ ]i at the wound edge that spread as a calcium wave (8.3 µm/s) away from the wound edge with gradually diminishing rate of rise and amplitude. The amplitude and area under the curve of [Ca2+ ]i flux was increased in high (1.2 mM) [Ca2+ ]o media. 18α-glycyrrhetinic acid (18αGA), a gap-junction inhibitor or hexokinase, an ATP scavenger, blocked the wound-induced calcium wave, dependent in part on [Ca2+ ]o . Wounding in a high [Ca2+ ]o increased nuclear factor of activated T-cells (NFAT) but not NFkB activation, assessed by dual-luciferase receptor assays compared to unwounded cells. Treatment with 18αGA or the store-operated channel blocker GSK-7975A inhibited wound-induced NFAT activation, whereas treatment with hexokinase did not. Real-time cell migration analysis, measuring wound closure rates over 24 h, revealed that 18αGA essentially blocked wound closure whereas hexokinase and GSK-7975A showed relatively minimal effects. Together these data indicate that while both gap-junction communication and ATP release from damaged cells are important in regulating the wound-induced calcium wave, long-term transcriptional and functional responses are dominantly regulated by gap-junction communication.

Extracellular Vesicles from Activated Dermal Fibroblasts Stimulate Hair Follicle Growth Through Dermal Papilla-Secreted Norrin.

Dermal papilla cells (DPCs) play a pivotal role in the regulation of hair follicle (HF) growth, formation, and cycling, mainly through paracrine mechanisms. In the last decade, extracellular vesicles (EVs) have been recognized as a new paracrine mechanism that can modify the physiological state of recipient cells by transferring biological material. Herein, we investigated the effect of EVs isolated from stimulated human dermal fibroblasts (DFs) on DPC activation and HF growth. We found that these EVs (st-EVs) enhanced HF growth ex vivo. Comparative transcriptomic analysis on DPCs identified specific activation of the NDP gene, encoding the non-Wnt ligand Norrin. We found that Norrin was secreted by st-EVs-stimulated DPCs activating in a noncell autonomous manner ß-catenin pathway in follicular keratinocytes (human HF keratinocyte [HHFK]) and hair growth ex vivo. Although Norrin-specific receptor Frizzled4 was barely detected in HHFK, we found its presence in DF-EVs. Accordingly, DF-EVs provided Frizzled4 to potentiate Norrin effects ex vivo. Our study identifies DF-EVs as efficient activators of DPCs and Norrin as a novel modulatory player in HF physiopathology. Stem Cells 2019;37:1166-1175.

Hierarchical patterning modes orchestrate hair follicle morphogenesis.

Two theories address the origin of repeating patterns, such as hair follicles, limb digits, and intestinal villi, during development. The Turing reaction-diffusion system posits that interacting diffusible signals produced by static cells first define a prepattern that then induces cell rearrangements to produce an anatomical structure. The second theory, that of mesenchymal self-organisation, proposes that mobile cells can form periodic patterns of cell aggregates directly, without reference to any prepattern. Early hair follicle development is characterised by the rapid appearance of periodic arrangements of altered gene expression in the epidermis and prominent clustering of the adjacent dermal mesenchymal cells. We assess the contributions and interplay between reaction-diffusion and mesenchymal self-organisation processes in hair follicle patterning, identifying a network of fibroblast growth factor (FGF), wingless-related integration site (WNT), and bone morphogenetic protein (BMP) signalling interactions capable of spontaneously producing a periodic pattern. Using time-lapse imaging, we find that mesenchymal cell condensation at hair follicles is locally directed by an epidermal prepattern. However, imposing this prepattern's condition of high FGF and low BMP activity across the entire skin reveals a latent dermal capacity to undergo spatially patterned self-organisation in the absence of epithelial direction. This mesenchymal self-organisation relies on restricted transforming growth factor (TGF) ß signalling, which serves to drive chemotactic mesenchymal patterning when reaction-diffusion patterning is suppressed, but, in normal conditions, facilitates cell movement to locally prepatterned sources of FGF. This work illustrates a hierarchy of periodic patterning modes operating in organogenesis.

Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes.

Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332.

Genome-wide association study in alopecia areata implicates both innate and adaptive immunity.

Alopecia areata (AA) is among the most highly prevalent human autoimmune diseases, leading to disfiguring hair loss due to the collapse of immune privilege of the hair follicle and subsequent autoimmune attack. The genetic basis of AA is largely unknown. We undertook a genome-wide association study (GWAS) in a sample of 1,054 cases and 3,278 controls and identified 139 single nucleotide polymorphisms that are significantly associated with AA (P

Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth.

De novo organ regeneration has been observed in several lower organisms, as well as rodents; however, demonstrating these regenerative properties in human cells and tissues has been challenging. In the hair follicle, rodent hair follicle-derived dermal cells can interact with local epithelia and induce de novo hair follicles in a variety of hairless recipient skin sites. However, multiple attempts to recapitulate this process in humans using human dermal papilla cells in human skin have failed, suggesting that human dermal papilla cells lose key inductive properties upon culture. Here, we performed global gene expression analysis of human dermal papilla cells in culture and discovered very rapid and profound molecular signature changes linking their transition from a 3D to a 2D environment with early loss of their hair-inducing capacity. We demonstrate that the intact dermal papilla transcriptional signature can be partially restored by growth of papilla cells in 3D spheroid cultures. This signature change translates to a partial restoration of inductive capability, and we show that human dermal papilla cells, when grown as spheroids, are capable of inducing de novo hair follicles in human skin.

Dermal white adipose tissue: a new component of the thermogenic response.

Recent literature suggests that the layer of adipocytes embedded in the skin below the dermis is far from being an inert spacer material. Instead, this layer of dermal white adipose tissue (dWAT) is a regulated lipid layer that comprises a crucial environmental defense. Among all the classes of biological molecules, lipids have the lowest thermal conductance and highest insulation potential. This property can be exploited by mammals to reduce heat loss, suppress brown adipose tissue activation, reduce the activation of thermogenic programs, and increase metabolic efficiency. Furthermore, this layer responds to bacterial challenge to provide a physical barrier and antimicrobial disinfection, and its expansion supports the growth of hair follicles and regenerating skin. In sum, this dWAT layer is a key defensive player with remarkable potential for modifying systemic metabolism, immune function, and physiology. In this review, we discuss the key literature illustrating the properties of this recently recognized adipose depot.

Defining dermal adipose tissue.

Here, we explore the evolution and development of skin-associated adipose tissue with the goal of establishing nomenclature for this tissue. Underlying the reticular dermis, a thick layer of adipocytes exists that encases mature hair follicles in rodents and humans. The association of lipid-filled cells with the skin is found in many invertebrate and vertebrate species. Historically, this layer of adipocytes has been termed subcutaneous adipose, hypodermis and subcutis. Recent data have revealed a common precursor for dermal fibroblasts and intradermal adipocytes during development. Furthermore, the development of adipocytes in the skin is independent from that of subcutaneous adipose tissue development. Finally, the role of adipocytes has been shown to be relevant for epidermal homoeostasis during hair follicle regeneration and wound healing. Thus, we propose a refined nomenclature for the cells and adipose tissue underlying the reticular dermis as intradermal adipocytes and dermal white adipose tissue, respectively.

Human hair follicle dermal sheath and papilla cells support keratinocyte growth in monolayer coculture.

Traditional skin grafting techniques are effective but limited methods of skin replacement. Autologous transplantation of rapidly cultured keratinocytes is successful for epidermal regeneration, but the current gold-standard technique requires mouse fibroblast feeders and serum-rich media, with serum-free systems and dermal fibroblast (DF) feeders performing relatively poorly. Here, we investigated the capacity of human hair follicle dermal cells to act as alternative supports for keratinocyte growth. Dermal papilla (DP) dermal sheath (DS), DF and 3T3 cells were used as inactivated feeder cells for human keratinocyte coculture. Under conditions favouring dermal cells, proliferation of keratinocytes in the presence of either DS or DP cells was significantly enhanced compared with DF cells, at levels comparable to keratinocytes cultured under gold-standard conditions. Secreted protein acidic and rich in cysteine (SPARC) expression increased DS and DP cells relative to DFs; however, further experiments did not demonstrate a role in keratinocyte support.

Human hair follicle dermal cells and skin fibroblasts show differential activation of NF-κB in response to pro-inflammatory challenge.

The underlying mechanism of immune privilege in hair follicle cell dermal papilla (DP) and sheath (DS) populations is not well understood, and the responsiveness of hair follicle dermal cells to pro-inflammatory challenge presently remains unknown. In this work, we describe acute NF-κB activation in human DS, DP and dermal fibroblast (DF) cells challenged with TNF-alpha and IL1-beta. In contrast, the DS and DP cells revealed an unexpected tolerance to bacterial LPS challenge relative to DF cells. Understanding follicle cell responses to typical pro-inflammatory stimuli is critical for diseases where collapse of hair follicle immune privilege is observed, and to further applications in autologous stem cell/wound healing therapeutics.

Modelling the hair follicle dermal papilla using spheroid cell cultures.

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.

Dynamic expression of Syndecan-1 during hair follicle morphogenesis.

Syndecan-1 is a cell-surface heparan-sulphate proteoglycan that is involved in growth factor regulation, cell adhesion, proliferation, differentiation, blood coagulation, lipid metabolism, as well as tumour formation. In this study, investigation of discrete LCM captured dermal cells by semi-quantitative RT-PCR revealed Syndecan-1 mRNA transcripts were expressed only in the dermal condensation (DC) within this skin compartment during murine pelage hair follicle (HF) morphogenesis. Further immunofluorescence studies showed that, during early skin development, Syndecan-1 was expressed in the epidermis while being absent from the mesenchyme. As HF morphogenesis began ( approximately E14.5) Syndecan-1 expression was lost from the epithelial compartment of the HF and activated in HF mesenchymal cells. This Syndecan-1 expression profile was consistent between different hair follicle types including primary and secondary pelage, vibrissa, and tail hair follicles. Furthermore we show by using gene targeted mice lacking Syndecan-1 expression that Syndecan-1 is not required for follicle initiation and development.

A highly enriched niche of precursor cells with neuronal and glial potential within the hair follicle dermal papilla of adult skin.

Skin-derived precursor cells (SKPs) are multipotent neural crest-related stem cells that grow as self-renewing spheres and are capable of generating neurons and myelinating glial cells. SKPs are of clinical interest because they are accessible and potentially autologous. However, although spheres can be readily isolated from embryonic and neonatal skin, SKP frequency falls away sharply in adulthood, and primary sphere generation from adult human skin is more problematic. In addition, the culture-initiating cell population is undefined and heterogeneous, limiting experimental studies addressing important aspects of these cells such as the behavior of endogenous precursors in vivo and the molecular mechanisms of neural generation. Using a combined fate-mapping and microdissection approach, we identified and characterized a highly enriched niche of neural crest-derived sphere-forming cells within the dermal papilla of the hair follicle of adult skin. We demonstrated that the dermal papilla of the rodent vibrissal follicle is 1,000-fold enriched for sphere-forming neural crest-derived cells compared with whole facial skin. These "papillaspheres" share a phenotypic and developmental profile similar to that of SKPs, can be readily expanded in vitro, and are able to generate both neuronal and glial cells in response to appropriate cues. We demonstrate that papillaspheres can be efficiently generated and expanded from adult human facial skin by microdissection of a single hair follicle. This strategy of targeting a highly enriched niche of sphere-forming cells provides a novel and efficient method for generating neuronal and glial cells from an accessible adult somatic source that is both defined and minimally invasive.

An improved method of human keratinocyte culture from skin explants: cell expansion is linked to markers of activated progenitor cells.

Human keratinocyte primary cultures are commonly established by tissue dissociation and often rely on feeder cell supports and culture medium that is not defined. Further, contamination by unwanted fibroblasts can be problematic. Here, we developed a skin explant method for growing primary keratinocytes that was rapid, simple, and reliably generated keratinocyte cultures free of fibroblast contamination. The process capitalized on the observation that fibroblasts migrate out of adult skin explants later than epidermal cells, allowing the early harvesting of keratinocytes by trypsinization. When grown subsequently in defined medium in the absence of feeder cells, the explant-derived cells grew rapidly and could be cultured for multiple passages. Immunofluorescence microscopy revealed that a high percentage of cells harvested from the explant outgrowths expressed K15, while very few expressed the differentiation marker K10. Cells that were stained while migrating out from explants strongly expressed markers associated with progenitor cells, including p63, K15 and CD133, and displayed intense K6 expression, indicative of activated keratinocytes in wound-healing epidermis. By replenishing the explants with fresh medium after harvesting, further epidermal outgrowths could be obtained, offering the possibility of greatly increased keratinocyte yields for clinical applications.

Exogen involves gradual release of the hair club fibre in the vibrissa follicle model.

Exogen is a distinct phase of the hair cycle describing the process by which the hair club fibre is shed from the follicle. This process is difficult to study in human skin and little is known about the mechanisms involved in the release of club fibres. We sought an alternative model system to study exogen in more detail, and therefore utilised the vibrissa system on the rodent mystacial pad. The time at which a vibrissa club hair will be lost can be predicted, based on the relative lengths of the new growing fibre and old club fibre. This timing phenomenon was exploited to investigate the club fibre within the follicle as it approaches final release, revealing key changes in the adhesive state of the club fibre within the epithelial sac as it approached release. We propose that exogen should be subdivided to represent variations in the club fibre status.

Keratin 10 (K10) is expressed suprabasally throughout the limbus of embryonic and neonatal rat corneas, with interrupted expression in the adult limbus.

The corneal epithelium is continuously replaced by epithelial stem cells located in the basal layer of the limbus, located at the margin of the cornea. Studying how the stem cell niche is established at the limbus during development of the eye may lead to better understanding and treatments for diseases associated with limbal deficiencies. Using two highly specific commercially available antibodies, K10 was consistently detected suprabasally throughout the developing limbal epithelium of late gestation (20.5 dpc) and neonatal rat corneas, with interrupted expression in adult rat limbal epithelium. RT-PCR confirmed K10 expression at the transcript level in embryonic, neonatal and adult rat eyes. We have identified a time point where early stages of limbal development may be facilitated by the suprabasal expression of K10.

Dynamic expression of the zinc-finger transcription factor Trps1 during hair follicle morphogenesis and cycling.

Mutations in the gene encoding the zinc finger transcription factor TRPS1 result in tricho-rhino-phalangeal syndrome, characterized by craniofacial and skeletal abnormalities, and sparse scalp hair. In this study, Trps1 was identified by microarray hybridization analysis as having a complex pattern of spatiotemporal regulation in murine skin during morphogenesis. During early skin development, Trps1 expression decreased in the epidermis while simultaneously increasing in the dermis. Trps1 was specifically expressed in the nuclei of mesenchymal cells during hair follicle morphogenesis. An analysis of Trps1 expression during postnatal murine hair follicle cycling revealed that the protein localized to the nuclei of dermal papillae cells during telogen and anagen. Additionally, we found that Trps1 consistently localized to the nuclei of dermal papillae cells and the highly proliferative epithelial cells of mouse, rat and human hair follicles.

C/EBPalpha identifies differentiating preadipocytes around hair follicles in foetal and neonatal rat and mouse skin.

Previous studies have described a close anatomical association between hair follicles and subcutaneous adipocytes, yet little is known about the developmental origin of this preadipocyte population. Many transcription factors controlling adipogenesis in cell culture have been described; however, the molecular events governing the process of adipogenesis in rodent skin in vivo are largely unknown. In this study, we investigated the onset and progression of adipocyte differentiation in the skin of foetal and newborn rats and mice. We first analysed the temporo-spatial expression pattern of the transcription factor C/EBPalpha, a key player in adipocyte differentiation. Oil red O staining was then used to identify the presence of lipid within mature adipocytes in the same skin samples. In both species, nuclear staining of C/EBPalpha was first seen in cells around and below the bases of fully formed hair follicles in foetal dermis between 2 and 3 days before birth. Over time, increasing numbers of cells became labelled with C/EBPalpha, predominantly located between, rather than below, the hair follicles. Oil red O staining followed exactly the same pattern seen with the C/EBPalpha antibody, but with a delay of 12-24 h, and histomorphometry showed that the C/EBPalpha labelled cells matured into lipid filled adipocytes. These data show that C/EBPalpha is a useful developmental marker of preadipocytes in vivo. The close developmental association and physical proximity between the lower follicle and surrounding preadipocytes leads us to postulate that follicles control local adipogenic events, via signalling or by contributing to the preadipocyte pool.

Tissue engineering of human hair follicles using a biomimetic developmental approach.

Human skin constructs (HSCs) have the potential to provide an effective therapy for patients with significant skin injuries and to enable human-relevant drug screening for skin diseases; however, the incorporation of engineered skin appendages, such as hair follicles (HFs), into HSCs remains a major challenge. Here, we demonstrate a biomimetic approach for generation of human HFs within HSCs by recapitulating the physiological 3D organization of cells in the HF microenvironment using 3D-printed molds. Overexpression of Lef-1 in dermal papilla cells (DPC) restores the intact DPC transcriptional signature and significantly enhances the efficiency of HF differentiation in HSCs. Furthermore, vascularization of hair-bearing HSCs prior to engraftment allows for efficient human hair growth in immunodeficient mice. The ability to regenerate an entire HF from cultured human cells will have a transformative impact on the medical management of different types of alopecia, as well as chronic wounds, which represent major unmet medical needs.