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.

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.

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.

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.

Hair Follicle Dermal Cells Support Expansion of Murine and Human Embryonic and Induced Pluripotent Stem Cells and Promote Haematopoiesis in Mouse Cultures.

In the hair follicle, the dermal papilla (DP) and dermal sheath (DS) support and maintain proliferation and differentiation of the epithelial stem cells that produce the hair fibre. In view of their regulatory properties, in this study, we investigated the interaction between hair follicle dermal cells (DP and DS) and embryonic stem cells (ESCs); induced pluripotent stem cells (iPSCs); and haematopoietic stem cells. We found that coculture of follicular dermal cells with ESCs or iPSCs supported their prolonged maintenance in an apparently undifferentiated state as established by differentiation assays, immunocytochemistry, and RT-PCR for markers of undifferentiated ESCs. We further showed that cytokines that are involved in ESC support are also expressed by cultured follicle dermal cells, providing a possible explanation for maintenance of ES cell stemness in cocultures. The same cytokines were expressed within follicles in situ in a pattern more consistent with a role in follicle growth activities than stem cell maintenance. Finally, we show that cultured mouse follicle dermal cells provide good stromal support for haematopoiesis in an established coculture model. Human follicular dermal cells represent an accessible and readily propagated source of feeder cells for pluripotent and haematopoietic cells and have potential for use in clinical applications.

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.

What Lies Beneath: Wnt/ß-Catenin Signaling and Cell Fate in the Lower Dermis.

Dermal cell populations are markedly heterogeneous, and they have the capacity to differentiate into dynamic and complex dermal cell compartments. However, the regulatory processes that govern the establishment of each dermal subset remain unknown. Mastrogiannaki et al. provide evidence of Wnt/ß-catenin signaling controlling adipogenic differentiation in the developing reticular dermis. They also show that overexpression of localized Wnt converts dermal adipose cells into a distinct fibroblast subtype, which leads to fibrosis and disrupted hair follicle cycling. These findings highlight the multifaceted roles of Wnt signaling in the normal development and pathology of skin, including the establishment of dermal identity. Further understanding of Wnt involvement and uncovering the roles of specific Wnt ligands could be useful for discovering new therapeutic targets in treating fibrosis-related disorders.

Cbx4 maintains the epithelial lineage identity and cell proliferation in the developing stratified epithelium.

During development, multipotent progenitor cells establish lineage-specific programmers of gene activation and silencing underlying their differentiation into specialized cell types. We show that the Polycomb component Cbx4 serves as a critical determinant that maintains the epithelial identity in the developing epidermis by repressing nonepidermal gene expression programs. Cbx4 ablation in mice results in a marked decrease of the epidermal thickness and keratinocyte (KC) proliferation associated with activation of numerous neuronal genes and genes encoding cyclin-dependent kinase inhibitors (p16/p19 and p57). Furthermore, the chromodomain- and SUMO E3 ligase-dependent Cbx4 activities differentially regulate proliferation, differentiation, and expression of nonepidermal genes in KCs. Finally, Cbx4 expression in KCs is directly regulated by p63 transcription factor, whereas Cbx4 overexpression is capable of partially rescuing the effects of p63 ablation on epidermal development. These data demonstrate that Cbx4 plays a crucial role in the p63-regulated program of epidermal differentiation, maintaining the epithelial identity and proliferative activity in KCs via repression of the selected nonepidermal lineage and cell cycle inhibitor genes.