Year: 2009
Type: article
Abstract: Stem cells possess the salient features of multipotency and self-renewal, enabling them with the ability to differentiate into the multiple cell types required to generate new tissue throughout the development and the lifetime of the organism. Their maintenance and activation during homeostasis and in response to injury provide resiliency to the organism, while their dysregulation can lead to the tissue compromise and dysfunction that underlie aging and tumorigenesis. Large strides have been made to understand how stem cells are regulated in an effort to develop novel treatments in the fields of cancer, aging and regenerative medicine. To this end, several animal and organ models have been vital to the synthesis of principles explaining adult stem cell maintenance and differentiation. The hair follicle (HF) has emerged into the forefront as one of the most illustrative and accessible models to examine adult stem cell regulation in mammals, attributed to its ability to predictably cycle through well-characterized phases of degeneration (catagen), rest (telogen) and regeneration (anagen) throughout life. The ability of the follicle to maintain its regenerative capacity rests upon resident stem cells that lie in a region called the bulge, located at the base of the upper permanent portion of the follicular outer root sheath (ORS). 1 Similar to many other somatic stem cells, bulge cells are slow cycling in nature. This feature has permitted their initial identification and isolation as label-retaining cells (LRCs) that can retain a pulse of nucleotide label following a long chase period and is frequently regarded as a defining characteristic of the HF stem cell. 1,2 In addition, the availability of several immunohistochemical markers, including keratin-15 (K15) and CD34, that specifically label murine follicular stem cells has given researchers the ability to carefully examine the signals required for adult stem cell activation and renewal. 3,4 Subsequently, different groups have utilized these tools to label and track the fate of bulge cells and their progeny during homeostasis and following epithelial injury. These studies demonstrate that the bulge serves as a repository of long-lived multipotent stem cells, imparted with the capacity to differentiate into all cell types that constitute the lower cyclic portion of the HF, as well as the interfollicular epidermis during wound repair. 5–9 Since then, the explosion of genetic and molecular tools available to target and profile specific HF cell populations in mice has fueled the rapid growth of information regarding the molecular mechanisms governing follicular morphogenesis and the hair cycle. Additionally, the identification of other stem cell markers has provided further resolution of the bulge and has revealed that the bulge is composed of distinct subpopulations. 4,10–14 Despite these advancements, our understanding of how HF stem cells are initially generated and what cells constitute the HF stem cell niche is still incomplete. In particular, it is unclear if immunohistochemically distinct bulge populations represent stem cells with different lineage commitment and self-renewing potential. Furthermore, even less is known about how other cell types, including dermal fibroblasts, regulate their identity and behavior during development and the hair cycle. This two-part review will focus on some of the recent studies that have examined these questions and have resurrected earlier seminal efforts to define the germinative origin of the anagen follicle. In addition to label-retention studies, several experimental tools have permitted the characterization of stem cells and the molecular mechanisms that regulate them. Although a comprehensive discussion of the available assays is beyond the scope and purpose of this review, we briefly descibe here a few of the techniques commonly used to study HF stem cells. An important advancement in developmental biology has been the creation of mice that inducibly and irreversibly label specific cell types. This model has been critical for in vivo fate-mapping, demonstrating the lineage potential of specific cell populations. Here, transgenic mice designed to express a detectable reporter (e.g. LacZ/ß-galactosidase or a fluorescent protein) driven by the ubiquitous Rosa26 promoter are crossed with mice that express a chimeric Cre recombinase fused to a mutated hormone-binding domain (estrogen or progesterone receptor), whose expression is driven by a cell-type-specific promoter (Fig. 1). The reporter gene contains a stop signal flanked by two loxP sites, requiring Cre-mediated recombination at the loxP sequences to permit reporter expression. This occurs only once the chimeric Cre protein (CreER or CrePR) localizes to the nucleus after hormone (Tamoxifen or RU486) stimulation, yielding stable expression of the reporter. As the recombined locus is heritable, induced cells and their progeny will express the reporter, allowing for lineage tracing. This system has been invaluable for demonstrating the multipotency of stem cells in vivo and was used to show that K15+ bulge cells expressing CrePR give rise to all HF cell types. 8 In vivo lineage-tracing. In this example, mice expressing both the lacz reporter under the ubiquitous Rosa26 promoter and CrePR under the K15 promoter undergo Cre-mediated removal of the STOP signal when exposed to RU486. This permits LacZ/ß-galactosidase (ß-gal) expression, which can be visualized histologically. All K15+ cells temporally exposed to RU486 and their progeny will express ß-gal. Another method used to demonstrate lineage potential is the reconstitution assay. Briefly, this experiment entails isolating specific cultured or fresh populations of epidermal keratinocytes derived from various sources (e.g. transgenic, retrovirally transduced) and mixing them with neonatal dermal fibroblasts followed by implantation into the back skins or kidney capsules of immunodeficient mice. Engraftments are later analyzed for HF formation and growth. Although this assay may avert the costs and time needed to generate transgenic mice, it is unclear how well this model recapitulates in vivo cell-fate potential. Colony-forming assays are based upon the ability of stem cells to form large holoclones in culture that can be passaged indefinitely, while committed cells terminally differentiate after a finite number of proliferative cycles. Despite their caveats, these and other tools, including microarray gene analysis of specific HF populations, have fostered our understanding and characterization of stem cells in hair morphogenesis and regeneration. In mice, the HF is identified as a wave beginning at E14.5 (Fig. 2). At this stage, the underlying mesenchyme provides a signal to the epidermis to initiate appendage specification. The first identifiable HF structure is the placode, which expresses several markers, many of which are critical for HF morphogenesis. Most notably, regulators of Wnt/ß-catenin, ectodysplasin receptor (EDAR), fibroblast growth factor receptor (FGFR) and bone morphogenetic protein receptor (BMPR) signaling play important roles at this stage to dictate HF specification and patterning. 15,16 Subsequent reciprocal epithelial-mesenchymal interactions follow to promote HF downgrowth and differentiation and simultaneous maturation of the closely associated dermal papilla (DP), which becomes enveloped by proliferating matrix cells. Matrix cells terminally differentiate into the various layers comprising the inner root sheath (IRS) and hair shaft. The ORS is continuous with the epidermal basal layer and grows downward, separated from the IRS by the companion layer. The sebaceous gland develops once the HF reaches maturity and is largely maintained by a unipotent source of progenitors. 17 The interaction between the DP and follicular epithelium is critical for regulating hair development during ontogeny and stem cell behavior throughout the postnatal hair cycle. Hair follicle (HF) morphogenesis. Reciprocal epithelial-mesenchymal signals (red arrows) guide development of the HF and maturation of the dermal papilla (DP), which remains closely associated with the follicle base postnatally. It is felt that the dermis provides ‘the first dermal signal’ to direct follicular specification and HF placode formation. In turn, specified follicular cells provide a reciprocal signal to the dermis to initiate formation of the DP. In mice, the first growth phase of pelage follicles ends at ∼P17 when catagen begins, which lasts 3–4 days. First telogen begins at ∼P21 and lasts 3 days. Similar histochemical and morphological changes occur during de novo HF neogenesis in adult mice. Putative bulge stem cells (green cells), Sox9, T-cell factor-3 (Tcf3), nuclear factor of activated T cells-1 (NFATc1), lymphoid enhancer-binding factor-1 (Lef-1) and LIM homeobox protein-2 (Lhx2) are transcription factors. It has been unclear how and when HF stem cells are specified, as both HF-restricted K15 and CD34-positive cells do not appear until the initiation of postnatal hair cycles. Consequently, embryonic hair morphogenesis was felt by some to occur independently of HF stem cells. Using more recently identified stem cell markers (e.g. Sox9, NFATc1) and label-retaining studies, Nowak et al. have recently shown that putative HF stem cells can be identified early during hair morphogenesis at the placode/germ stage (Fig. 2). 18 In support of a previous study 19, these cells localize to the upper ORS during development and give rise to the characteristic bulge population identified at first telogen. In addition, they lend cells to the growing follicle during development, suggesting that these presumptive HF stem cells are not just reserved for fueling postnatal hair cycles. In the absence of embryonic Sox9 expression, HFs prematurely degenerate during the first growth phase, owing to the progressive loss of matrix cells. One possible explanation is that Sox9 expression is required for the maintenance of HF stem cells, which contribute terminally differentiating cells to the matrix. However, it is unclear if the lack of putative stem cells in itself explains this phenotype, as Sox9 expression extends beyond the label-retaining region. Additionally, it is still unknown if Sox9-expressing cells of the label-retaining region function as veritable stem cells. Definitive lineage-tracing experiments will be important to determine if these cells can give rise to all HF cell types during development. Nonetheless, this study demonstrates the origination of a label-retaining population early during development that gives rise to the bulge population postnatally. How these cells are specified and their derivation during development remain unknown. Until recently, the ability to generate mature pilosebaceous units de novo in adult mice had only previously been demonstrated following genetic manipulation (e.g. epidermal overexpression of stabilized ß-catenin). 5,20,21 However, the recent demonstration of adult HF neogenesis after full-thickness skin wounding has shown that the inductive properties of the skin can be reclaimed during wound healing. 22 Interestingly, it is possible that typically unipotent interfollicular epidermal keratinocytes may undergo HF specification in adult mice, as bulge-derived HF cells do not significantly contribute to nascent HFs in this study. Moreover, neogenic HFs generate new non-bulge-derived HF stem cells that allow them to cycle independently of surrounding HFs from intact skin. Precisely how wound repair orchestrates HF neogenesis is under current investigation. Understanding how the embryo establishes an HF stem cell population will be a prerequisite for elucidating how adult skin can generate new follicular stem cells. This first part of the review is intended to serve as a preface and background for the following second portion, which will focus on recent articles that have refined our definition of the bulge and have highlighted the heterogeneity of the HF stem cell niche. Peggy Myung, MD, PhD 1 Thomas Andl, PhD 2 and Mayumi Ito, PhD 3 We thank Drs. Stephen Somach and Arlene Rosenberg for their mentorship and for providing critical review of these manuscripts.
Source: Journal of Cutaneous Pathology
Authors: Peggy Myung, Thomas Andl, Mayumi Ito
Language: English
Topic: Hair Growth and Disorders
Subfield: Urology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W4241169995