George Cotsarelis

Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells

Despite the obvious importance of epithelial stem cells in tissue homeostasis and tumorigenesis, little is known about their specific location or biological characteristics. Using 3H-thymidine labeling, we have identified a subpopulation of corneal epithelial basal cells, located in the peripheral cornea in a region called limbus, that are normally slow cycling, but can be stimulated to proliferate in response to wounding and to a tumor promotor, TPA. No such cells can be detected in the central corneal epithelium, suggesting that corneal epithelial stem cells are located in the limbus. A comparison of various types of epithelial stem cells revealed a common set of features, including their preferred location, pigment protection, and growth properties, which presumably play a crucial role in epithelial stem cell function.

β-Catenin Controls Hair Follicle Morphogenesis and Stem Cell Differentiation in the Skin

beta-Catenin is an essential molecule in Wnt/wingless signaling, which controls decisive steps in embryogenesis. To study the role of beta-catenin in skin development, we introduced a conditional mutation of the gene in the epidermis and hair follicles using Cre/loxP technology. When beta-catenin is mutated during embryogenesis, formation of placodes that generate hair follicles is blocked. We show that beta-catenin is required genetically downstream of tabby/downless and upstream of bmp and shh in placode formation. If beta-catenin is deleted after hair follicles have formed, hair is completely lost after the first hair cycle. Further analysis demonstrates that beta-catenin is essential for fate decisions of skin stem cells: in the absence of beta-catenin, stem cells fail to differentiate into follicular keratinocytes, but instead adopt an epidermal fate.

Capturing and profiling adult hair follicle stem cells

The hair follicle bulge possesses putative epithelial stem cells. Characterization of these cells has been hampered by the inability to target bulge cells genetically. Here, we use a Keratin1-15 (Krt1-15, also known as K15) promoter to target mouse bulge cells with an inducible Cre recombinase construct or with the gene encoding enhanced green fluorescent protein (EGFP), which allow for lineage analysis and for isolation of the cells. We show that bulge cells in adult mice generate all epithelial cell types within the intact follicle and hair during normal hair follicle cycling. After isolation, adult Krt1-15-EGFP-positive cells reconstituted all components of the cutaneous epithelium and had a higher proliferative potential than Krt1-15-EGFP-negative cells. Genetic profiling of hair follicle stem cells revealed several known and unknown receptors and signaling pathways important for maintaining the stem cell phenotype. Ultimately, these findings provide potential targets for the treatment of hair loss and other disorders of skin and hair.

Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding

The mammalian hair follicle is a complex ‘mini-organ’ thought to form only during development; loss of an adult follicle is considered permanent. However, the possibility that hair follicles develop de novo following wounding was raised in studies on rabbits, mice and even humans fifty years ago. Subsequently, these observations were generally discounted because definitive evidence for follicular neogenesis was not presented. Here we show that, after wounding, hair follicles form de novo in genetically normal adult mice. The regenerated hair follicles establish a stem cell population, express known molecular markers of follicle differentiation, produce a hair shaft and progress through all stages of the hair follicle cycle. Lineage analysis demonstrated that the nascent follicles arise from epithelial cells outside of the hair follicle stem cell niche, suggesting that epidermal cells in the wound assume a hair follicle stem cell phenotype. Inhibition of Wnt signalling after re-epithelialization completely abrogates this wounding-induced folliculogenesis, whereas overexpression of Wnt ligand in the epidermis increases the number of regenerated hair follicles. These remarkable regenerative capabilities of the adult support the notion that wounding induces an embryonic phenotype in skin, and that this provides a window for manipulation of hair follicle neogenesis by Wnt proteins. These findings suggest treatments for wounds, hair loss and other degenerative skin disorders.

Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss.

Developmental abnormalities, cancer, and premature aging each have been linked to defects in the DNA damage response (DDR). Mutations in the ATR checkpoint regulator cause developmental defects in mice (pregastrulation lethality) and humans (Seckel syndrome). Here we show that eliminating ATR in adult mice leads to defects in tissue homeostasis and the rapid appearance of age-related phenotypes, such as hair graying, alopecia, kyphosis, osteoporosis, thymic involution, fibrosis, and other abnormalities. Histological and genetic analyses indicate that ATR deletion causes acute cellular loss in tissues in which continuous cell proliferation is required for maintenance. Importantly, thymic involution, alopecia, and hair graying in ATR knockout mice were associated with dramatic reductions in tissue-specific stem and progenitor cells and exhaustion of tissue renewal and homeostatic capacity. In aggregate, these studies suggest that reduced regenerative capacity in adults via deletion of a developmentally essential DDR gene is sufficient to cause the premature appearance of age-related phenotypes.

Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment

BACKGROUND The mammalian hair represents an unparalleled model system to understand both developmental processes and stem cell biology. The hair follicle consists of several concentric epithelial sheaths with the outer root sheath (ORS) forming the outermost layer. Functionally, the ORS has been implicated in the migration of hair stem cells from the stem cell niche toward the hair bulb. However, factors required for the differentiation of this critical cell lineage remain to be identified. Here, we describe an unexpected role of the HMG-box-containing gene Sox9 in hair development. RESULTS Sox9 expression can be first detected in the epithelial component of the hair placode but then becomes restricted to the outer root sheath (ORS) and the hair stem cell compartment (bulge). Using tissue-specific inactivation of Sox9, we demonstrate that this gene serves a crucial role in hair differentiation and that skin deleted for Sox9 lacks external hair. Strikingly, the ORS acquires epidermal characteristics with ectopic expression of GATA3. Moreover, Sox9 knock hair show severe proliferative defects and the stem cell niche never forms. Finally, we show that Sox9 expression depends on sonic hedgehog (Shh) signaling and demonstrate overexpression in skin tumors in mouse and man. CONCLUSIONS We conclude that although Sox9 is dispensable for hair induction, it directs differentiation of the ORS and is required for the formation of the hair stem cell compartment. Our genetic analysis places Sox9 in a molecular cascade downstream of sonic hedgehog and suggests that this gene is involved in basal cell carcinoma.

The longest telomeres: a general signature of adult stem cell compartments

Identification of adult stem cells and their location (niches) is of great relevance for regenerative medicine. However, stem cell niches are still poorly defined in most adult tissues. Here, we show that the longest telomeres are a general feature of adult stem cell compartments. Using confocal telomere quantitative fluorescence in situ hybridization (telomapping), we find gradients of telomere length within tissues, with the longest telomeres mapping to the known stem cell compartments. In mouse hair follicles, we show that cells with the longest telomeres map to the known stem cell compartments, colocalize with stem cell markers, and behave as stem cells upon treatment with mitogenic stimuli. Using K15-EGFP reporter mice, which mark hair follicle stem cells, we show that GFP-positive cells have the longest telomeres. The stem cell compartments in small intestine, testis, cornea, and brain of the mouse are also enriched in cells with the longest telomeres. This constitutes the description of a novel general property of adult stem cell compartments. Finally, we make the novel finding that telomeres shorten with age in different mouse stem cell compartments, which parallels a decline in stem cell functionality, suggesting that telomere loss may contribute to stem cell dysfunction with age.

The human hair follicle immune system: cellular composition and immune privilege.

The immunology of the hair follicle, its relationship with the ‘skin immune system’ and its role in hair diseases remain biologically intriguing and clinically important. In this study, we analysed the immunoreactivity patterns of 15 immunodermatological markers to determine the cellular composition and immune privilege of the human hair follicle immune system in anagen VI (growth phase). The most prominent cells located in or around the hair follicle were Langerhans cells, CD4+ or CD8+ T cells, macrophages and mast cells, whereas B cells, natural killer cells and γδ T cells were found very rarely. Langerhans cells (CD1a+, major histocompatibility complex, MHC class II+), and T cells (CD4+ or CD8+) were predominantly distributed in the distal hair follicle epithelium, whereas macrophages (CD68+, MHC class II+) and mast cells (Giemsa+) were located in the perifollicular connective tissue sheath. Transmission electron microscopy confirmed low numbers of immune cells in the proximal hair follicle epithelium, and very few macrophages and Langerhans cells were seen in the dermal papilla. Melanophages were observed in the connective tissue sheath and dermal papilla. MHC class I (HLA‐A, ‐B, ‐C) and β2‐microglobulin immunoreactivity was found on most skin cells, but was substantially reduced on isthmus keratinocytes and virtually absent in the proximal hair follicle epithelium. Apart from the absence of Fas ligand immunoreactivity, the sharply reduced numbers of T cells and Langerhans cells, and the virtual absence of MHC class I expression all suggest that the anagen proximal hair follicle constitutes an area of immune privilege within the hair follicle immune system, whose collapse may be crucial for the pathogenesis of alopecia areata.

Towards a molecular understanding of hair loss and its treatment

Most common forms of hair loss (alopecia) are caused by aberrant hair follicle cycling and changes in hair follicle morphology. However, current treatments for alopecia do not specifically target these processes. We are now beginning to identify the molecules and molecular pathways that control normal hair follicle formation, cycling and growth. In parallel, new techniques are being developed for delivering molecules to hair follicles. Here, we outline the characteristics of common hair loss diseases, and discuss ways in which recent advances in hair follicle biology could be translated into effective therapies for these conditions.

Review of hair follicle dermal cells

Hair follicle stem cells in the epithelial bulge are responsible for the continual regeneration of the hair follicle during cycling. The bulge cells reside in a niche composed of dermal cells. The dermal compartment of the hair follicle consists of the dermal papilla and dermal sheath. Interactions between hair follicle epithelial and dermal cells are necessary for hair follicle morphogenesis during development and in hair reconstitution assays. Dermal papilla and dermal sheath cells express specific markers and possess distinctive morphology and behavior in culture. These cells can induce hair follicle differentiation in epithelial cells and are required in hair reconstitution assays either in the form of intact tissue, dissociated freshly prepared cells or cultured cells. This review will focus on hair follicle dermal cells since most therapeutic efforts to date have concentrated on this aspect of the hair follicle, with the idea that enriching hair-inductive dermal cell populations and expanding their number by culture while maintaining their properties, will establish an efficient hair reconstitution assay that could eventually have therapeutic implications.