Bulge Stem Cells Research Articles

Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin

Skin hair follicles (HF) contain bulge stem cells (SC) that regenerate HFs during hair cycles, and repair skin epithelia following injury. As natural aging is associated with decreased skin repair capacity in humans, we have investigated the impact of age on human scalp HF bulge cell number and function. Here, we isolated human bulge cells, characterized as CD200+/KRT15+/KRT19+ cells of the HF, by dissection-combined CD200 selection in young and aged human skin. Targeted transcriptional profiling indicates that KRT15, KRT19, Dkk3, Dkk4, Tcf3, S100A4, Gas1, EGFR and CTGF/CCN2 are also preferentially expressed by human bulge cells, compared to differentiated HF keratinocytes (KC). Our results demonstrate that aging does not alter expression or localization of these HF SC markers. In addition, we could not detect significant differences in HF density or bulge cell number between young and aged human scalp skin. Interestingly, hedgehog (Hh) signaling is activated in human bulge cells in vivo, and down-regulated in differentiated HF KCs, both in young and aged skin. In addition, activation of Hh signaling by lentivirus-mediated overexpression of transcription factor Gli1 induces transcription of HF SC markers KRT15, KRT19, and Gas1, in cultured KCs. Together with previously reported knock-out mouse results, these data suggest a role for Hh signaling in maintaining bulge cell phenotype in young and aged human skin.

Nestin in Human Skin: Exclusive Expression in Intramesenchymal Skin Compartments and Regulation by Leptin

Cutaneous nestin+ cells are of substantial interest in regenerative medicine. However, the location of nestin+ cells in situ remains controversial. We therefore sought to determine their location in female human scalp skin, using stringently controlled immunohistochemical techniques, Western blot analysis, and in situ hybridization and complementing those techniques with relative and quantitative reverse transcriptase-PCR of enzymatically digested or laser-capture microdissected human hair follicle (HF) compartments. We show here that the immunoreactivity (IR) patterns obtained with anti-nestin antibodies are highly dependent on the tissue-fixation and immunohistochemical methods used. NESTIN mRNA could not be detected within HF-associated epithelial cells in situ or in RNA extracts of the microdissected HF epithelium. Instead, NESTIN transcripts were found only in intramesenchymal skin compartments. Individual cells showing both, specific nestin IR and NESTIN mRNA were detectable in the connective-tissue sheaths of human HFs, sebaceous and sweat glands. Moreover, stimulation of organ-cultured human scalp skin with the adipokine leptin increased the number of nestin+ cells in these intramesenchymal skin locations, whereas no specific nestin IR could be induced by leptin within the HF epithelium, including the bulge. Therefore, nestin expression at the gene and protein levels in human scalp skin is restricted to the periappendage mesenchyme and can be stimulated by leptin.

Spatiotemporal expression pattern of neuroepithelial stem cell marker nestin suggests a role in dermal homeostasis, neovasculogenesis, and tumor stroma development: A study on embryonic and adult human skin

Whereas keratinocytic bulge stem cells are well characterized, comparably little is known about cutaneous mesenchymal stem cells. The follicular connective tissue sheath is proposed as a niche for dermal stem cells. Because the neuroepithelial stem cell marker nestin represents a marker for mesenchymal stem cells in various tissues, our aim was to characterize its spatiotemporal expression pattern in the skin with special reference to the follicular mesenchyme. We studied immunohistochemically nestin expression over the course of human cutaneous embryogenesis, in postnatal skin, in scalp wounds, and in the peritumoral stroma of basal cell carcinomas and compared its expression with that of other known mesenchymal markers. Nestin is expressed throughout the entire early embryonic dermis but confined later during development to the follicular connective tissue sheath, where it can also be found in postnatal human hair follicles. Its expression is up-regulated in scalp wounds and the nestin-positive cells seem to originate from the follicular mesenchyme. Nestin is also expressed in a thin layer of fibroblasts in the immediate vicinity of basal cell carcinomas. The examination for nestin expression of scalp wounds is considered preliminary, because we examined scalp wounds representing re-excisions of previously diagnosed neoplasms from which we had no exact time table available as to when the original excision took place. We propose that nestin functions as a stem cell marker of the follicular mesenchyme and has a major regulatory role in dermal homeostasis, cutaneous neovasculogenesis, and tumor stroma development.

Canine hair‐follicle keratinocytes enriched with bulge cells have the highly proliferative characteristic of stem cells

Homeostasis of the epidermis and skin appendages is maintained by tissue-specific stem cells. In mice and humans, two populations of epithelial stem cells have been identified: one in the basal layer of the interfollicular epidermis and another in the bulge area of hair follicles. However, our understanding of canine epithelial stem cells is extremely limited. In this study, in vitro colony-forming assays were performed to locate highly proliferative keratinocytes in canine skin. Their phenotypic resemblance to epithelial stem cells in other species was also assessed. When equal numbers of epidermal or hair-follicle keratinocytes were cultured, colonies derived from follicular keratinocytes were significantly larger both in total numbers and size, than those derived from epidermal keratinocytes. In addition, immunoreactivity for CD34, a putative bulge stem-cell marker in the mouse, was predominantly detected in follicular keratinocytes. Thus in dogs, follicular keratinocytes were distinct from epidermal keratinocytes in proliferative capacity and CD34 expression. Using microdissection, highly proliferative keratinocytes were located within the middle portion of hair follicles, including the bulge area. Immunohistochemical study revealed that keratin 15, an established marker of bulge stem cells in mice and humans, was also predominantly expressed in the canine bulge area. Flow cytometry analysis revealed high numbers of keratin-15-positive cells in the highly proliferative keratinocyte compartment. Of note, keratin 15(high) cells possessed the phenotypic characteristics of putative stem cells. This study represents the first in vitro identification and isolation of highly proliferative canine keratinocytes, which represent candidate epithelial stem cells.

Necl2 regulates epidermal adhesion and wound repair

Differential expression of cell adhesion molecules regulates stem cell location, self-renewal and lineage selection under steady state conditions and during tissue repair. We show that the intercellular adhesion protein nectin-like molecule 2 (Necl2) is highly expressed in bulge stem cells of adult human and mouse hair follicles. Overexpression of Necl2 in cultured human keratinocytes led to upregulation of calcium/calmodulin-associated Ser/Thr kinase (CASK), increased calcium-independent intercellular adhesion, and inhibition of cell motility and in vitro wound healing. Although the rate of cell proliferation was reduced, terminal differentiation was unaffected. To assess the role of Necl2 in vivo, we examined the epidermis of Necl2-null mice and developed transgenic mice that expressed Necl2 in the basal layer of murine epidermis. Necl2 overexpression led to a reduction in S-phase cells and an increase in quiescent cells retaining DNA label in the bulge. Although epidermal homeostasis appeared normal in both transgenic and knockout mice, wound healing was markedly delayed. Necl2 overexpression resulted in reduced proliferation and increased levels of CASK and E-cadherin at the leading edge of healing wounds, consistent with its effects in culture. Our results demonstrate that Necl2 is involved in regulating epidermal stem cell quiescence and location.

Expression and function of glycogen synthase kinase-3 in human hair follicles

Beta-catenin is involved in the hair follicle morphogenesis and stem cell differentiation, and inhibition of glycogen synthase kinase-3 (GSK-3) increases beta-catenin concentration in the cytoplasm. To examine the effects of GSK-3 inhibition on the hair follicle epithelium, we first examined the expression of GSK-3 in plucked human hair follicles by RT-PCR and found GSK-3 expression in hair follicles. Western blotting with a GSK-3beta-specific antibody, Y174, also demonstrated GSK-3beta expression in the follicles. Moreover, GSK-3beta immunostaining with Y174 showed that GSK-3beta colocalized with hair follicle bulge markers. Contrary to GSK-3beta, GSK-3 alpha was widely expressed throughout the follicles when immunostained with a specific antibody, EP793Y. We then investigated the influence of GSK-3 inhibition. A GSK-3 inhibitor, BIO, promoted the growth of human outer root sheath cells, which could be cultured for up to four passages. The BIO-treated cells exhibited smaller and more undifferentiated morphology than control cells. Moreover, in organ culture of plucked human hair, outer root sheath cells in the middle of a hair follicle proliferated when cultured with BIO. These results indicate that GSK-3beta is expressed in hair bulge stem cells and BIO promotes the growth of ORS cells, possibly by regulating the GSK-3 signaling pathway.

Defining the hair follicle stem cell (Part I)

Defining the hair follicle stem cell (Part I)

Mysteries of salt and pepper

Mysteries of salt and pepper

Human keratinocytes derived from the bulge region of hair follicles are refractory to differentiation

Human keratinocyte strains derived from the bulge region of plucked human follicles were successfully established from all 43 donors (age 24-76) regardless of the age and gender. The total cell number, number of population doublings and population doubling time were similar among the strains. These bulge-derived keratinocytes, BDKs, expressed keratin family genes specific to basal cell layers of the epidermis. They also expressed CD34, one of the bulge stem cell marker genes. The growth behavior and positivity of CD34 indicate that BDKs contain stem cells. BDKs were cultured until confluency or treated with CaCl2 to induce differentiation. Morphology and expression of keratin family genes in BDKs before and after differentiation induction with CaCl2 were similar to those of epidermal keratinocytes obtained from skin biopsies (NHEKs). However, expression levels of keratin-10, a prickle cell layer marker, in CaCl2-treated BDKs were lower than those in CaCl2-treated NHEKs. Higher expression of integrin-alpha6, a basal cell layer marker, was also noted in BDKs than in NHEKs after differentiation induction. Expression of stem cell marker genes other than CD34, including CD200, Sox2 and NANOG, was about the same at confluency in both cells, but significantly higher in BDKs than NHEKs after differentiation. These results indicate that BDKs were more refractory to differentiation than NHEKs. We then examined Wnt signaling inhibitor genes, DKK-3 and WIF-1 that function as tumor suppressors. DKK-3 expression decreased in both BDKs and NHEKs after CaCl2-induced differentiation. Expression of WIF-1 decreased 50% in BDKs one day after CaCl2 treatment and remained low, but was induced 1.7 times in NHEKs one day after CaCl2 treatment and further induced thereafter (>2.5 times), suggesting that WIF-1 may be involved in maintaining the differentiation-refractory status of BDKs. Since cancer stem cells in the skin have been reported to be similar to bulge-derived stem cells, our BDK strains may be of use in studying characteristics of cancer stem cells of the epidermis.

Notch signaling in bulge stem cells is not required for selection of hair follicle fate

Notch signaling plays an important role in hair follicle maintenance, and it has been suggested that Notch is also required for follicular fate selection by adult hair follicle stem cells in the bulge. Here we demonstrate that, on the contrary, Notch signaling in bi-potential bulge stem cells or their uncommitted descendents acts to suppress the epidermal fate choice, thus ensuring follicular fate selection. To examine the role of Notch signaling in adult hair follicle stem cells, we used a Krt1-15-CrePR1 transgenic mouse line to delete Rbpj or all Notch proteins specifically in the bulge stem cells. We conclusively determined that in the absence of Notch signaling, bulge stem cell descendents retain their capacity to execute the follicular differentiation program but fail to maintain it owing to their genetic deficiency. The defect in terminal differentiation caused the diversion of Notch-deficient hair follicles to epidermal cysts, and the presence of wild-type cells could not prevent this conversion. Importantly, our analysis revealed that a functional Notch signaling pathway was required to block bulge stem cells from migrating into, and assuming the fate of, interfollicular epidermis. Taken together, our findings yield detailed insight into the function of Notch signaling in hair follicle stem cells and reveal the mechanism of the replacement of Notch-deficient adult hair follicles by epidermal cysts.

The River of Stem Cells

In this issue of Cell Stem Cell, Greco et al. (2009) characterize the hair germ as a novel stop between bulge stem cell and transient amplifying cells during hair regeneration. The work implies stem cell states can be regulated to form different numbers of intermediate stops, depending on physiological requirements.

A Two-Step Mechanism for Stem Cell Activation during Hair Regeneration

Hair follicles (HFs) undergo cyclic bouts of degeneration, rest, and regeneration. During rest (telogen), the hair germ (HG) appears as a small cell cluster between the slow-cycling bulge and dermal papilla (DP). Here we show that HG cells are derived from bulge stem cells (SCs) but become responsive quicker to DP-promoting signals. In vitro, HG cells also proliferate sooner but display shorter-lived potential than bulge cells. Molecularly, they more closely resemble activated bulge rather than transit-amplifying (matrix) cells. Transcriptional profiling reveals precocious activity of both HG and DP in late telogen, accompanied by Wnt signaling in HG and elevated FGFs and BMP inhibitors in DP. FGFs and BMP inhibitors participate with Wnts in exerting selective and potent stimuli to the HG both in vivo and in vitro. Our findings suggest a model where HG cells fuel initial steps in hair regeneration, while the bulge is the engine maintaining the process.

Bone Marrow-Derived Cells Are Not the Origin of the Cancer Stem Cells in Ultraviolet-Induced Skin Cancer

Several lines of evidence have demonstrated that various cancers are derived from cancer stem cells (CSCs), which are thought to originate from either tissue stem or progenitor cells. However, recent studies have suggested that the origin of CSCs could be bone marrow-derived cells (BMDCs); for example, gastric cancer, which follows persistent gastric inflammation, appears to originate from BMDCs. Although our previous research showed the capability of BMDCs to differentiate into epidermal keratinocytes, it has yet to be determined whether skin CSCs originate from BMDCs. To assess the possibility that BMDCs could be the origin of CSCs in skin squamous cell carcinoma (SCC), we used a mouse model of UVB-induced skin SCC. We detected a low percentage of BMDCs in the lesions of epidermal dysplasia (0.59%), SCC in situ (0.15%), and SCC (0.03%). Furthermore, we could not find any evidence of clonal BMDC expansion. In SCC lesions, we also found that most of the BMDCs were tumor-infiltrating hematopoietic cells. In addition, BMDCs in the SCC lesions lacked characteristics of epidermal stem cells, including expression of stem cell markers (CD34, high alpha6 integrin) and the potential retention of BrdU label. These results indicate that BMDCs are not a major source of malignant keratinocytes in UVB-induced SCC. Therefore, we conclude that BMDCs are not the origin of CSCs in UVB-induced SCC.

Have Hair Follicle Stem Cells Shed Their Tranquil Image?

In a recent issue of Nature Genetics, Jaks et al. (2008) demonstrate that hair follicle cells expressing the intestinal stem cell marker Lgr5 are hair follicle epithelial stem cells. In contrast to the established bulge stem cell population, Lrg5(+) cells are actively cycling and reside in part outside the bulge.

The multipotency of adult vibrissa follicle stem cells

Several studies focused on the characterization of bulge keratinocytes have proved that they are multipotent stem cells, being recruited not only to regenerate the hair follicle itself, but also the sebaceous gland and the epidermis. However, due to the difficulty in preparing transplantable cell sheets harvested with conventional enzymatic digestion, there is still no direct evidence of the bulge stem cells’ multipotency. Whether they can respond to adult dermal papilla (DP) signals in recombination experiments also remains unclear. In this study, we addressed this problem by culturing and detaching intact bulge keratinocyte sheets from thermo-responsive culture dishes, only by reducing its temperature. When sheets of mass cultured bulge keratinocytes isolated from rat vibrissa follicles were recombined with fresh adult DPs and sole skin dermis in vivo, regeneration of epidermis and sebaceous gland-like structures, and formation of hair bulb with differentiating inner root sheath and hair cuticle were observed within 3 weeks. However, regardless the expression of stem cells markers like CD34, SA1004 and SA1006, no structures were observed when cloned bulge keratinocytes were used to prepare cell sheets and recombinants, revealing the possible existence of monoclonal stem cells within the bulge region. This report is the first to succeed in harvesting adult bulge keratinocyte sheets. Using these sheets it is demonstrated that bulge stem cells directly respond to adult DP signals to induce hair bulb formation in vivo.

Involvement of the bulge region in primary scarring alopecia

While the pathogenesis of most scarring alopecias is poorly understood, one recent study indicates destruction of follicular stem cells as a possible mechanism in lichen planopilaris, the prototypic scarring alopecia. The aim of this cross-sectional study was to ascertain the target of inflammation and to more precisely characterize the inflammatory infiltrate in various stages of primary scarring alopecias. Immunohistochemical studies were performed using a panel of antibodies that included anti-cytokeratin 15, an antibody that specifically targets follicular bulge stem cells and CD4, CD8, CD1a and human leukocyte antigen-DR to characterize the inflammatory infiltrate. Our data showing absence of follicular bulge stem cells in cases with moderate to heavy inflammation suggest involvement of the bulge region in 'early' active stages of primary scarring alopecia. The paucity of CD8+ T cells in the inflammatory infiltrate in the majority of these cases argues against a cell-mediated cytotoxic destruction of follicular bulge stem cells. Preservation of CK15+ cells in 'late' fibrotic stages of primary scarring alopecia further supports this and implies that the irreversible loss of hair follicles, the sine qua non of primary scarring alopecia, is not necessarily a consequence of T cell-mediated destruction of follicular bulge stem cells.

Limbal microenvironment can induce transdifferentiation of hair follicle stem cells into corneal epithelial‐like cells

Abstract Purpose To investigate the plasticity of murine vibrissa hair follicle (HF) stem cells regarding differentiation into corneal epithelial‐like cells through modulation by limbus‐specific microenvironmental factors. Methods HFs were isolated and the dissociated bulge stem cells enriched by clonal expansion, and subcultivated on various extracellular matrices (collagen type IV, laminin‐1, laminin‐5, fibronectin) in DMEM/F12 medium supplemented with different conditioned media (CMs). CMs were derived from central and peripheral corneal fibroblasts and limbal stromal fibroblasts. Growth potential and cellular phenotype were evaluated by light and electron microscopy, real‐time‐PCR and immunohistochemistry using antibodies against stem cell (CK15, integrin α6) and differentiation markers (CK12) or (CK10). Results Laminin‐5 and collagen IV promoted rapid cell adhesion, proliferation, and generation of confluent, regularly arranged epitheloid cell sheets, whereas laminin‐1 and fibronectin adversely affected these cell properties. Addition of CMs differentially influenced cellular phenotype and differentiation. Limbal fibroblast CM induced differentiation of cuboid cells showing ultrastructural characteristics of a corneal epithelial phenotype. Moreover, it markedly increased the number of CK12 immunopositive cells while decreasing expression of CK10 opposed to the other CMs. Conclusion Hair follicle stem cells are capable of differentiating into corneal epithelial‐like cells in vitro when exposed to a limbus‐specific microenvironment. Therefore, the HF may be an alternative therapeutic source of multipotent stem cells for generation of autologous epithelial cell sheets for ocular surface reconstruction.

Dlx3 is a crucial regulator of hair follicle differentiation and cycling

Dlx homeobox transcription factors regulate epidermal, neural and osteogenic cellular differentiation. Here, we demonstrate the central role of Dlx3 as a crucial transcriptional regulator of hair formation and regeneration. The selective ablation of Dlx3 in the epidermis results in complete alopecia owing to failure of the hair shaft and inner root sheath to form, which is caused by the abnormal differentiation of the cortex. Significantly, we elucidate the regulatory cascade that positions Dlx3 downstream of Wnt signaling and as an upstream regulator of other transcription factors that regulate hair follicle differentiation, such as Hoxc13 and Gata3. Colocalization of phospho-Smad1/5/8 and Dlx3 is consistent with a regulatory role for BMP signaling to Dlx3 during hair morphogenesis. Importantly, mutant catagen follicles undergo delayed regression and display persistent proliferation. Moreover, ablation of Dlx3 expression in the telogen bulge stem cells is associated with a loss of BMP signaling, precluding re-initiation of the hair follicle growth cycle. Taken together with hair follicle abnormalities in humans with Tricho-Dento-Osseous (TDO) syndrome, an autosomal dominant ectodermal dysplasia linked to mutations in the DLX3 gene, our results establish that Dlx3 is essential for hair morphogenesis, differentiation and cycling programs.

Increased expression of an epidermal stem cell marker, cytokeratin 19, in cutaneous squamous cell carcinoma

Cytokeratin 19 (CK19) has been considered to be a putative marker for epidermal stem cells in the hair follicle bulge. Cumulative reports have shown that epidermal stem cells play an important role in skin carcinogenesis. However, to date there has been no report on the clinical alteration of the stem cells in squamous cell carcinoma (SCC). To investigate alteration of the stem cells and proliferating cells and to assess their relationship and potential contribution to SCC. Thirty paraffin-embedded neoplastic skin lesions, consisting of 10 cases each of actinic keratosis (AK), Bowen disease (BD) and SCC, were examined immunohistologically for CK19 and Ki-67. Positive reactivity for CK19 was seen in 30% of AK, 50% of BD and 80% of SCC lesions. There was significantly higher expression levels of CK19 in SCC than in AK and BD (P < 0.05). In addition, BD lesions harboured a significantly higher number of CK19-positive cells than did AK lesions (P < 0.05). There were significant differences in Ki-67 labelling indices between AK and BD and between AK and SCC (P < 0.001), but not between BD and SCC (P > 0.05). Furthermore, a serial section comparison study showed that there was a minor population of cells co-expressing CK19 and Ki-67 in a subset of the tumour cells of SCC samples. The percentage of CK19+ cells significantly correlated with that of Ki67+ cells in all examined neoplastic skin lesions. These results suggest that CK19 expression may be associated with the retention of stem cell characteristics or a state that is uncommitted to terminal squamous differentiation.

The Roots of Hair Growth

DEVELOPMENT Adult hair follicles sustain repeated cycles of hair loss and regrowth. Stem cells reside within a small niche, called the bulge, located in the upper part of the hair follicle. These stem cells are responsible for driving this cycle of growth and can repopulate follicles and surrounding epidermis damaged by wounding. However, the first hair follicles in mice arise from the developing epidermis, not from preexisting bulge stem cells, and these first-time hair follicles do not exhibit the conspicuous bulge that only becomes apparent some weeks after birth. Nowak et al. have analyzed the origins of the hair follicle stem cell niche in developing mice. The stem cells of the bulge are in fact established much earlier than previously suspected and begin to form in the embryo. These cells, which are characterized by expression of the transcription factor Sox9, not only contribute to the formation of the initial hair follicle, but also give rise to the adult bulge stem cells that are responsible for the maintenance of the hair follicle itself. Ablation of Sox9 left the embryos without bulge cells, and the mice never grew any hair and did not have any sebaceous glands. Moreover, the skin did not repair epidermal wounds well when Sox9 was missing. These results implicate Sox9 in establishment of the hair follicle stem cell population and show that early stem cells can contribute to skin morphogenesis before assuming their role as adult stem cells. — PJH Cell Stem Cell 3 , 33 (2008).