Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog

Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients.     

Multiple potential roles of thymosin β4 in the growth and development of hair follicles

The hair follicle (HF) is an important mini-organ of the skin, composed of many types of cells. Dermal papilla cells are important signalling components that guide the proliferation, upward migration and differentiation of HF stem cell progenitor cells to form other types of HF cells. Thymosin β4 (Tβ4), a major actin-sequestering protein, is involved in various cellular responses and has recently been shown to play key roles in HF growth and development. Endogenous Tβ4 can activate the mouse HF cycle transition and affect HF growth and development by promoting the migration and differentiation of HF stem cells and their progeny. In addition, exogenous Tβ4 increases the rate of hair growth in mice and promotes cashmere production by increasing the number of secondary HFs (hair follicles) in cashmere goats. However, the molecular mechanisms through which Tβ4 promotes HF growth and development have rarely been reported. Herein, we review the functions and mechanisms of Tβ4 in HF growth and development and describe the endogenous and exogenous actions of Tβ4 in HFs to provide insights into the roles of Tβ4 in HF growth and development.     

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle.As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90⁺ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.Key words: CD90, mesenchymal stem cells, hair follicle, dog.The hair follicle represents an important stem cell niche in the skin. It contains dermal and epithelial stem populations that display distinct properties and localization. While epithelial stem cells reside in the middle region of the hair follicle outer root sheath (Schneider et al., 2009; Lyle et al., 1998; Cotsarelis et al., 1990), dermal stem cells are located in the dermal sheath (DS) (Jahoda, 2003; Jahoda and Reynolds, 2001).The dermal sheath, or fibrous root sheath, is a layer of dense connective tissue that extends along the hair follicle, from the bulb up to the infundibulum. In the anagen hair follicle, it is comprised of mesenchymal cells located among collagen and elastic fibres.The cells are flat and elongated while collagen fibres form a circular inner layer and a longitudinal outer layer in the lower part of hair follicle (VonTscharner and Suter, 1994; Jahoda et al., 1992). At the base of the hair follicle, the DS is connected to the dermal papilla (Scott et al., 2000). The basement membrane, or glassy membrane, separates the DS from the epithelial component of the hair follicle (Scott et al., 2000).Follicular dermal stem cells have a mesenchymal origin and share many properties common to bone marrow-derived mesenchymal stem cells (MSCs) (Hoogduijn et al., 2006). They express the MSC cell-surface marker CD90, show a high colony forming unit ability and can differentiate into several mesenchymal lineages, such as osteoblasts, adipocytes, chondrocytes and myocytes (Hoogduijn et al., 2006; Jahoda et al., 2003). They also express neuroprogenitor markers (Hoogduijn et al., 2006) and, finally, they can repopulate the haematopoietic system (Lako et al., 2002). In the literature, we can find different information about stem cell localization: the whole dermal sheath, the peri-bulbar dermal sheath, the dermal papilla (Hoogduijn et al., 2006, McElwee et al., 2003, Gharzi et al., 2003, Jahoda et al., 2003.)CD90 (Thy-1) is a small GPI-anchored protein localized in the outer leaflet of the cell membrane (Low and Kincade, 1985). This protein is present in a large number of tissues and cells, even if a great species variation has been described (Mansour Haeryfar, 2004; Tokugawa et al., 1997; McKenzle and Fabre, 1981). CD90 plays a role in cell-cell interaction events, including intracellular adhesion and cell recognition during development (Saalbach et al., 2000; Morris, 1985), and is considered an important stem cell marker; for this last reason it is commonly used to identify mesenchymal stem cells in vitro (Kern et al., 2007; Yoshimura et al., 2006; Le Blanc and Ringdén, 2006; Pittenger et al., 1999). Furthermore, it has been identified in other kinds of stem cells such as haematopoietic progenitor cells (Craig et al., 1993) and hepatic progenitor cells in the human fetal liver (Masson et al., 2006).The hair follicle is the focus of increasing interest because it contains well defined stem cell populations that exhibit various developmental properties. We retain that in dogs, as already demonstrated in other species (Hoogduijn et al., 2006; Zhang et al., 2006; Jahoda et al., 2003; Lako et al., 2002), this organ may be a suitable and accessible source for both epithelial and mesenchymal stem cells that may be isolated and in vitro cultured. Since it is possible to take skin samples without injuring the patient, we chose the hair follicle to study and identify stem cells with the future purpose of using them in regenerative medicine.Dogs are affected by several skin diseases and some of them may be related to alterations of somatic stem cells. We retain that the study of hair follicle stem cell biology may improve our knowledge of etiology and pathogenesis of these skin diseases.In previous works we investigated the stem cells in dog hair follicles; we identified the location of putative epithelial stem cells at the isthmus and described the bulge-like region (Pascucci et al., 2006; Mercati et al., 2008). To the authors’ knowledge, there are no data available neither concerning the localization of DS stem cells nor concerning the expression of CD90 in the hair follicle as regards the canine species. Therefore, in this study, we described the morphological characteristics of DS cells and examined the immunohistochemical localization of CD90 protein in dog hair follicles with both light and transmission electron microscopy. The aim of our study is to observe the dermal sheath cells encompassing the hair follicle and to determine where CD90⁺ cells reside. CD90 is one of the main markers used to identify mesenchymal stem cells and it has been observed in stem cells isolated from the dermal sheath of hair follicles (Hoogduijn et al.,2006). For this reason, we suppose that CD90 protein can help us to identify the hair follicle dermal stem compartment in dog.     

Hair follicle renewal: organization of stem cells in the matrix and the role of stereotyped lineages and behaviors

Hair follicles (HFs) are renewed via multipotent stem cells located in a reservoir (the bulge); however, little is known about how they generate multi-tissue HFs from a proliferative zone (the matrix). To address this issue, we temporally induced clonal labeling during HF growth. Challenging the prevailing hypothesis, we found that the matrix contains restricted self-renewing stem cells for each inner structure. These cells are located around the dermal papilla forming a germinative layer. They occupy different proximodistal sectors and produce differentiated cells along the matrix radial axis via stereotyped lineages and cell behavior. By contrast, the outer layer of HFs displays a mode of growth involving apoptosis that coordinates the development of outer and inner structures. HF morphology is therefore determined by the organization of cell fates along the proximodistal axis and by cell behavior along the radial (lateral) axis in the matrix. Thus, our studies suggest that fate and behavior are organized by two systems (uncoupled), and this uncoupling may represent a fundamental way to simplify morphogenesis.     

Stem Cell-Associated Marker Expression in Canine Hair Follicles

Functional hair follicle (HF) stem cells (SCs) are crucial to maintain the constant recurring growth of hair. In mice and humans, SC subpopulations with different biomarker expression profiles have been identified in discrete anatomic compartments of the HF. The rare studies investigating canine HF SCs have shown similarities in biomarker expression profiles to that of mouse and human SCs. The aim of our study was to broaden the current repertoire of SC-associated markers and their expression patterns in the dog. We combined analyses on the expression levels of CD34, K15, Sox9, CD200, Nestin, LGR5 and LGR6 in canine skin using RT-qPCR, the corresponding proteins in dog skin lysates, and their expression patterns in canine HFs using immunohistochemistry. Using validated antibodies, we were able to define the location of CD34, Sox9, Keratin15, LGR5 and Nestin in canine HFs and confirm that all tested biomarkers are expressed in canine skin. Our results show similarities between the expression profile of canine, human and mouse HF SC markers. This repertoire of biomarkers will allow us to conduct functional studies and investigate alterations in the canine SC compartment of different diseases, like alopecia or skin cancer with the possibility to extend relevant findings to human patients.     

Disruption of Smad4 in Mouse Epidermis Leads to Depletion of Follicle Stem Cells

Follicle stem cells (SCs) residing in the bulge region of a hair follicle (HF) can give rise to multiple lineages during the hair cycle and wound healing. The activation and self-renewal of follicle SCs must be tightly regulated to maintain the HF and epidermal homeostasis. Here we show that, in young mice, disruption of epidermal Smad4, the common mediator of transforming growth factor-β (TGF-β) signaling, stimulated the activation of follicle SCs, leading to hyperplasia of interfollicular epidermis (IFE), HFs, and sebaceous glands (SGs). Increased proliferation of follicle SCs ultimately exhausted the SC niche, indicated by the loss of bromodeoxyuridine (BrdU) label–retaining cells (LRCs), loss of keratin 15 (K15), and CD34 expression. In addition, the colony-forming efficiency of Smad4 mutant keratinocytes was significantly decreased. Increased nuclear localization of β-catenin and increased expression of c-Myc were correlated with the overactivation and depletion of follicle SCs. We concluded that Smad4 plays a pivotal role in follicle SC maintenance.     

Hair follicle stem cells: walking the maze

The discovery of epithelial stem cells (eSCs) in the bulge region of the outer root sheath of hair follicles in mice and man has encouraged research into utilizing the hair follicle as a therapeutic source of stem cells (SCs) for regenerative medicine, and has called attention to the hair follicle as a highly instructive model system for SC biology. Under physiological circumstances, bulge eSCs serve as cell pool for the cyclic regeneration of the anagen hair bulb, while they can also regenerate the sebaceous gland and the epidermis after injury. More recently, melanocyte SCs, nestin+, mesenchymal and additional, as yet ill-defined "stem cell" populations, have also been identified in or immediately adjacent to the hair follicle epithelium, including in the specialized hair follicle mesenchyme (connective tissue sheath), which is crucial to wound healing. Thus the hair follicle and its adjacent tissue environment contain unipotent, multipotent, and possibly even pluripotent SC populations of different developmental origin. It provides an ideal model system for the study of central issues in SC biology such as plasticity and SC niches, and for the identification of reliable, specific SC markers, which distinguish them from their immediate progeny (e.g. transient amplifying cells). The current review attempts to provide some guidance in this growing maze of hair follicle-associated SCs and their progeny, critically reviews potential or claimed hair follicle SC markers, highlights related differences between murine and human hair follicles, and defines major unanswered questions in this rapidly advancing field.     

Differential expression of stem cell markers in human follicular bulge and interfollicular epidermal compartments

Although skin contains a number of stem cell repositories, their characterization has been hindered by a lack of specific markers and an unclear in vivo localization. In this study, we whole mounted single human scalp hair follicles and examined their profiles using in situ immunohistochemistry and multicolor immunofluorescence in search of markers to distinguish between stem cells residing in the interfollicular epidermis (IFE) and bulge. Our study revealed that expression of several biomarkers localized uniquely to the basal IFE (CD34 and CD117), bulge region (CD200), or both (CK15, CD49f, and CD29). In addition, we found that both basal IFE and bulge stem cells did not express CD71 or CD24 suggesting their potential utility as negative selection markers. Dermal papilla but not basal IFE or bulge stem cells expressed CD90, making it a potential positive selection marker for dermal hair follicle stem cells. The markers tested in this study may enable pursuit of cell sorting and purification strategies aimed at determining each stem cell population’s unique molecular signature.     

The human hair follicle contains two distinct K19 positive compartments in the outer root sheath: a unifying hypothesis for stem cell reservoir?

Up to now, the localization of stem cells in human anagen hair follicle relied on three complementary approaches; namely, detection of slow cycling cells, detection of high colony forming cells, and differential immunohistochemical staining. These techniques, however, gave conflicting results since stem cells were localized either as long label retaining cells in the so-called bulge area or as high colony forming cells in the lower third of the follicle. In the present study we investigated the expression of cytokeratin 19, a marker for putative stem cell-containing epithelial compartments, in order to characterize stem cell distribution in the human hair follicle throughout the hair cycle. We found that anagen human hair follicles contain two distinct reservoirs for stem cells located in the upper and lower thirds of the follicle. These two reservoirs fuse during the catagentelogen transition phase and individualize again in the newly forming anagen hair follicle.     

β1 Integrin Signaling Maintains Human Epithelial Progenitor Cell Survival In Situ and Controls Proliferation,Apoptosis and Migration of Their Progeny

β1 integrin regulates multiple epithelial cell functions by connecting cells with the extracellular matrix (ECM). While β1 integrin-mediated signaling in murine epithelial stem cells is well-studied, its role in human adult epithelial progenitor cells (ePCs) in situ remains to be defined. Using microdissected, organ-cultured human scalp hair follicles (HFs) as a clinically relevant model for studying human ePCs within their natural topobiological habitat, β1 integrin-mediated signaling in ePC biology was explored by β1 integrin siRNA silencing, specific β1 integrin-binding antibodies and pharmacological inhibition of integrin-linked kinase (ILK), a key component of the integrin-induced signaling cascade. β1 integrin knock down reduced keratin 15 (K15) expression as well as the proliferation of outer root sheath keratinocytes (ORSKs). Embedding of HF epithelium into an ECM rich in β1 integrin ligands that mimic the HF mesenchyme significantly enhanced proliferation and migration of ORSKs, while K15 and CD200 gene and protein expression were inhibited. Employing ECM-embedded β1 integrin-activating or -inhibiting antibodies allowed to identify functionally distinct human ePC subpopulations in different compartments of the HF epithelium. The β1 integrin-inhibitory antibody reduced β1 integrin expression in situ and selectively enhanced proliferation of bulge ePCs, while the β1 integrin-stimulating antibody decreased hair matrix keratinocyte apoptosis and enhanced transferrin receptor (CD71) immunoreactivity, a marker of transit amplifying cells, but did not affect bulge ePC proliferation. That the putative ILK inhibitor QLT0267 significantly reduced ORSK migration and proliferation and induced massive ORSK apoptosis suggests a key role for ILK in mediating the ß1 integrin effects. Taken together, these findings demonstrate that ePCs in human HFs require β1 integrin-mediated signaling for survival, adhesion, and migration, and that different human HF ePC subpopulations differ in their response to β1 integrin signaling. These insights may be exploited for cell-based regenerative medicine strategies that employ human HF-derived ePCs.     

Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen

The lowermost portion of the resting (telogen) follicle consists of the bulge and secondary hair germ. We previously showed that the progeny of stem cells in the bulge form the lower follicle and hair, but the relationship of the bulge cells with the secondary hair germ cells, which are also involved in the generation of the new hair at the onset of the hair growth cycle (anagen), remains unclear. Here we address whether secondary hair germ cells are derived directly from epithelial stem cells in the adjacent bulge or whether they arise from cells within the lower follicle that survive the degenerative phase of the hair cycle (catagen). We use 5-bromo-2'-deoxyuridine to label bulge cells at anagen onset, and demonstrate that the lowermost portion of the bulge collapses around the hair and forms the secondary hair germ during late catagen. During the first six days of anagen onset bulge cells proliferate and self-renew. Bulge cell proliferation at this time also generates cells that form the future secondary germ. As bulge cells form the secondary germ cells at the end of catagen, they lose expression of a biochemical marker, S100A6. Remarkably, however, following injury of bulge cells by hair depilation, progenitor cells in the secondary hair germ repopulate the bulge and re-express bulge cell markers. These findings support the notion that keratinocytes can "dedifferentiate" to a stem cell state in response to wounding, perhaps related to signals from the stem cell niche. Finally, we also present evidence that quiescent bulge cells undergo apoptosis during follicle remodeling in catagen, indicating that a subpopulation of bulge cells is not permanent.     

Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles

We previously demonstrated that keratin 15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin 15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 mo of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland, and hair follicle lineages. Intact beta-catenin dependent signaling, which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation.     

Epithelial stem cells: stepping out of their niche

In this issue of Cell, have shown that two subpopulations of cells exist within the hair follicle stem cell niche. Despite being partially differentiated, clonal populations of suprabasal bulge region cells can regenerate skin and hair follicles as well as a new stem cell niche. The findings suggest that early lineage commitments of epithelial cells in the hair follicle may be reversible.