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Kobayashi K, Rochat A, Barrandon YSegregation of keratinocyte colony-forming cells in the bulge of the rat vibrissa. Proc Natl Acad Sci USA 90:7391-7395
Abstract
The epidermis and its related appendages such as the hair follicle constitute the epithelial compartment of the skin. The exact location and distribution of the keratinocyte colony-forming cells within the epidermis or its appendages are unknown. We report that in the rat vibrissa, keratinocyte colony-forming cells are highly clustered in the bulge-containing region. Approximately 95% of the total colonies formed in culture from fractionated vibrissae were in this location and fewer than 4% were located in the matrix area of the follicle. Finer dissection of the bulge-containing region located the colony-forming cells in the small part containing the bulge itself. The segregation of keratinocyte colony-forming cells in the bulge confirms the hypothesis that the bulge is the reservoir of the stem cells responsible for the long-term growth of the hair follicle and perhaps of the epidermis as well.
- ... Rat vibrissae follicles were isolated by using modified methods described by Sieber-Blum and Kobayashi[33]. Briefly, the whisker pads were removed and placed in Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM/F12; 3:1) supplemented with 100 U penicillin and 100 mg streptomycin per mL. ...... It has been well known that the bulge area of hair follicles comprise the cells with stem cell properties[3334]. Hair follicle stem cells are characterized by high proliferation potential, slow cycling and label retaining[3435], which fulfilling the required criteria for a stem cell population. ...Emerging studies of treating spinal cord injury (SCI) with adult stem cells led us to evaluate the effects of transplantation of hair follicle stem cells in rats with a compression-induced spinal cord lesion. Here, we proposed a hypothesis that rat hair follicle stem cell transplantation can promote the recovery of injured spinal cord. Compression-induced spinal cord injury was induced in Wistar rats in this study. The bulge area of the rat vibrissa follicles was isolated, cultivated and characterized with nestin as a stem cell marker. 5-Bromo-2'-deoxyuridine (BrdU) labeled bulge stem cells were transplanted into rats with spinal cord injury. Immunohistochemical staining results showed that some of the grafted cells could survive and differentiate into oligodendrocytes (receptor-interacting protein positive cells) and neuronal-like cells (βIII-tubulin positive cells) at 3 weeks after transplantation. In addition, recovery of hind limb locomotor function in spinal cord injury rats at 8 weeks following cell transplantation was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale. The results demonstrate that the grafted hair follicle stem cells can survive for a long time period in vivo and differentiate into neuronal- and glial-like cells. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury.
- ... Demonstration of the difference in proliferation potential between the stem cells and the transient amplifying cells. (a) Reproduced with permission from [24, Figure 2]. Difference in clonogenicity between the bulge cells and bulb cells of HFs. ...... Cotsarelis et al. [4] found that stem cells are enriched in the mouse HF bulge but not elsewhere in the follicle including the bulb. Kobayashi et al. [24] reported that the bulge region of rat vibrissa contains 95% of the clonogenic keratinocytes present in an anagen rat follicle, whereas the hair bulb contained the remaining 5% (Figure 10(a)). These results demonstrate that HF stem cells located in the bulge have the potential to proliferate more extensively than those found in the cyclic regeneration component including the bulb. ...We have made comparisons between hair follicles (HFs) and antler units (AUs)-two seemingly unrelated mammalian organs. HFs are tiny and concealed within skin, whereas AUs are gigantic and grown externally for visual display. However, these two organs share some striking similarities. Both consist of permanent and cyclic/temporary components and undergo stem-cell-based organogenesis and cyclic regeneration. Stem cells of both organs reside in the permanent part and the growth centres are located in the temporary part of each respective organ. Organogenesis and regeneration of both organs depend on epithelial-mesenchymal interactions. Establishment of these interactions requires stem cells and reactive/niche cells (dermal papilla cells for HFs and epidermal cells for AUs) to be juxtaposed, which is achieved through destruction of the cyclic part to bring the reactive cells into close proximity to the respective stem cell niche. Developments of HFs and AUs are regulated by similar endocrine (particularly testosterone) and paracrine (particularly IGF1) factors. Interestingly, these two organs come to interplay during antlerogenesis. In conclusion, we believe that investigators from the fields of both HF and AU biology could greatly benefit from a comprehensive comparison between these two organs.
- ... Hair follicles, which go through continuous cycles of regeneration (anagen), involution (catagen), and resting (telogen) phases are maintained by distinct multipotent stem cells located in their upper, constant region (the bulge) (Kobayashi et al., 1993;Rochat et al., 1994). During homeostasis, these stem cells only renew the epithelial cell lineages of the hair follicle; however, if a wound needs to be healed, they can participate to the regeneration of the interfollicular epidermis (Ito et al., 2005;Claudinot et al., 2005). ...... In the rat, the bulge region of whisker follicles (specialized sensory hairs of the snout) contains clonogenic keratinocytes that initiate progressively growing colonies (Kobayashi et al., 1993). ...Regenerative medicine aims at using stem cells to restore or establish lost, damaged, diseased, or aging tissues and organs function. The transplantation of cultured epidermal autograft (CEA) has saved the lives of many burned patients over the last 30 years, but the resulting epidermis is far from perfect, and clinical outcomes remain unpredictable. The engraftment of cultured epidermal stem cells is poorly understood, and, as a result, suboptimal. Using a large animal model, the pig, we recapitulated the clinical settings and outcomes of human CEA transplantation. With this model, we study the engraftment process, and we demonstrate that cultured epidermal stem cells often, but not always, favor the choice of differentiation over self-renewal when transplanted on full thickness wounds. Differences in this early fate choice are likely to explain the variability of clinical outcomes. We developed a system of in vitro live cell imaging (LCI) to study the fate choices of multiple individual cells in a microenvironment that is much more controlled than the grafting bed of a burned patient. We demonstrate that keratinocytes have a very heterogeneous behavior in vitro. With a fluorescent reporter of cell cycle progression (FUCCI), we show the influence of the cell cycle on early fate choices of cultured keratinocytes. By introducing a ROCK inhibitor in the culture medium, we show that keratinocyte’s early fate choices can be impacted so that they show greater clonogenicity and growth potential. The unique combination of large animal CEA transplantation with individual cell LCI demonstrate the critical role of early fate choice, both for epidermal stem cell engraftment and in vitro behavior. The possibility to influence the early fate choices of stem cells by modifying the microenvironment has a great potential to improve the engraftment and the ex vivo amplification of stem cells, the two limiting steps of cell replacement therapy.
- ... Our results of rat hair follicle bulge area culture also supported this hypothesis [31]. Morphology-based manual microdissection was used to isolate the bulge cells from the hair follicle [32, 33]. The method of isolation was based on the migration of cells from the cultured bulge region. ...Background: The seladin-1 (selective Alzheimer disease indicator-1), also known as DHCR24, is a gene found to be down-regulated in brain region affected by Alzheimer disease (AD). Whereas, hair follicle stem cells (HFSC), which are affected in with neurogenic potential, it might to hypothesize that this multipotent cell compartment is the predominant source of seladin-1. Our aim was to evaluate seladin-1 gene expression in hair follicle stem cells. Methods: In this study, bulge area of male Wistar rat HFSC were cultured and then characterized with Seladin-1 immunocytochemistry and flow cytometry on days 8 to 14. Next, 9-11-day cells were evaluated for seladin-1 gene expression by real-time PCR. Results: Our results indicated that expression of the seladin-1 gene (DHCR24) on days 9, 10, and 11 may contribute to the development of HFSC. However, the expression of this gene on day 11 was more than day 10 and on 10th day was more than day 9. Also, we assessed HFSC on day 14 and demonstrated these cells were positive for β-ш tubulin, and seladin-1 was not expressed in this day. Conclusion: HFSC express seladin-1 and this result demonstrates that these cells might be used to cell therapy for AD in future.
- ... When cultured on TCP in medium with or without serum, HDFs behaved individually and retained typical spindle-like morphologies, while HaCat cells tended to form colonies and their proliferation was dependent on both serum and cell seeding densities. As the keratinocyte colonies formed within epidermis, epidermal appendages, or recreated in 2D cell cultures, are subject to auto-regulation [28,29], the requirement of HaCat cells on high seeding density for colony formation and active proliferation can be explained as the genetically definable quantitative trait [30]. Within slow growing colonies, keratinocytes were observed to pack loosely, this could lead to the adjacent keratinocytes not forming the necessary coherence through the desmosomal junctions [31]; while the successful culture using high initial densities could be due to the short distances and extensive intercellular junctions between the cultivated keratinocytes [32]. ...A generic research platform with 2-dimensional (2D) cell culture technology, a 3-dimensional (3D) in vitro tissue model, and a scaled-down cell culture and imaging system in between, was utilized to address the problematic issues associated with the use of serum in skin tissue engineering. Human dermal fibroblasts (HDFs) and immortalized keratinocytes (HaCat cells) mono- or co-cultured in serum or serum-free medium were compared and analyzed via the platform. It was demonstrated that serum depletion had significant influence on the attachment of HaCat cells onto tissue culture plastic (TCP), porous substrates and cellulosic scaffolds, which was further enhanced by the pre-seeded HDFs. The complex structures formed by the HDFs colonized within the porous substrates and scaffolds not only prevented the seeded HaCat cells from filtering through the open pores, but also acted as cellular substrates for HaCat cells to attach onto. When mono-cultured on TCP, both HDFs and HaCat cells were less proliferative in medium without serum than with serum. However, both cell types were successfully co-cultured in 2D using serum-free medium if the initial cell seeding density was higher than 80,000 cells/cm² (with 1:1 ratio). Based on the results from 2D cultures, co-culture of both cell types on modular substrates with small open pores (125 μm) and cellulosic scaffolds with open pores of varying sizes (50-300 µm) were then conducted successfully in serum-free medium. This study demonstrated that the generic research platform had great potential for in-depth understanding of HDFs and HaCat cells cultivated in serum-free medium, which could inform the processes for manufacturing skin cells or tissues for clinical applications.
- ... The authors also demonstrated the presence of skirt-like projections in small vellus hair but not in large vellus hairs. The APM-bulge connection persists throughout the hair growth cycle and has been suggested to play an important role in morphogenesis and renewal of hair follicles.[30313233] ...The arrector pili muscle (APM) consists of a small band of smooth muscle that connects the hair follicle to the connective tissue of the basement membrane. The APM mediates thermoregulation by contracting to increase air-trapping, but was thought to be vestigial in humans. The APM attaches proximally to the hair follicle at the bulge, a known stem cell niche. Recent studies have been directed toward this muscle's possible role in maintaining the follicular integrity and stability. This review summarizes APM anatomy and physiology and then discusses the relationship between the follicular unit and the APM. The potential role of the APM in hair loss disorders is also described, and a model explaining APM changes in hair loss is proposed.
- ... The location of stem cells in the hair follicle is better characterized; the follicular bulge is a well-known stem cell niche [13, 14]. Bulge cells have an undifferentiated structure [13], have greater in vitro growth ability compared with other cells in the epidermis and follicle [15], and are multipotent [16][17][18][19][20]. They proliferate transiently to initiate hair growth in the early stages of anagen or after being exposed to external simulation [2,[21][22][23]. ...http://www.hindawi.com/journals/sci/2016/1286315/ Inconsistent with the view that epidermal stem cells reside randomly spread along the basal layer of the epidermal rete ridges, we found that epidermal cells expressing stem cell markers in nonglabrous skin exist in direct connection with the distal end of the arrector pili muscle. The epidermal cells that express stem cell markers consist of a subpopulation of basal keratinocytes located in a niche at the lowermost portion of the rete ridges at the distal arrector pili muscle attachment site. Keratinocytes in the epidermal stem cell niche express K15, MCSP, and α 6 integrin. α5 integrin marks the distal end of the APM colocalized with basal keratinocytes expressing stem cell markers located in a well-protected and nourished environment at the lowermost point of the epidermis; these cells are hypothesized to participate directly in epidermal renewal and homeostasis and also indirectly in wound healing through communication with the hair follicle bulge epithelial stem cell population through the APM. Our findings, plus a reevaluation of the literature, support the hierarchical model of interfollicular epidermal stem cell units of Fitzpatrick. This new view provides insights into epidermal control and the possible involvement of epidermal stem cells in nonmelanoma skin carcinogenesis.
- The hair follicle is a dynamic structure which contains different niches for stem cells, therefore; it has been considered as valuable and rich sources of stem cells, due to easy access, multipotency and non-oncogenic properties. In the present study, the differentiation capacities of hair follicle stem cells into bone cells on the natural collagen scaffolds were investigated. The stem cells were extracted from the hair follicle bulge area of male Wistar rats’ whisker and cultured until 3rd passage, then osteogenic differentiations were induced by culturing the cells in the specific osteogenic medium. After 3 weeks, the differentiation parameters, including morphological changes, levels of calcification and expression of the bone specific genes were detected. The hydrogel preparation and scaffold fabrication was carried out using the extracted collagen and was studied by scanning electron microscope. Comparison of the stem cells’ growth and changes on the scaffold and non-scaffold conditions showed that, in the both situation, the cells revealed differentiation signs of osteocytes, including large and cubic morphology with a star-shaped nucleus. Staining by Alizarin-red and Von-Kossa methods showed the presence of red and black calcium mass on the scaffold. Expression of the osteopontin and alkaline phosphatase genes confirmed the differentiation. Considerable porosity in the surface of the scaffold was recorded by scanning electron microscopy, which made it convenient for cells’ attachment and growth. The data showed that the bulge stem cells possess significant capacity for osteoblastic differentiation and collagen scaffolds were found to be an appropriate matrix for growth and differentiation of the cell.
- Follicular stem cells and their progeny are responsible for the cyclical renewal of hair follicles and maintenance of the hair coat. The understanding of pathways involved in this process is essential to elucidate the pathogenetic mechanisms of primary alopecia. Stem cells and their direct descendants are located in the bulge region of the isthmus of hair follicles. Although these cells have been studied extensively in mice and humans, data for canine isthmic keratinocyte activation and proliferation are not available. The aim was to establish an accurate and reliable in vitro system to study the growth potential of canine isthmic keratinocytes. We assessed the colony-promoting capability of a commercially available canine-specific medium, CELLnTEC (CnT-09), compared with a well-established home-made medium, complete FAD (cFAD). The CnT-09 medium is specific for the growth of canine keratinocytes, while the cFAD medium can support growth and colony formation of keratinocytes from several species. Skin biopsies were obtained from 15 recently euthanized dogs of various breeds with no skin abnormalities. The isthmic region of compound hair follicles was isolated by microdissection and cell growth monitored using several parameters with colony-forming assays. The CnT-09 and cFAD media provided similar growth as measured by the total number and size of colonies, as well as rate of cell differentiation. The commercial canine-specific CnT-09 medium was comparable to the home-made cFAD medium in supporting the growth and proliferation of canine follicular keratinocytes in vitro. The CnT-09 medium should be a viable alternative growth medium for molecular studies of alopecic disorders in dogs. © 2014 ESVD and ACVD.
- Background and Objectives Hair follicles are located at the interface of the external and internal environments and their cycling has been shown to be regulated by intra- and extra-follicular factors. The aim of this study is to examine whether or how hair follicles respond to visible light.Study Design/Materials and Methods We examined the effect of 3 mW red (630 nm, 1 J/cm2), 2 mW green (522 nm, 1 J/cm2), and 2 mW blue light (463 nm, 1 J/cm2) on telogen in mice for 3 weeks. The photobiologic effects of red light on cell proliferation of outer root sheath keratinocytes and dermal papilla cells were studied in vitro.ResultsWe found that red light accelerated anagen entry faster than green and blue light in mice. Red light irradiation stimulated the proliferation of both outer root sheath keratinocytes and dermal papilla cells in a dose-dependent manner by promoting cell cycle progression. This stimulative effect was mediated via extracellular signal-regulated kinase phosphorylation in both cells. In a co-culture condition, dermal papilla cells irradiated by red light further enhanced keratinocyte proliferation, suggesting enhanced epithelial-mesenchymal interaction. In search for factors that mediated this paracrine effect, we found fibroblast growth factor 7 was upregulated in both mRNA and protein levels. The stimulative paracrine effect on keratinocytes was significantly inhibited by neutralizing antibody against fibroblast growth factor 7.Conclusions These results suggest that hair follicles respond to visible light in vivo. Red light may promote physiological telogen to anagen transition by directly stimulating outer root sheath keratinocytes and indirectly by enhancing epithelial-mesenchymal interaction in vitro. Lasers Surg. Med. © 2014 Wiley Periodicals, Inc.
- Differentiation of hair follicle stem cells (HFSCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. The hair follicle bulge area is reported as a putative source of new stem cell population for many years. In vitro studies have implicated neural differentiation of HFSCs. Here, we report the identification and purification of CD34 + cells from hair follicle by magnetic activated cell sorting (MACS). We next determined the cytotoxic effects of all-trans retinoic acid (RA) by using cell viability assays. Moreover, the neural differentiation potential of CD34 + cells was evaluated in the presence of RA, serum-free condition, and neural differentiation medium (NDM) treatments by using immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). Our results showed that the isolated CD34 + stem cells were 12% of the total cells in the bulge area, and the neural cells derived from the stem cells expressed nestin, microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein (GFAP). Interestingly, all the neural induction media supported neuronal differentiation most effectively, but treatment with serum-free medium significantly increased the number of GFAP-positive glial cells. Moreover, increasing RA concentration (≥10 μM) leads to increased cell death in the cells, but a lower concentration of RA (1 μM) treatment results in a decrease in CD34-expressing stem cells. These findings show an instructive neuronal effect of three neural induction media in HFSCs, indicating the important role of this induction media in the specification of the stem cells toward a neural phenotype.
- In this review article I explore the suitability of human epidermal neural crest stem cells (hEPI-NCSC) for translational medicine. hEPI-NCSC are multipotent somatic stem cells that are derived from the embryonic neural crest. hEPI-NCSC are located in the bulge of hair follicles where they persist postnatally and into adulthood. Because of their location in the hairy skin and their migratory behavior, hEPI-NCSC can be easily isolated as a highly pure population of stem cells without the need for purification. Furthermore they can be expanded ex vivo into millions of stem cells, they do not form tumors in vivo, and they can undergo directed differentiation into crest and noncrest-derived cell types of clinical relevance. Taken together, these characteristics make hEPI-NCSC attractive candidates for cell-based therapies, drug discovery, and disease modeling. Birth Defects Research (Part C), 2014. © 2014 Wiley Periodicals, Inc.
- Facial skin replacement after burns or other injuries has proved to be a challenging task in wound healing. The application of laboratory-based tissue-expansion techniques is a potential solution to the problem of surface area cover. Considerable progress has been made in approaches to these techniques, including allograft and autograft skin transplantation to replace skin temporarily or permanently. Methods for handling skin wounds, such as transplantation, tissue engineering, and, more recently, stem cell ...
- β-catenin signaling is required for hair follicle development and regeneration which are involved in the resuscitation of hair follicle stem cells (HFSCs). To further characterize the role of β-catenin in the regulation of proliferation of HFSCs, the β-catenin expression was measured in the defined stages of hair follicle cycle and the proliferative potency was determined by using an in vitro cell growth assay. Our results showed that activation of β-catenin correlated with HFSCs proliferation, which appeared to be mediated by the nuclear translocation of stabilized β-catenin and the activation of responsible cell cycle genes (cyclin D1 and p21). In addition, PI3K/Akt pathway was also involved in the HFSCs proliferation, partly regulated by β-catenin signaling pathway. These results demonstrate that β-catenin is an essential factor in the regulation of HFSCs proliferation via PI3K/Akt pathway and might be a potential therapeutic target for the regulation of the yield of keratinocytes from HFSCs.
- Human skin maintains the ability to regenerate during adulthood, as it constantly renews itself throughout adult life, and the hair follicle (HF) undergoes a perpetual cycle of growth and degeneration. The study of stem cells (SCs) in the epidermis and skin tissue engineering is a rapidly emerging field, where advances have been made in both basic and clinical research. Advances in basic science include the ability to assay SCs of the epidermis in vivo, identification of an independent interfollicular epidermal SC, and improved ability to analyze individual SCs divisions, as well as the recent hair organ regeneration via the bioengineered hair follicular unit transplantation (FUT) in mice. Advances in the clinic include recognition of the importance of SCs for wound repair and for gene therapy in inherited skin diseases, for example epidermolysis bullosa. The study of the HF stem cells (HFSCs) started by identification of epidermal SC in the HF bulge as quiescent "label retaining cells". The research of these cells emerged rapidly after the identification of bulge cell molecular markers, such as keratin 15 (K15) and CD34 in mice and CD200 in humans, which allowed the isolation and characterization of bulge cells from follicles. This paper provides an overview of the current knowledge on epidermal SCs in the HF describing their essential characteristics and the control of follicle SCs fate, their role in alopecia, as well as their use in tissue engineering.
- Recent evidence supports and reinforces the concept that environmental cues may reprogramme somatic cells and change their natural fate. In the present review, we concentrate on environmental reprogramming and fate potency of different epithelial cells. These include stratified epithelia, such as the epidermis, hair follicle, cornea and oesophagus, as well as the thymic epithelium, which stands alone among simple and stratified epithelia, and has been shown recently to contain stem cells. In addition, we briefly discuss the pancreas as an example of plasticity of intrinsic progenitors and even differentiated cells. Of relevance, examples of plasticity and fate change characterize pathologies such as oesophageal metaplasia, whose possible cell origin is still debated, but has important implications as a pre-neoplastic event. Although much work remains to be done in order to unravel the full potential and plasticity of epithelial cells, exploitation of this phenomenon has already entered the clinical arena, and might provide new avenues for future cell therapy of these tissues.
- Introduction: Stem cells are found in different portions of the body, including the bone marrow, these cells are capable of differentiated into bone, ligament, muscle, nerve, pancreatic beta cells and etc. Thus, these cells can treat degenerative diseases such as brain injuries, spinal cord injuries, and other disease. Given the lack of research on various electromagnetic field associated with the hair follicle stem cell differentiation onto neuronal cells In this study, we evaluated the effects of electromagnetic fields on hair follicle stem cells to neuron. Materials & Methods: 6 rats were selected .anesthetized and sterilized in their upper lip hair follicle cells under hood apart and separate them from the membrane. Then cultured in DMEM. After a week the cells were exposed to electromagnetic field characteristics(1-1.8 and 2.4 MT) with a period 7-10-14 and finally evaluated by immune histochemistry using antibodies specific for nestin were. Results: Electromagnetic field characteristics 1.8 in a period 10 day and 2.4 MT in a period 14 day cause cells express nestin. And other intensity and day cant cause express nestin. Conclusion: Electromagnetic field with different characteristic can cause differentiated hair follicle stem cells into neuron like cells. © 2015 AENSI Publisher All rights reserved. To Cite This Article: Manouchehr Safari, Laya Ghahari, Majid Kasaei., The effect of electromagnetic fields on the differentiation of hair follicles stem cells into nerve cells in Adult Male Rats. Adv. Environ. Biol., 9(3), 925-929, 2015
- Androgenic alopecia (AGA) is the most common hair loss condition in men and women. Hair loss is caused by follicle miniaturization, which is largely irreversible beyond a certain degree of follicular regression. In contrast, hair loss in telogen effluvium (TE) is readily reversible. The arrector pili muscle (APM) connects the follicle to the surrounding skin. To compare histopathological features of the APM in AGA and TE. Archival blocks of 4 mm scalp punch biopsies from 8 patients with AGA and 5 with TE were obtained. New 4 mm biopsies from 5 normal cases were used as controls. Serial 7 μm sections were stained with a modified Mason's trichrome. "Reconstruct" software was used to construct and evaluate three-dimensional images of the follicle and APM. The APM degenerated and was replaced by adipose tissue in all AGA specimens. Remnants of the APM remained attached to the hair follicle. There was no fat in the normal skin specimens. Fat was seen in 2 of 5 TE specimens, but could be attributed to these patients also showing evidence of AGA. Quantitative analysis showed that muscle volume decreased and fat volume increased significantly (P<0.05) in AGA compared to controls. APM degeneration and replacement with fat in AGA has not previously been described. The underlying mechanism remains to be determined. However we speculate that this phenomenon might be related to depletion of stem or progenitor cells from the follicle mesenchyme, explaining why AGA is treatment resistant. This article is protected by copyright. All rights reserved.
- Hair follicles are skin appendages of the mammalian skin that have the ability to periodically and stereotypically regenerate in order to continuously produce new hair over our lifetime. The ability of the hair follicle to regenerate is due to the presence of stem cells that along with other cell populations and non-cellular components, including molecular signals and extracellular material, make up a niche microenvironment. Mounting evidence suggests that the niche is critical for regulating stem cell behavior and thus the process of regeneration. Here we review the literature concerning past and current studies that have utilized mouse genetic models, combined with other approaches to dissect the molecular and cellular composition of the hair follicle niche. We also discuss our current understanding of how stem cells operate within the niche during the process of tissue regeneration and the factors that regulate their behavior.
- Purpose: The effects of ionizing radiations on somatic stem cells largely remain to be studied. Hair follicles are self-renewing structures that reconstitute themselves throughout the hair cycle, which is comprised of the following phases: Anagen (growth), catagen (regression) and telogen (resting), suggesting the presence of their own stem cells. Materials and methods: The whole bodies of C57BL/10JHir mice in the 1st telogen phase were irradiated with γ-rays. Mice were examined for effects on hair follicles, including their number, morphology and pigmentation in the 2nd anagen phase. Results: Decreased hair follicle density and induction of curved hair follicles were observed in the dermal skin of irradiated mice. In addition to these keratinocyte-derived anomalies, melanocyte-derived anomalies including white hair and hypopigmented hair bulbs were found. The decrease in hair follicle density and the increase in the frequency of hypopigmented hair bulbs were dependent on the dose of γ-rays. Conclusions: These results suggest that γ-rays damage stem cells and progenitors for keratinocytes and melanocytes, thereby affecting the structure and character of regenerated hair follicles. The density of hair follicles and pigment production in hair bulbs are established as criteria for the effects of γ-rays on the hair cycle.
- The maintenance and repair of many adult tissues are ensured by stem cells (SCs), which reside at the top of the cellular hierarchy of these tissues. Functional assays, such as in vitro clonogenic assays, transplantation and in vivo lineage tracing, have been used to assess the renewing and differentiation potential of normal SCs. Similar strategies have suggested that solid tumours may also be hierarchically organized and contain cancer SCs (CSCs) that sustain tumour growth and relapse after therapy. In this Opinion article, we discuss the different parallels that can be drawn between adult SCs and CSCs in solid tumours.
- Although vibrissae hair follicles (VHFs) have long been a key research model in the life sciences, their immune system (IS) is essentially unknown. Therefore, we have characterized basic parameters of the VHF-IS of C57BL/6J mice by quantitative (immuno-)histomorphometry. Murine anagen VHF harbour few CD4+ and CD8+ T cells in the distal mesenchyme and sinuses but hardly any gamma-delta T cells in their distal epithelium. MHC class II+ Langerhans cells are seeded in the VHF infundibulum, which is also surrounded by MHC class II+ and CD11b+ cells (macrophages). The number of Langerhans cells then declines sharply in the VHF bulge, and the VHF bulb lacks MHC class II+ cells. Mast cells densely populate the VHF connective tissue sheath, where they strikingly cluster around the bulge. Both the bulge and the bulb of VHF display signs of immune privilege, that is, low MHC class I and MHC class II expression and local immunoinhibitor expression (CD200, TGFβ1). This immunophenotyping study fills an important gap in the immunobiology of murine skin and identifies differences between the IS of VHF, mouse pelage and human terminal HFs. This facilitates utilizing murine VHF as a versatile organ culture model for general immunology and immune privilege research in situ.
- Objective: Transplants of multipotent stem cells have been shown to have a neuropro- tective effect after central nervous system injury. The bulge region of the hair follicle has been reported as a putative source of hair follicle stem cells (HFSC) for many years; however, few studies have documented the properties of bulge derived cells in vitro until now. This study was conducted to isolate and culture bulge cells from rat hair follicles and to determine the morphological and biological features of the cultured cells. Materials and Methods: The bulge region of the rat whisker was isolated and cul- tured in Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12) sup- plemented with epidermal growth factor (EGF), cholera toxin. Dissociated bulge stem cells were differentiated on coated substrates together with NT-3. The morphological and biological features of cultured bulge cells were observed by light microscopy and immunocytochemistry methods. Results: Our results showed that newly proliferated cells could be observed on the 4th day after explantation. The expression of a neural progenitor marker, nestin, was seen before differentiation of the bulge cells. The differentiated cells expressed βIII-Tubulin and RIP, which are the markers of neural and glial lineages. Conclusion: The results indicated that the bulge cells cultured from the rat hair follicle had the characteristics of stem cells and could differentiate into neural and glial lineages.
- Despite the importance of integrins in epithelial cell biology surprisingly little is known about their regulation. It is known that they form hemidesmosomes (HDs), are actively involved in cell contacts during cell migration/invasion, and are key signaling molecules for survival and growth. However, there has been a distinct lack of understanding about what controls the dynamic integrin localization during cell activation and movement. Growth factors, such as EGF, are elevated during wound healing and carcinoma invasion leading to phosphorylation of ITGβ4 and the disassembly of the HD and mobilization of ITGβ4 to actin-rich protrusions. More recently the phosphorylation of a novel site on ITGβ4 (S1424) was found to be distinctly enriched on the trailing edge of migrating cells, suggesting a possible mechanism for the dissociation of ITGβ4 from HDs. Arrestin family member proteins are involved in the regulation of cell surface proteins and vesicular trafficking. In this study, we find that over-expression of arrestin family member ARRDC3 causes internalization and proteosome-dependent degradation of ITGβ4, while decreased levels of ARRDC3 stabilizes ITGβ4 levels. These results lead us to a new mechanism of ITGβ4 internalization, trafficking and degradation. During migration, ARRDC3 co-localizes with ITGβ4 on the lagging edge of cells but has a distinct distribution on the leading edge of cells. Additional immuno co-precipitation experiments demonstrate that ARRDC3 preferentially binds to ITGβ4 when phosphorylated on S1424. Using confocal microscopy, we show that the expression pattern of ARRDC3 on the lagging edge of a migrating cell is identical to the expression pattern of ITGβ4-pS1424. We demonstrate that ARRDC3 expression represses cell proliferation, migration, invasion, growth in soft agar and tumorigenicity. Collectively, our data reveals that ARRDC3 is a negative regulator of β4 integrin and demonstrates how this new pathway impacts biologic processes in stem cell and cancer biology. Additionally, as ARRDC3 is highly expressed in several tissues and conserved across species, our results are likely to be translated to other models.
- Observations in thyroid patients and experimental animals show that the skin is an important target for the thyroid hormones. We have previously shown that deletion in mice of the thyroid hormone nuclear receptors TRα1 and TRβ (the main thyroid hormone binding isoforms), results in impaired epidermal proliferation, hair growth and wound healing. Stem cells located at the bulges of the hair follicles are responsible for hair cycling and contribute to the regeneration of the new epidermis after wounding. Therefore a reduction in the number or function of the bulge stem cells could be responsible for this phenotype. Bulge cells show increased levels of epigenetic repressive marks, can retain bromodeoxyuridine labeling for a long time and have colony formation efficiency (CFE) in vitro. Here, we demonstrate that mice lacking TRs do not have a decrease of the bulge stem cell population. Rather, they show an increase of label-retaining cells (LRCs) in the bulges and enhanced CFE in vitro. A reduced activation of stem cells leading to their accumulation in the bulges is indicated by a strongly reduced response to mobilization by 12-O-tetradecanolyphorbol-13-acetate (TPA). Altered function of the bulge stem cells is associated with aberrant activation of Smad signaling leading to a reduced nuclear accumulation of β-catenin, which is crucial for stem cell proliferation and mobilization. LRCs of TR deficient mice also show increased levels of epigenetic repressive marks. We conclude that thyroid hormone signaling is an important determinant of the mobilization of stem cells out of their niche in the hair bulge. These findings correlate with the skin defects observed in mice and with the alterations found in human thyroid disorders. © 2015 by The American Society for Cell Biology.
- Somatic stem cells replenish many tissues throughout life to repair damage and to maintain tissue homeostasis. Stem cell function is frequently described as following a hierarchical model in which a single master cell undergoes self-renewal and differentiation into multiple cell types and is responsible for most regenerative activity. However, recent data from studies on blood, skin and intestinal epithelium all point to the concomitant action of multiple types of stem cells with distinct everyday roles. Under stress conditions such as acute injury, the surprising developmental flexibility of these stem cells enables them to adapt to diverse roles and to acquire different regeneration capabilities. This paradigm shift raises many new questions about the developmental origins, inter-relationships and molecular regulation of these multiple stem cell types.
- There is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs. We investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract. Immunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, β1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models. These data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.
- This study investigated the potential hair regrowth effects associated with a plant extract of Perilla frutescens, which was selected due to its putative hair regrowth activity. Extracts were prepared from dried P. frutescens suspended in distilled water, where the resultant aqueous suspension was fractionated sequentially using hexane, ethyl acetate, n-butanol, and distilled water. We observed that the n-butanol fraction resulted in the highest hair regrowth activity. The n-butanol soluble fraction of P. frutescens extract (BFPE) was further separated using AB-8 macroporous resin and silica gel chromatography to obtain rosmarinic acid (RA), which demonstrated effective hair growth regeneration potential. BFPE also showed in vivo anti-androgenic activity following the use of a hair growth assay in testosterone-sensitive male C57Bl/6NCrSlc mice. Furthermore, the effects of cell viability promotion were investigated following an in vitro analysis in primary hair follicle fibroblast cells (PHFCs) treated with RA. The results suggested that RA was the active compound in P. frutescens that triggers hair growth, and RA could be a potential therapeutic agent for the promotion of hair growth and prevention of androgenetic alopecia (AGA).
- Stem cells (SC) for the corneal epithelium are located exclusively at the limbus, i.e., the anatomic junction between the cornea and conjunctiva. Limbal epithelial SC are the ultimate source of regeneration of the entire corneal epithelium under normal and injured states. The epithelial phenotype of the limbal basal epithelium does not express corneal epithelial-specific keratin3, keratin 12 and connexin 43. As shown in cell-cycle kinetic studies, some portions of the limbal basal epithelial cells are slow-cycling and label-retaining. Other studies further confirmed limbal epithelial SC have greater growth potential in explant cultures and higher clonogenicity when cocultured on 3T3 fibroblast-feeder layers than corneal epithelial cells, and their proliferative potential is resistant to tumor-promoting phorbol esters.
- This chapter will focus on the basic and fundamental ideas of how to design an affinity-based drug delivery system. First, the various types of delivery mechanisms and the mathematics that govern these mechanisms will be discussed to reveal how affinity-based mechanisms are unique. Next, the most common affinity mechanisms will be described, with biological examples. Finally, the appropriate design of an affinity-based drug delivery system for specific applications will be expounded in detail. Literature examples will be used to explain general approaches to affinity mechanisms. Description of polymer-based drug delivery mechanisms As new materials and delivery systems are developed, polymer-delivery-based applications are becoming attractive because of their versatility in adapting the delivery system for specific applications [1–10]. These polymer systems can be designed with various release mechanisms: (1) a diffusion-controlled mechanism, in which the release of the drug into the body is achieved by means of a concentration gradient; (2) an erosion-controlled mechanism, in which the release of the drug is achieved by means of the polymer degrading in the body; and (3) an affinity-controlled mechanism, in which the release of the drug is achieved by means of an equilibrium relationship between the drug and polymer. See Figure 23.1 for a visual qualitative representation of these three drug delivery systems.
- The hair follicle (HF) is a skin appendage providing physical barrier to protect mammals from external stimuli. HF has been attracting great interest as an object of developmental and stem cell research, however, little attention has been paid to HF in the light of immunology. Previous observations supported that HF might enjoy immune privilege by suppressing immune-related antigen expression and producing immunosuppressive molecules, indicating HF possesses intrinsic machinery to escape from unwanted immune response. The recent discovery that HF segments differentially express chemokines, both chemoattractive and possibly suppressive, suggested that HF is not merely an innocent bystander but may play previously unrecognized roles in the regulation of immune reactions in skin. In this chapter, basic knowledge of hair follicle morphology and physiology is summarized. The similarities and differences between mouse pelage and human scalp hair follicles is presented, which is crucial for translational research. Finally, immunological features of hair follicle segments is discussed.
- Proximally the arrector pili muscle (APM) attaches to the follicular stem cell niche in the bulge but the distal properties remain comparatively unclear to date. Methods: A novel method employing an F-actin probe, phalloidin, is employed to visualize the APM anatomy. Phalloidin staining of the APM was validated by comparison with conventional antibodies/stains and generating three-dimensional reconstructions. The proximal attachment of the APM to the bulge in 8 patients with androgenic alopecia was studied using Masson Trichrome stain. Results: Phalloidin visualized extensive branching of the APM. The distal end of the human APM exhibits a unique "C" shaped structure connecting to the dermalepidermal junction. The proximal APM attachment demonstrated to be lost or extremely miniaturised in androgenic alopecia. Conclusion: The unique shape, location and attachment sites of the APM suggest a significant role for this muscle in maintaining follicular integrity. Proximally, the APM encircles the follicular unit and only attaches to the primary hair follicle in the bulge. This attachment is lost in irreversible hair loss. Exhibiting an arborized morphology as it ascends toward the epidermis. The muscle anchors to the basement membrane.
- BNC2 is an extremely conserved zinc finger protein with important functions in the development of craniofacial bones and male germ cells. Because disruption of the Bnc2 gene in mice causes neonatal lethality, the function of the protein in adult animals has not been studied. Until now BNC2 was considered to have a wider tissue distribution than its paralog, BNC1, but the precise cell types expressing Bnc2 are largely unknown. We identify here the cell types containing BNC2 in the mouse and we show the unexpected presence of BNC1 in many BNC2-containing cells. BNC1 and BNC2 are colocalized in male and female germ cells, ovarian epithelial cells, sensory neurons, hair follicle keratinocytes and connective cells of organ capsules. In many cell lineages, the two basonuclins appear and disappear synchronously. Within the male germ cell lineage, BNC1 and BNC2 are found in prospermatogonia and undifferentiated spermatogonia, and disappear abruptly from differentiating spermatogonia. During oogenesis, the two basonuclins accumulate specifically in maturing oocytes. During the development of hair follicles, BNC1 and BNC2 concentrate in the primary hair germs. As follicle morphogenesis proceeds, cells possessing BNC1 and BNC2 invade the dermis and surround the papilla. During anagen, BNC1 and BNC2 are largely restricted to the basal layer of the outer root sheath and the matrix. During catagen, the compartment of cells possessing BNC1 and BNC2 regresses, and in telogen, the two basonuclins are confined to the secondary hair germ. During the next anagen, the BNC1/BNC2-containing cell population regenerates the hair follicle. By examining Bnc2(-/-) mice that have escaped the neonatal lethality usually associated with lack of BNC2, we demonstrate that BNC2 possesses important functions in many of the cell types where it resides. Hair follicles of postnatal Bnc2(-/-) mice do not fully develop during the first cycle and thereafter remain blocked in telogen. It is concluded that the presence of BNC2 in the secondary hair germ is required to regenerate the transient segment of the follicle. Postnatal Bnc2(-/-) mice also show severe dwarfism, defects in oogenesis and alterations of palatal rugae. Although the two basonuclins possess very similar zinc fingers and are largely coexpressed, BNC1 cannot substitute for BNC2. This is shown incontrovertibly in knockin mice expressing Bnc1 instead of Bnc2 as these mice invariably die at birth with craniofacial abnormalities undistinguishable from those of Bnc2(-/-) mice. The function of the basonuclins in the secondary hair germ is of particular interest.
- 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.
- Keratinocytes comprise the bulk of the epithelium, undergo keratinization and form dead superficial layers of the skin. These superficial keratinized cells are continuously replaced by cells derived from mitotic cells in the lowest layer of the epidermis (basal layer). The proliferation and differentiation of keratinocytes is regulated by many factors including hormones, growth factors and cytokines. Melanocytes are cells in the basal layers of the epidermis that do not keratinize but can produce melanin pigments. Keratinocytes and melanocytes migrate from the epidermis to the dermis to form pigmented hairs. Mammalian hair is formed by three kinds of cell, namely keratinocytes and melanocytes derived from the epidermis and fibroblasts consisting of the dermal papilla. Cellular interactions between keratinocytes and melanocytes/fibroblasts are important for the development and maintenance of hair. Keratinocyte stem cells and melanocyte stem cells locate in the bulge of hair follicles and produce new proliferating and differentiating keratinocytes and melanocytes at the new hair growth cycle. The proliferation and differentiation of keratinocytes appear to be regulated by their own autocrine factors as well as by paracrine factors derived from melanocytes and fibroblasts. Keratinocytes also produce and release proliferation- and differentiation-stimulating factors towards melanocytes and fibroblasts. Thus, in addition to the autocrine factors, the paracrine factors derived from the tissue environment such as melanocytes and fibroblasts are thought to play an important role in the regulation of keratinocyte function as well as maintenance of skin homeostasis.
- Although the loss of scalp hair is distressing and many medical treatments focus on its restoration, the removal of body hair has been adopted since ancient times. Beauty standards, which r eflect the culture of each society, have been presenting the depilated body as absolutely desirable. Through the ages various methods of hair removal have been used depending on the requirements of the individuals. In recent years, Laser and Intense Pulse Light devices have been considered as the most promising solution for excess hair growth, without excluding the efficacy of other methods to induce satisfactory epilatory results. The enzyme-based hair removal method has received little recognition even though experimental and clinical data support its efficacy to provide long term or even permanent epilation. The present review presents these data and examines the likelihood of considering the aforementioned method as ideal.
- Hair follicle stem cells (HFSCs) are able to differentiate into neurons and glial cells. Distinct microRNAs (miRNAs) regulate the proliferation and differentiation of HFSCs. However, the exact role of miR‐124 in the neural differentiation of HFSCs has not been elucidated. HFSCs were isolated from mouse whisker follicles. miR‐9, let‐7b, and miR‐124, Ptbp1 , and Sox9 expression levels were detected by real‐time polymerase chain reaction (RT‐PCR). The influence of miR‐124 transfection was evaluated using immunostaining. We demonstrated that miR‐124 and let‐7b expression levels were significantly increased after the neural differentiation. Sox9 and Ptbp1 were identified as the target of miR‐124 in the HFSCs. During neural differentiation and miR‐124 mimicking, Ptbp1 and Sox9 levels were decreased. Moreover, the miR‐124 overexpression increased MAP2 (58.43 ± 11.26) and NeuN (48.34 ± 11.15) proteins expression. The results demonstrated that miR‐124 may promote the differentiation of HFSCs into neuronal cells by targeting Sox9 and Ptbp1.
- The adult body harbors powerful reservoirs of stem cells that enable tissue regeneration under homeostatic conditions or in response to disease or injury. The hair follicle is a readily accessible mini organ within the skin and contains stem cells from diverse developmental origins that are shown to have surprisingly broad differentiation potential. In this chapter, we discuss the biology of the hair follicle with particular emphasis on the various stem cell populations residing within the tissue. We summarize the existing knowledge on putative hair follicle stem cell markers, the differentiation potential, and technologies to isolate and expand distinct stem cell populations. We also discuss the potential of hair follicle stem cells for drug and gene delivery, tissue engineering, and regenerative medicine. We propose that the abundance of stem cells with broad differentiation potential and the ease of accessibility make the hair follicle an ideal source of stem cells for gene and cell therapies. © 2013 Springer Science+Business Media New York. All rights reserved.
- Hair follicle stem cells (HFSCs) possess fascinating self-renewal capacity and multipotency, which play important roles in mammalian hair growth and skin wound repair. Although HFSCs from other mammalian species have been obtained, the characteristics of ovine HFSCs, as well as the methods to isolate them have not been well addressed. Here, we report an efficient strategy to obtain multipotent ovine HFSCs. Through microdissection and organ culture, we obtained keratinocytes that grew from the bulge area of vibrissa hair follicles, and even abundant keratinocytes were harvested from a single hair follicle. These bulge-derived keratinocytes are highly positive for Krt15, Krt14, Tp63, Krt19 and Itga6; in addition to their strong proliferation abilities in vitro, these keratinocytes formed new epidermis, hair follicles and sebaceous glands in skin reconstitution experiments, showing that these are HFSCs from the bulge outer root sheath. Taken together, we developed an efficient in vitro system to enrich ovine HFSCs, providing enough HFSCs for the investigations about the ovine hair cycle, aiming to promote wool production in the future.
- The principal goal of agriculture is the production of high quality, safe and affordable food for an ever-increasing population worldwide. Furthermore, agricultural growers and producers have the additional constraints of economic profitability and sustainability. Looking at the negative environmental impact of chemical fertilizers, the use of beneficial soil microorganisms for sustainable and safe agriculture has increased globally during the last couple of decades. Plant growth promoting rhizobacteria are naturally occurring soil bacteria that assertively colonize plant roots and benefit plants by providing growth factors. Stressrelieving and antagonistic rhizobacteria might be useful in formulating new inoculants with combinations of different mechanisms of action, leading to a competent use for biocontrol strategies to improve cropping systems.
- This chapter reviews present knowledge of keratinocyte stem cells of the cornea, epidermis, and hair follicle. All external surfaces of the body, including the skin and cornea, are covered by a stratified squamous epithelia composed of keratinocytes. These cells synthesize tissue-restricted keratin intermediate filament proteins, and form a specialized submembrane structure known as the comified envelop in the final stages of differentiation that is composed of covalently crosslinked proteins, including involucrin and loricrin. Stem cells exist in specific locations within a given tissue, and comprise a small percentage of the total cell population. They are ultrastructurally unspecialized, with a large nuclear-to-cytoplasmic ratio and few organelles. Stem cells have a proliferative potential that exceeds an individual's lifetime. Because stem cells cycle slowly, and represent only a small percentage of a cellular population, an intermediate group of more rapidly proliferating cells exists, that form clonal expansions resulting in the final, differentiated cell population. The stem cells of the corneal epithelium are located in a narrow section of the peripheral cornea, bordering the bulbar conjunctiva, termed the limbus. In this chapter, nine characteristics of stem cells derived initially from studies of the hematopoietic system are used as a framework to discuss keratinocyte populations.
- Conflict-of-interest: The authors declare that there are no conflicts of interest. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. Abstract Hair follicle stem cells (HFSCs) normally give rise to keratinocytes, sebocytes, and transient amplifying progenitor cells. Along with the capacity to proliferate rapidly, HFSCs provide the basis for establishing a putative source of stem cells for cell therapy. HFSCs are multipotent stem cells originating from the bulge area. The importance of these cells arises from two important characteristics, distinguishing them from all other adult stem cells. First, they are accessible and proliferate for long periods. Second, they are multipotent, possessing the ability to differentiate into mesodermal and ectodermal cell types. In addition to a developmental capacity in vitro , HFSCs display an ability to form differentiated cells in vivo. During the last two decades, numerous studies have led to the development of an appropriate culture condition for producing various cell lineages from HFSCs. Therefore, these stem cells are considered as a novel source for cell therapy of a broad spectrum of neurodegenerative disorders. This review presents the current status of human, rat, and mouse HFSCs from both the cellular and molecular biology and cell therapy perspectives. The first section of this review highlights the importance of HFSCs and in vitro differentiation, while the final section emphasizes the significance of cell differentiation in vivo .
- Human epidermis represents a large reservoir of stem cells that continue to self-renew throughout life. Stem cells are essential for skin regeneration and for repair after wounding. They allow long-term culture of keratinocytes that produce large sheets of epidermis to cover extensive burns, thus being lifesaving for these patients. Furthermore, stem cells can be expanded in culture, genetically modified to correct the gene deficiency in genetic skin diseases. This chapter will describe the most recent data on stem cell biology and the potential medical applications of these cells.
- Transcriptional regulation is fundamentally important for the progression of tissue stem cells through different stages of development and differentiation. Mammalian skin epidermis is an excellent model system to study such regulatory mechanisms due to its easy accessibility, stereotypic spatial arrangement, and availability of well-established cell type/lineage differentiation markers. Moreover, epidermis is one of the few mammalian tissues the stem cells of which can be maintained and propagated in culture to generate mature cell types and a functional tissue (reviewed in [1]), offering in vitro and ex vivo platforms to probe deep into the underlying cell and molecular mechanisms of biological functions.
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Heid, H. W., Werner, E. & Franke, W. W. (1986) Differentiation 32, 101-119.- The events in epidermal development are distinctive for each trimester. During the first trimester, the epidermis changes from a single-layered covering to a stratified epithelium. Many of the structural changes that occur establish cellular integration and support to stabilize this barrier between the embryo and its environment. The morphologic and physiologic evidence, indicates, however, that the epidermis is not a functional barrier to diffusible compounds during this period, a time when the environment is comparable and perhaps important in providing substances needed for development. This is also a period in which all of the primordia of the epidermal appendages are initiated and the immigrant cells begin to migrate in among the keratinocytes. In the second trimester, the system is established and the pattern for the adult epidermis is laid out. Further development and differentiation are required to refine the pattern into a functional system which will acquire the properties of the adult tissue. The work of the second trimester then is to expand and elaborate certain structures and to change and modify others. The functional state is altered as glands become secretory and the epidermis differentiates into a functional barrier, first by regression of the periderm and secondly with keratinization of the epidermis proper. Exclusion of the amniotic fluid occurs at a time when this formerly compatible environment becomes hostile. The epidermis, thus formed in nearly its final form, ceases, new developmental events and subsists throughout the third trimester until near birth in basically the same condition. Most of the data to support these generalizations have come from an impressive body of morphologic information. From the present review, it should be quite apparent that the quantitative and functional aspects of epidermal development are areas where inquiry is needed so that speculation may be substantiated by data and replaced with fact.
- A study was made on the stem cell concepts. When we are talking about stem cells, we do not necessarily, and certainly not always talk about hemopoietic stem cells. There are many kinds of stem cells in the adult organism and there is one definition only which can apply to all these stem cells, and that is: a self-maintaining capacity. This means that stem cells can maintain their numbers for at least one life span of the organism. This means that 'stemness' implies an extensive self-maintaining capacity, and we mean self-maintaining capacity for many cell cycles, which in the mouse in most cases of 200 cell cycles, and in man - especially with a hemopoietic or intestinal stem cell system - well in excess of a thousand cell cycles. Every cell in the body, after the zygote and the first few cleavage divisions, is already a differentiated cell. That is: stem cells in the late embryonic crypt stem cells, skin epithelial stem cells or liver stem cell, are already differentiated cells. It is true that there are some stem cells which can give rise to further differentiated products, but not all stem cells do so. The author has given in his paper some of his ideas, about G(o) and genetic housekeeping, proliferation control and cell interaction, which like all concepts are pure fiction until such time that appropriate experimental evidence corroborates them. But at least, with the hemopoietic stem cells assay methods are available and with a little bit of clear thinking, clear definition - according to the author - and proper use of the available assays, the ideas can be tested. (E. Szirmai - Stuttgart) - Stuttgart, GFR)
- Morphological changes which occur in the growth cycle of the rat vibrissal follicle during the transitional period between consecutive anagen phases are described. In contrast with pelage hair follicles, there is no shortening of the follicle, no formation of a papilla 'rest' and no close synchrony between club differentiation and follicle regression. Telogen is therefore considered to occur after loss of the matrix of the hair bulb and maximal diminution of the dermal papilla to a small aggregation of cells. These difference are discussed in relation to current nomenclature of the hair cycle and the function of the vibrissal follicle.
- Human diploid epidermis epidermal cells have been successfully grown in serial culture. To initiate colony formation, they require the presence of fibroblasts, but proliferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct density. When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human fibroblasts is largely suppressed, but epidermal cells grow from single cells into colonies. Each colony consists of keratinocytes ultimately forming a stratified squamous epithelium in which the dividing cells are confined to the lowest layer(s). Hydrocortisone is added to the medium, since in secondary and subsequent subcultures it makes the colony morphology more oderly and distinctive, and maintains proliferation at a slightly greater rate. Under these culture conditions, it is possible to isolate keratinocyte clones free of viable fibroblasts. Like human diploid fibroblasts, human diploid keratinocytes appear to have a finite culture lifetime. For 7 strains studied, the culture lifetime ranged from 20-50 cell generations. The plating efficiency of the epidermal cells taken directly from skin was usually 0.1-1.0%. On subsequent transfer of the cultures initiated from newborns, the plating efficiency rose to 10% or higher, but was most often in the range of 1-5% and dropped sharply toward the end of their culture life. The plating efficiency and culture lifetime were lower for keratinocytes of older persons.
- A microcolony technique is described for measuring epidermal cell survival 3 days after whole-body X-irradiation. This assay provides a cell D0 value of 233 +/- 11 rad and a zero-dose extrapolate of 1-23 x 10(4) cells/cm2 for mice irradiated in oxygen 20 hours after hair plucking. The microcolony cellularity had an apparent doubling-time of 25 hours which may be an upper limit at least for some clones. The clones appeared to fragment continually and form new daughter clones, suggesting that few would form macroscopic nodules. Many of the clones were also apparently associated with hair follicles.
- The growth of hair from the mystacial vibrissal follicles of C3H mice and Wistar rats has been measured for up to seven cycles. Normally growth in length and thickness was regular and little affected by age or sex. Plucking vibrissae at any stage during the growing period was followed by the appearance of a new vibrissa 8-11 days later. Plucking when growth had ceased had no effect on the time of appearance of the subsequent cycle. New whiskers emerging after plucking grew at the normal rate. Withholding food slowed the growth of vibrissae within 1 day. Normal growth was re-established 3 days after return to full diet.
- The mammalian hair follicle is a treasure waiting to be discovered by more molecular geneticists. How can a tiny cluster of apparently uniform epithelial cells, adjacent to a tiny cluster of uniform mesenchymal cells, give rise to five or six concentric cylinders, each of which is composed of cells of a distinctive type that synthesize their own distinctive set of proteins? There is now evidence that several growth factors, cell adhesion molecules and other molecules play important roles in the regulation of this minute organ.
- A novel 150 kd protein expressed on the surface of mesenchymal cells of mouse embryonic tissues was identified. A monoclonal antibody to this molecule inhibited various processes of epithelial morphogenesis, such as hair follicle growth and lung epithelial tubular formation, in organ cultures of these tissues. Sequence analysis of cDNA encoding this protein revealed that it had 289 amino acids with a hydrophobic stretch at the C-terminus. NIH 3T3 cells transfected with the cDNA of this protein expressed the exogenous 150 kd protein on their surface. When lung epithelial cells were cocultured with these transfected cells, they showed normal tubular morphogenesis, but not with untransfected NIH 3T3 cells. These results indicate that this protein, termed epimorphin, plays a central role in epithelial-mesenchymal interactions.
- We report for the first time the successful maintenance and growth of human hair follicles in vitro. Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermo-subcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watchmakers' forceps. Isolated hair follicles maintained free-floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinised hair shaft, and was not associated with the loss of hair follicle morphology. [methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labelling of keratins showed that their patterns of synthesis did not change with maintenance. The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-beta 1 has a negative growth-regulatory effect on hair follicles in vitro and that EGF mimics the in vivo depilatory effects that have been reported in sheep and mice.
- Germinative epidermal cells in the lower end bulb region of anagen hair follicles are highly active, and give rise to hair fibres through rapid proliferation and complex differentiation. They have often been termed hair follicle stem cells, but owing to difficulties in isolation and identification their properties have previously only been clearly documented in vivo. We aimed to isolate and culture germinative cells in vitro, and used microdissection methods to dissect a small but identifiable group of cells from complete follicles. Transmission electron microscopy confirmed that the isolated cells were identical to germinative epidermal cells in situ. SDS-PAGE was used to show that they did not have the same protein composition as epidermis from their immediate proximity (overlying hair matrix), or from other follicular (outer root sheath) and interfollicular (skin basal) regions. Moreover, the germinative cells were found to display morphology and in vitro behaviour that distinguished them from comparative epidermal cells. When cultured in media and on substrata normally conducive to epidermal cell growth they remained in a quiescent state, and did not divide or differentiate. In contrast to other epidermal cells that formed typical pavement-like arrangements, germinative cells remained uniformly small, round and closely packed. However, when cultured in association with hair follicle dermal papilla cells they were radically stimulated into proliferative and aggregative behaviour. Furthermore, they were able to form organotypic-like structures, and exceptionally for skin-derived cell recombinations, a distinct basal lamina at the papilla-germinative cell junction. These results provide evidence that hair follicle germinative cells have intriguing properties that distinguish them from other follicular epidermis. The finding that they can be activated by dermal papilla cells reflects the intimate nature of the papilla-germinative cell relationship in situ, and should facilitate research into hair growth control mechanisms. The nature of germinative cells is discussed in the wider context of hair follicle stem-cell terminology.
- The Journal of Investigative Dermatology publishes basic and clinical research in cutaneous biology and skin disease.
- Inconsistent with the view that hair follicle stem cells reside in the matrix area of the hair bulb, we found that label-retaining cells exist exclusively in the bulge area of the mouse hair follicle. The bulge consists of a subpopulation of outer root sheath cells located in the midportion of the follicle at the arrector pili muscle attachment site. Keratinocytes in the bulge area are relatively undifferentiated ultrastructurally. They are normally slow cycling, but can be stimulated to proliferate transiently by TPA. Located in a well-protected and nourished environment, these cells mark the lower end of the "permanent" portion of the follicle. Our findings, plus a reevaluation of the literature, suggest that follicular stem cells reside in the bulge region, instead of the lower bulb. This new view provides insights into hair cycle control and the possible involvement of hair follicle stem cells in skin carcinogenesis.
- Keratinocytes isolated from human epidermis and subsequently cultured may form clones if they are 11 micron or less in diameter but are irreversibly committed to further enlargement and terminal differentiation if they are 12 micron or more in diameter. When a founding cell of 11 micron or less forms a small rapidly growing clone in culture, the cells of that clone are able to found new colonies even when their diameter is as great as 20 micron. As the clone becomes larger and grows more slowly, the maximal size of its clonogenic cells is reduced toward that of the epidermis. A cultured cell of up to 20 micron in diameter can, when it divides, give rise to clonogenic progeny smaller than itself, thus reversing the process of enlargement. Cells larger than 20 micron cannot divide and therefore cannot be rescued from terminal differentiation. It is concluded that when keratinocytes multiply rapidly, they extend reversibly the maximal size at which they are capable of generating clones into the range usually characteristic of terminally differentiating cells. It is proposed that this mechanism enables the keratinocyte to accommodate an increased rate of multiplication to its need to attain a large size during terminal differentiation.
- Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co-expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation.
- Living hair-forming cells (trichocytes) were obtained from basal portions of human, bovine and ovine hair-follicles, free from contaminations of root-sheath epithelia. Their intermediate filament (IF) cytoskeleton was studied by gel electrophoresis of the native, i.e. non-S-carboxymethylated polypeptides, by peptide-map analysis of the individual components, by reconstitution experiments and by immunological methods. The IF protein complement of trichocytes from all three species is characterized by a very similar set of eight highly conserved alpha-keratin polypeptides, comprising four members of the basic (type II; Mr 56,500-60,000) and four members of the acidic (type I; Mr 41,000-44,000) cytokeratin subfamily. None of these eight trichocyte alpha-keratin polypeptides, which form heterotypic complexes and IF in vivo and in vitro, is identical to any of the epithelial cytokeratins of the same species. All the trichocyte-specific cytokeratins are native polypeptides encoded by different mRNAs, as demonstrated by in vitro translation of hair follicle mRNA. The same polypeptides are also found in mature hairs, although with different patterns of modification. Our study provides the first analysis of the native unmodified alpha-keratin polypeptides of trichocytes and hairs and therefore allows a direct comparison of these with the epithelial cytokeratins and other IF proteins from the same species. These findings indicate that, during fetal hair-follicle formation, the differentiation of trichocytes from epithelial cells involves a complete cessation of the synthesis of epithelial cytokeratins and a marked induction of the synthesis of a complex set of trichocyte-specific cytokeratins.
- Colony-forming human epidermal cells are heterogeneous in their capacity for sustained growth. Once a clone has been derived from a single cell, its growth potential can be estimated from the colony types resulting from a single plating, and the clone can be assigned to one of three classes. The holoclone has the greatest reproductive capacity: under standard conditions, fewer than 5% of the colonies formed by the cells of a holoclone abort and terminally differentiate. The paraclone contains exclusively cells with a short replicative lifespan (not more than 15 cell generations), after which they uniformly abort and terminally differentiate. The third type of clone, the meroclone, contains a mixture of cells of different growth potential and is a transitional stage between the holoclone and the paraclone. The incidence of the different clonal types is affected by aging, since cells originating from the epidermis of older donors give rise to a lower proportion of holoclones and a higher proportion of paraclones.
- We have investigated keratin and keratin mRNA expression during (1) differentiation of stem cells into epidermis and hair follicles and (2) morphogenesis of follicles. Our results indicate that a type I keratin K14 is expressed early in embryonal basal cells. Subsequently, its expression is elevated in the basal layer of developing epidermis but suppressed in developing matrix cells. This difference represents an early and major biochemical distinction between the two diverging cell types. Moreover, because expression of this keratin is not readily influenced by extracellular regulators or cell culture, it suggests a well-defined and narrow window of development during which an irreversible divergence in basal and matrix cells may take place. In contrast to K14, which is expressed very early in development and coincident with basal epidermal differentiation, a hair-specific type I keratin and its mRNA is expressed late in hair matrix development and well after follicle morphogenesis. Besides providing an additional developmental difference between epidermal and hair matrix cells, the hair-specific keratins provide the first demonstration that keratin expression may be a consequence rather than a cause of cell organization and differentiation.
- A transglutaminase-catalyzed cross-linking process characteristic of keratinocytes leads to the formation of the insoluble corneocyte envelope. The essentials of this process take place in vitro in a reconstituted system derived from subcellular fractions. A particulate fraction containing membrane-bound envelope precursor proteins and the enzyme transglutaminase is combined with cytosolic proteins; when the enzyme is activated by Ca++, cytosolic proteins are removed from solution and cross-linked to particulate proteins. This interaction is cell-type-specific, since particulates derived from fibroblasts and also containing transglutaminase activity cannot substitute for those of keratinocytes. Involucrin, a cytosolic protein known to be a precursor of the envelope, is more efficiently cross-linked than other cytosolic proteins. The cross-linking of proteins of the particulate fraction (membrane proteins) is promoted by the presence of involucrin.
- We have developed a culture system for detecting and isolating rare hypoxanthine phosphoribosyltransferase-deficient mutants of human epidermal keratinocytes. A thioguanine-resistant variant, 3T3M1, of the Swiss mouse fibroblast line 3T3 was used as a feeder layer to support clonal growth of mutant keratinocytes. A near-diploid, epidermal squamous cell carcinoma line, SCC-13Y, was used as a prototype to determine mutagen treatment conditions, plating density, and phenotypic expression time for maximum mutant recovery. To extend this system to normal keratinocytes, we improved the culture conditions by adding insulin, adenine, and Ham's nutrient mixture F-12, which increased colony-forming efficiencies to 30% in early passage and made feasible the detection of rare mutants in normal epidermal keratinocyte populations. We have quantitated mutation in SCC-13Y and three strains of normal human epidermal keratinocytes after exposure to polycyclic aromatic hydrocarbons, which are activated to their mutagenic forms by cellular mixed-function oxidases. 7,12-Dimethylbenz[a]anthracene and benzo[a]pyrene caused almost no cytotoxicity, but induced thioguanine-resistant mutants at frequencies as much as 50-fold higher than the spontaneous frequency of approximately 10(-6). The mutants were aminopterin-sensitive and possessed no measurable hypoxanthine phosphoribosyltransferase activity; their behavior was indistinguishable from that of keratinocytes cultured from individuals with Lesch-Nyhan syndrome. This mutagenesis assay system should also be applicable to other feeder layer-dependent human epithelial cell types, such as urothelial, mammary, and tracheal epithelial cells.
- The volumes of the dermal papilla, matrix and fully grown hair of vibrissal follicles of rats and mice were measured. Dermal papillae were surgically removed from rats' vibrissal follicles and a small new dermal papilla was always reformed. The lower third of rats and mice follicles, containing the whole of the dermal papilla was amputated and very small dermal papillae were reformed. Follicles containing small dermal papillae produced small hairs and a constant positive linear relationship of 7.0 was found between the volume of the dermal papilla and the volume of the full-grown hair for all cases studied.
- The Journal of Investigative Dermatology publishes basic and clinical research in cutaneous biology and skin disease.
