Regulation of melanocyte stem cells in the pigmentation of skin and its appendages: Biological patterning and therapeutic potentials

Skin evolves essential appendages and indispensable types of cells that synergistically insulate the body from environmental insults. Residing in the specific regions in the skin such as epidermis, dermis and hair follicle, melanocytes perform an array of vital functions including defending the ultraviolet radiation and diversifying animal appearance. As one of the adult stem cells, melanocyte stem cells in the hair follicle bulge niche can proliferate, differentiate and keep quiescence to control and coordinate tissue homeostasis, repair and regeneration. In synchrony with hair follicle stem cells, melanocyte stem cells in the hair follicles undergo cyclic activation, degeneration and resting phases, to pigment the hairs and to preserve the stem cells. Disorder of melanocytes results in severe skin problems such as canities, vitiligo and even melanoma. Here, we compare and summarize recent discoveries about melanocyte in the skin, particularly in the hair follicle. A better understanding of the physiological and pathological regulation of melanocyte and melanocyte stem cell behaviours will help to guide the clinical applications in regenerative medicine.     

Perivascular localization of dermal stem cells in human scalp

In mammalian skin, the existence of stem cells in the dermis is still poorly understood. Previous studies have indicated that mesenchymal stem cells (MSCs) are situated as pericytes in various mammalian tissues. We speculated that the human adult dermis also contains MSC-like cells positive for CD34 at perivascular sites similar to adipose tissue. At first, stromal cells from adult scalp skin tissues showed colony-forming ability and differentiated into mesenchymal lineages (osteogenic, chondrogenic and adipogenic). Three-dimensional analysis of scalp skin with a confocal microscope clearly demonstrated that perivascular cells were positive for not only NG2, but also CD34, immunoreactivity. Perivascular CD34-positive cells were abundant around follicular portions. Furthermore, CD34-positive cell fractions collected with magnetic cell sorting were capable of differentiating into mesenchymal lineages. This study suggests that dermal perivascular sites act as a niche of MSCs in human scalp skin, which are easily accessible and useful in regenerative medicine.     

毛囊间充质干细胞的研究进展

 毛囊间充质干细胞（HFMSCs）是一类具有自我再生能力、高度增殖潜能、可多向分化且来源丰富的慢周期标记滞留细胞,可促进表皮、毛囊和皮脂腺再生。得益于其来源丰富、易获得、可于体外扩增、无需基因操作、不会形成肿瘤和无伦理限制等特点,毛囊间充质干细胞在再生医学中具有重要优势。Wnt、Shh、Notch和BMP等信号通路之间的协同和拮抗作用在干细胞稳态调节、表皮发育和毛囊再生过程中发挥至关重要的作用,这些途径的失调可能导致毛发脱落或者肿瘤的发生。本文综述毛囊间充质干细胞的分类及其特异性生物标记物、毛囊间充质干细胞的活化以及影响毛发再生的生物分子途径,为毛发疾病的治疗提供新的线索和靶点。     

Aging in hair follicle stem cells and niche microenvironment

Hair graying and hair loss are prominent and common characteristics of the elderly population. In some individuals these processes can significantly impact their quality of life, leading to depression, anxiety and other serious mental health problems. Accordingly, there has been much interest in understanding the complex physiological changes within the hair follicle in the aging individual. It is now known that hair follicles represent a prototypical stem cell niche, where both micro‐ and macroenvironmental influences are integrated alongside stem cell–stem cell and stem cell–stem niche interactions to determine hair growth or hair follicle senescence. Recent studies have identified imbalanced stem cell differentiation and altered stem cell activity as important factors during hair loss, indicating new avenues for the development of therapeutic agents to stimulate hair growth. Here, we pull together the latest findings on the hair follicle stem cell niche and the multifactorial interactions underlying the various forms of hair loss.     

Exploring the role of stem cells in cutaneous wound healing

Abstract:  The skin offers a perfect model system for studying the wound healing cascade, which involves a finely tuned interplay between several cell types, pathways and processes. The dysregulation of these factors may lead to wound healing disorders resulting in chronic wounds, as well as abnormal scars such as hypertrophic and keloid scars. As the contribution of stem cells towards tissue regeneration and wound healing is increasingly appreciated, a rising number of stem cell therapies for cutaneous wounds are currently under development, encouraged by emerging preliminary findings in both animal models and human studies. However, we still lack an in-depth understanding of the underlying mechanisms through which stem cells contribute to cutaneous wound healing. The aim of this review is, therefore, to present a critical synthesis of our current understanding of the role of stem cells in normal cutaneous wound healing. In addition to summarizing wound healing principles and related key molecular and cellular players, we discuss the potential participation of different cutaneous stem cell populations in wound healing, and list corresponding stem cells markers. In summary, this review delineates current strategies, future applications, and limitations of stem cell-based or stem cell-targeted therapy in the management of acute and chronic skin wounds.     

Immunophenotyping of the human bulge region: the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche

Abstract:  Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR.
According to our experience, the most useful positive markers for human bulge cells turned out to be cytokeratin 15, cytokeratin 19 and CD200, but were not exclusive, while β1 integrin and Lhx2 IR were not upregulated by human bulge keratinocytes. Absent IR for CD34, connexin43 and nestin on human bulge cells may be exploited as negative markers. α6 integrin, fibronectin, nidogen, fibrillin-1 and latent transforming growth factor (TGF)-beta-binding protein-1 were expressed throughout the connective tissue sheath of human HFs. On the other hand, tenascin-C was upregulated in the bulge and may thus constitute a component of the bulge stem cell niche of human HFs.
These immunophenotyping results shed further light on the in situ expression patterns of claimed follicular 'stem cell markers' and suggest that not a single marker alone but only the use of a limited corresponding panel of positive and negative markers may offer a reasonable and pragmatic compromise for identifying human bulge stem cells in situ .     

Assessment of epidermal subpopulations and proliferation in healthy skin, symptomless and lesional skin of spreading psoriasis

BACKGROUND: The margin zone in spreading psoriatic lesions has frequently been used as a model to study the changes in epidermal proliferation, keratinization and inflammation during the transition from symptomless to lesional skin. However, the dynamics of the changes in the epidermal subpopulations-basal cells, transit amplifying cells and differentiated cells-have not been studied in the transition between symptomless and lesional skin. OBJECTIVES: To quantify in a dynamic model of the margin zone in psoriasis the characteristics of these subpopulations with respect to epidermal proliferation and differentiation. METHODS: From seven patients with active psoriasis, biopsies were taken from the distant uninvolved skin, outer margin, inner margin and centre of a spreading psoriatic plaque. Frozen sections were labelled immunofluorescently using direct immunofluorescence for Ki-67 and beta1 integrin and the Zenon labelling technique for keratin 6, 10 and 15. Digital photographs of the stained sections were quantitatively analysed. RESULTS: In the distant uninvolved skin the expression of beta1 integrin was decreased and keratin 15 expression was lost. In this area suprabasal cells expressed beta1 integrin and in the outer margin suprabasal cells expressed Ki-67. From the outer to the inner margin of the psoriasis plaque, which coincided with the appearance of the clinical lesion, there was a significant change in the various markers. The patchy expression of keratin 6 in the inner margin became homogeneous in the centre of the psoriasis plaque and here was also coexpression of keratin 6 and keratin 10 in a single cell. CONCLUSIONS: The present study provides additional evidence that the distant uninvolved skin has a prepsoriatic phenotype, which is the first step in a psoriatic cascade. The cascade between symptomless and lesional skin comprises first an abnormality in inflammation with involvement of beta1 integrin-dim cells (transit amplifying cells) subsequently eliciting an enlarged germinative compartment with increased recruitment of cycling epidermal cells and focal expression of proliferation-associated keratins, ultimately culminating in a more-or-less homogeneous epidermis with massive recruitment of cycling epidermal cells and proliferation-associated keratinization.     

Clues that mitochondria are involved in the hair cycle clock: MPZL3 regulates entry into and progression of murine hair follicle cycling

The molecular nature of the hair cycle clock (HCC), the intrinsic oscillator system that drives hair follicle (HF) cycling, remains incompletely understood; therefore, all relevant key players need to be identified. Here, we present evidence that implicates myelin protein zero-like 3 (MPZL3), a multifunctional nuclear-encoded mitochondrial protein known to be involved in epidermal differentiation, in HCC regulation. By analysing global Mpzl3 knockout (−/−) mice, we show that in the absence of functional MPZL3, mice commence HF cycling with retarded first catagen-telogen transition after normal postnatal HF morphogenesis. However, Mpzl3 −/− mice subsequently display strikingly accelerated HF cycling, i.e. a precocious telogen-to-anagen transition during the second hair cycle, compared to controls, suggesting that MPZL3 inhibits anagen entry. We also show that intrafollicular MPZL3 protein expression fluctuates in a hair cycle-dependent manner. In telogen HFs, MPZL3 is localized to the secondary hair germ, an epicentre of hair cycle regulation, where it partially co-localizes with P-cadherin. In early anagen HF, MPZL3 is localized immediately distal to the proximal hair matrix. These findings introduce the novel concept that mitochondria are more actively involved in hair cycle control than previously recognized and that MPZL3 plays a central role in the HCC.     

Human hair follicle dermal sheath and papilla cells support keratinocyte growth in monolayer coculture

Traditional skin grafting techniques are effective but limited methods of skin replacement. Autologous transplantation of rapidly cultured keratinocytes is successful for epidermal regeneration, but the current gold‐standard technique requires mouse fibroblast feeders and serum‐rich media, with serum‐free systems and dermal fibroblast (DF) feeders performing relatively poorly. Here, we investigated the capacity of human hair follicle dermal cells to act as alternative supports for keratinocyte growth. Dermal papilla (DP) dermal sheath (DS), DF and 3T3 cells were used as inactivated feeder cells for human keratinocyte coculture. Under conditions favouring dermal cells, proliferation of keratinocytes in the presence of either DS or DP cells was significantly enhanced compared with DF cells, at levels comparable to keratinocytes cultured under gold‐standard conditions. Secreted protein acidic and rich in cysteine (SPARC) expression increased DS and DP cells relative to DFs; however, further experiments did not demonstrate a role in keratinocyte support.     

Molecular basis for the rhino Yurlovo (hrrhY) phenotype: severe skin abnormalities and female reproductive defects associated with an insertion in the hairless gene

In 1989, mice bearing mutations at the hr (hairless) locus were first proposed as a model for the human hair growth disorder papular atrichia, since in both these mice and in corresponding patients, a complete hair loss develops due to disintegration of the normal follicle structure into dermal cysts and so-called utriculi. Recently, the human hairless gene was characterized, and pathogenetic mutations were found to be associated with a recessively inherited form atrichia with papular lesions; however, the functions of hr gene remain unclear. Allelic mutations in the murine hairless gene represent a potentially powerful tool to elucidate the role of the hairless gene protein product in hair follicle physiology. In 1980, several naked animals were discovered in a breeding colony of B10.R109/Y mice maintained in the Laboratory of Experimental Biological Models (L.E.B.M., Yurlovo, Moscow District, Russia). By cross breeding with hairles HRS/J hr/hr mice, this mutation was shown to be allelic with hairless. Here, we describe the molecular basis of the hr^rhY mutation in mice, which consists of a 13 bp insertion in exon 16 of the hr gene. Histological evaluation of Yurlovo mouse skin revealed some differences as compared to the hairless and rhino mutations, with the formation of dermal megacysts being the most specific peculiarity of the Yurlovo mutation. These results, together with previous studies of hr^rhY/hr^rhY mutant mice, suggest that the rhino Yurlovo (hr^rhY) mutation represents a third and potentially more severe variation of the hairless phenotype.     

Psoriasis and streptococci: the natural selection of psoriasis revisited

We have previously postulated that surviving invasive streptococcal infections may have been a factor in psoriasis becoming a common skin disease in some parts of the world. Many of the candidate genes linked to psoriasis are associated with the acquired or innate immune system, which are also important in host defence to invasive streptococcal infections. High rates of positive streptococcal throat swabs among patients with chronic plaque psoriasis suggest that they are efficient at internalizing/carrying β-haemolytic streptococci. Internalization of streptococci in the throat is dependent upon the transforming growth factor (TGF)-β/fibronectin/α5β1 integrin pathway. The immune cell Th17 and its related cytokine network are important in mucosal defence, being very effective against extracellular microbes but having little effect on intracellular organisms. The TGF-β/fibronectin/α5β1 integrin pathway and the Th17 cell network also appear to be operative in psoriasis, animal models of both TGF-β and α5β1 cutaneous overexpression being associated with characteristic psoriasis lesions. We postulate that some of the genotypic/phenotypic changes in different immunological pathways in psoriasis, including the acquired T-cell response, the innate immune response, the TGF-β/fibronectin/α5β1 integrin pathway and the Th17 cell system, confer protection against mortality during epidemics of invasive streptococcal infections, heightened efficiency in internalizing and allowing carriage of streptococci as well as predisposition to the development of psoriasis.     

Beta-papillomaviruses and psoriasis: an intra-patient comparison of human papillomavirus carriage in skin and hair

Background  Human papillomaviruses (HPVs) of the beta genus (β-PV), especially HPV5 and HPV36, are proposed to play a pathogenic role in psoriasis, but many previous studies have failed to control for potential confounders, including treatment.
Objectives  To re-examine the relationship between β-PV and psoriasis addressing limitations present in previous studies and analyse intra-patient concordance for carriage of HPV.
Methods  Plucked eyebrow hairs and forearm skin scrapes were collected from 20 newly diagnosed, previously untreated adult patients with psoriasis and 23 normal controls. A combination of type-specific and degenerate polymerase chain reaction methods was used to achieve comprehensive HPV DNA detection.
Results  The prevalence of HPV in hair and skin from psoriasis patients was higher than in controls (83·3% vs. 46·7%, respectively, P  <   0·03 corrected for age and clustering). HPV5 or HPV36 were not over-represented. The profile of diverse β-PV types was comparable in the two groups. Intra-patient concordance for HPV DNA at separate sites was high ( P  <   0·00001).
Conclusions  Our data do not support a specific causal role for HPV5 or HPV36 in psoriasis, but suggest that psoriatic skin may be more permissive for viral presence than normal skin. High intra-patient concordance for specific HPV types at separate sites, together with the ubiquity of HPV DNA in normal human skin, suggests that an individual becomes colonized with a particular β-PV profile presumably to the exclusion of other types. To what extent this HPV profile is then causal in the subsequent development of hyperproliferative skin disease is unknown.     

Co-culture of human melanocytes and keratinocytes in a skin equivalent model: effect of ultraviolet radiation

Melanocytes grown in pure monolayer culture lack the three-dimensional organization and many of the cellular interactions that exist in vivo. This can be partially overcome by growing melanocytes together with other epidermal cells in skin equivalent models. In this study skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in various ratios onto de-epidermized dermis. They were cultured in DMEM/Ham's F12 (31) for 3 days and then lifted to the air-liquid interface and maintained for 11 days. Histological examination revealed a structure that closely resembled human interfollicular epidermis. Melanocytes, identified by their dendritic appearance, positive dopa reaction and positive staining with a melanocyte-specific antibody (MEL5), were located in the basal layer. Melanin was seen both in melanocytes and in neighbouring keratinocytes. Whilst the skin equivalent became more pigmented following UV irradiation (total UVB 4760 J/m² over 3 days), the quantity and distribution of melanin at the light microscopic level appeared to be unchanged. However, the number and dendricity of melanocytes increased, as did their staining with dopa and MEL5. These results indicate that melanocytes are functional and capable of responding to UV irradiation.     

A specific combination of zeaxanthin,spermidine and rutin prevents apoptosis in human dermal papilla cells

Hair follicle (HF) regression is characterized by the activation of apoptosis in HF cells. Dermal papilla cells play a leading role in the regulation of HF development and cycling. Human follicular dermal papilla cells (HFDPC) were used to investigate the protective activities of rutin, sperimidine and zeaxanthine. HFDP cell incubation with staurosporine caused apoptosis, which was completely inhibited by exposure to rutin (2.2 μm ), spermidine (1 μm ) and zeaxanthin (80 μm ). These agents were much less effective when applied as single compounds. Moreover, treatment preserved the expression of anti‐apoptotic molecules such as Bcl‐2, MAP‐kinases and their phosphorylated forms. In conclusion, the investigated agents may represent an effective treatment for the prevention of apoptosis, one of the leading events involved in hair bulb regression.     

Human follicular stem cells: their presence in plucked hair and follicular cell culture

BACKGROUND AND OBJECTIVES: A considerable portion of the hair follicle remains attached to plucked hair and can be used for follicle cell culture. In this study we have phenotyped these cells in an attempt to identify the stem cell fraction. Reports in the literature have indicated that this cell population may be positive for cytokeratin (CK) 19. Because stem cells in general need to be protected from apoptosis, the presence of the apoptosis-suppressing Bcl-2 protein, together with the absence of the apoptosis-promoting Bax and the CK profile may be used as an indicator of the stem cell population in the hair follicle, and in cultures of hair follicle cells. METHODS: Hair follicles from skin biopsies and plucked hair were derived from the scalps of healthy volunteers. Follicular cells were cultured from the plucked hairs. These hair follicles, plucked hairs and cultured cells were examined for their CK profiles, which are indicative of the type of cell (basal/stem cells) and for their status with respect to the proliferation marker Ki-67, Bax and Bcl-2. RESULTS: We found coexpression for CK19 and Bcl-2, but not Bax in two distinct areas, localized in the upper and lower third of the follicle from both skin biopsies and plucked hairs, while proliferation markers were negative in these areas. CK19 and Bcl-2 were also coexpressed in combination in a fraction of the follicular cell culture. The skin basal cell marker CK14 could be found throughout the outer root sheath of the hair follicle from both skin biopsies and plucked hairs, as well as in the follicular cell culture. CONCLUSIONS: Thus, CK19/Bcl-2-positive and Bax-negative cells can be obtained from cells derived from plucked hair and are retained in cultures made from these cells. If this phenotype represents follicular stem cells, our finding endorses the assumption that stem cells are located in the bulge area of the hair follicle, as we did not find them in or near the dermal papilla.