Differentiation Of Melanocyte Stem Cells Research Articles

In vitro Differentiation of Melanocyte Stem Cells Derived from Vitiligo Patients into Functional Melanocytes

BACKGROUND: Melanocyte stem cells (MelSCs) residing in the hair follicle bulge act as melanocyte reservoir for skin and hair, and may serve as an autologous source for treating vitiligo. Therefore, the study aimed to evaluate the in vitro differentiation ability of MelSCs derived from vitiligo patients into melanin-producing melanocytes for potential cellular therapy.METHODS: MelSCs from the vitiliginous (V-MelSCs), non-vitiliginous (NV-MelSCs) regions of vitiligo patients, as well as from control subjects (C-MelSCs) were established for evaluating their differentiation potential into melanin producing cells. The differentiation abilities were compared at the cellular and molecular levels. MelSCs were differentiated in vitro into induced-melanocytes (iMCs) by supplementing the culture medium with melanogenic factors. iMCs were analyzed by quantitative polymerase chain reaction (qPCR) for the expression of key melanogenic markers, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT), microphthalmia-associated transcription factor (MITF), c-KIT and S100. iMCs were also stained with L-DOPA to assess TYR activity in cells. Intracellular melanin content in iMCs was evaluated and compared among the vitiligo and control groups.RESULTS: MelSCs induced into iMCs displayed morphological changes with longer dendrites and prominent nuclei. iMCs stained positive for L-DOPA with an average intracellular melanin content of 30 pg/cell. iMCs expressed key melanogenic genes and the relative expression did not differ significantly among the groups.CONCLUSION: NV-MelSCs were unaltered by disease pathogenesis and capable of differentiating into melanocytes compared to V-MelSCs. Hence, these cells might offer a reliable source of melanocytes for vitiligo repigmentation therapy by autologous cellular transplantation.KEYWORDS: melanocyte stem cells, differentiation, melanocytes, vitiligo, in vitro

Reversible Differentiation of Melanocyte Stem Cells: Designed to Last or Be Lost?

Reversible Differentiation of Melanocyte Stem Cells: Designed to Last or Be Lost?

Lenalidomide Augments Differentiation of Cultured Hair Follicle Derived Melanocyte Stem Cells Into Functional Melanocytes.

Melanocyte progenitors are embryonically derived from the neural crest and subsequently get localized in hair follicles and epidermis to provide hair and skin pigmentation. These progenitor cells in hair follicles repeatedly proliferate and differentiate to maintain pigmentation. Vitiligo, a pigmentary disorder, is associated with loss of melanocytes. Repigmentation of vitiligo lesions mainly depends upon the proliferation, migration and differentiation of melanocyte stem cells (MelSCs) into functional melanocytes. The current study is designed to check the efficacy of lenalidomide, an imide drug in the differentiation of MelSCs into functional melanocytes. The aim of the study is to check the effect of lenalidomide in the proliferation, migration of cultured hair follicle derived melanocyte stem cells and their differentiation into functional melanocytes. Primary culture of MelSCs was established from whisker hair of C57BL/6 mice. Proliferation and migration of cultured cells were done by MTT assay and Boyden's chamber migration assay, respectively. Effect of lenalidomide on the MelSCs differentiation was checked at gene level by qPCR and protein expression was checked by immunocytochemistry. A significant increase in the migration of MelSCs in comparison to control was also observed. Lenalidomide treatment significantly increased the expression of melanocyte specific genes in cultured MelSCs as compared to control. From the results we concluded that lenalidomide induce the proliferation and migration of MelSCs and accelerate the differentiation of MelSCs into functional melanocytes.

The possible role of Wnt/β-catenin signalling in vitiligo treatment.

Vitiligo is a common chronic skin disease which has an adverse impact on patients' life. Its pathogenesis is complex, involving autoimmunity and oxidative stress (OS). Autoimmunity leads to the loss of epidermal melanocytes and the formation of the depigmented patches of the disease. Treatment of vitiligo should control the exaggerated immune response to arrest the progress of active disease, and then promote melanocytes to repigmentation. Wnt/β-catenin signalling pathway has been of recent interest in vitiligo. Wnt/β-catenin signalling pathway is downregulated in vitiligo. Upregulation of Wnt/β-catenin signalling possibly control vitiligo autoimmune response by protecting melanocyte from OS damage, inhibiting CD8+ T cell effector cell differentiation and enhancing Treg. Wnt/β-catenin signalling plays a critical role in the melanocyte regeneration by driving the differentiation of melanocyte stem cells (McSCs) into melanocytes. Promoting Wnt/β-catenin signalling can not only arrest the progress of active disease of vitiligo but also promote repigmentation. Some of the main effective therapies for vitiligo are likely to work by activating Wnt/β-catenin signalling. Agents that can enhance the effect of Wnt/β-catenin signalling may become potential candidates for the development of new drugs for vitiligo treatment.

A commentary of “Stress turns hair white”: 10 remarkable discoveries from 2020 in Nature

This was the “News & Views” story in the journal Nature that was most popular online in 2020. The relative contribution of stress to greying of the hair is not known. Hair color is determined by melanocyte cells derived from melanocyte stem cells (MeSCs) located in a part of the hair follicle called the bulge. This paper, published in Nature in January 2020, was authored by Ya-Chieh Hsu's research group at Harvard University, with Bing Zhang as the first author [1]. Zhang et al. report that noradrenaline - a neurotransmitter molecule involved in the “fight or flight” response to stress — is released from neurons of the sympathetic nervous system. MeSC proliferation and differentiation increase markedly under extreme stress or on exposure to a high level of noradrenaline, resulting in a mass migration of melanocytes away from the bulge and leaving no remaining stem cells to replace them,① thus causing greying. This work by Zhang et al. provides a better understanding of how stress influences other stem cells as well as clues for finding ways to stop and reverse the effects of stress.①①Original source in Chinese: Ji Hu, Stress turns hair white, Bulletin of National Natural Science Foundation of China. 35 (2) (2021) 247-248

Melanogenesis Markers Expression in Premature Graying of Hair: A Cross-Sectional Study

Background: Studies on mice and aging human hair follicles provide compelling evidence that graying of hair results from premature differentiation of melanocyte stem cells in the niche/bulge. Objective: The aim of this study was to analyze whether differentiation of melanocyte stem cells is responsible for premature graying of hair (PGH). Methods: Twenty-five patients with PGH (n = 25) attending the dermatology department were recruited. Five unpigmented and 5 pigmented hairs were obtained per patient by separating individual follicles after 1 mm punch biopsies. The hairs were dissected at a distance of 2 mm from the bulb to separate the stem cells (upper segment – US) from the melanocytes (lower segment – LS). RNA was extracted from hair follicle US and LS, and expression of GP100, tyrosinase (TYR), and tyrosinase-related protein-1 (TYRP1) genes was quantified using Qiagen one-step RT-PCR kit. Results: We found melanogenesis gene expression in both temporary (US) and permanent (LS) segments of unpigmented and pigmented hair follicles. When compared between the US and LS of white hair, the expression of TYR and GP100 was much higher in US than LS, suggestive of melanogenesis in the bulge. Similarly, when compared between white and black US, the expression of all 3 genes was higher in white US than black US, although not statistically significant. Limitations: Low samples size and lack of data pertaining to the expression of genes at protein level are the limitations of current study. Conclusion: Even though this pilot study data yielded key information about the expression of GP100, TYR, and TYRP-1 at the mRNA level, further studies quantifying the expression of these genes at protein level are needed to provide additional clues to further address the results in detail.

Vitiligo: 30years to put together the puzzle pieces and to give rise to a new era of therapeutic options.

Vitiligo: 30years to put together the puzzle pieces and to give rise to a new era of therapeutic options.

In vitro differentiation of melanocyte stem cells derived from vitiligo patients into functional melanocytes

In vitro differentiation of melanocyte stem cells derived from vitiligo patients into functional melanocytes

Stress-associated ectopic differentiation of melanocyte stem cells and ORS amelanotic melanocytes in an ex vivo human hair follicle model.

Hair greying depends on the altered presence and functionality of hair follicle melanocytes. Melanocyte stem cells (MelSCs) reside in the bulge of hair follicles and give rise to migrating and differentiating progeny during the anagen phase. Ageing, genotoxic stress, redox stress and multiple behaviour-associated acute stressors have been seen to induce hair greying by depleting the MelSC pool, a phenomenon which is accompanied by ectopic pigmentation of these cells, followed by their depletion from the stem cell niche. This aberrant differentiation produces a state from which a return to stem cell-like quiescence appears to be lost. The cellular features of stress-induced hair greying have been extensively studied in murine models. Here, we describe a method to assess and quantify human hair follicle MelSC differentiation by measuring ectopically pigmented MelSCs in isolated human hair follicles exposed to specific stress signal mediators. Ionizing radiation, hydrogen peroxide and noradrenaline have been shown to cause hair greying in mice. We demonstrate here that isolated, ex vivo cultured human hair follicles exposed to these treatments display similar ectopic pigmentation within the bulge area which is accompanied by induction of differentiated melanocytic markers. This study suggests that as in murine models, stress signalling induces closely matching phenotypic changes in human hair follicles which can be monitored and studied as a surrogate model for early steps in human hair greying.

Regulating cell fate of human amnion epithelial cells using natural compounds: an example of enhanced neural and pigment differentiation by 3,4,5-tri-O-caffeoylquinic acid

Over the past years, Human Amnion Epithelial Cells (hAECs), a placental stem cell, are gaining higher attention from the scientific community as they showed several advantages over other types of stem cells, including availability, easy accessibility, reduced rejection rate, non-tumorigenicity, and minimal legal constraint. Recently, natural compounds are used to stimulate stem cell differentiation and proliferation and to enhance their disease-treating potential. A polyphenolic compound 3,4,5-Tri-O-Caffeoylquinic Acid (TCQA) has been previously reported to induce human neural stem cell differentiation and may affect melanocyte stem cell differentiation as well. In this study, TCQA was tested on 3D cultured hAECs after seven days of treatment, and then, microarray gene expression profiling was conducted of TCQA-treated and untreated control cells on day 0 and day 7. Analyses revealed that TCQA treatment significantly enriched pigment and neural cells sets; besides, genes linked with neurogenesis, oxidation–reduction process, epidermal development, and metabolism were positively regulated. Interestingly, TCQA stimulated cell cycle arrest-related pathways and differentiation signaling. On the other hand, TCQA decreased interleukins and cytokines expression and this due to its anti-inflammatory properties as a polyphenolic compound. Results were validated to highlight the main activities of TCQA on hAECs, including differentiation, cell cycle arrest, and anti-inflammatory. This study highlights the important role of hAECs in regenerative medicine and the use of natural compounds to regulate their fate.4ntr2z8GsC-Lz3nsa9SfYxVideo abstract

Identification of Gene Variants Associated with Melanocyte Stem Cell Differentiation in Mice Predisposed for Hair Graying.

Age-related hair graying is caused by malfunction in the regenerative potential of the adult pigmentation system. The retention of hair color over the life of an organism is dependent on the ability of the melanocyte stem cells and their progeny to produce pigment each time a new hair grows. Age-related hair graying is variable in association with genetic background suggesting that quantitative trait loci influencing this trait can be identified. Identification of these quantitative trait loci may lead to the discovery of novel and interesting genes involved in stem cell biology and/or melanogenesis. With this in mind we developed previously a sensitized, mouse modifier screen and discovered that the DBA/1J background is particularly resistant to melanocyte stem cell differentiation in comparison to the C57BL/6J background. Melanocyte stem cell differentiation generally precedes hair graying and is observed in melanocyte stem cells with age. Using quantitative trait loci analysis, we have now identified three quantitative trait loci on mouse chromosomes 7, 13, and X that are associated with DBA/1J-mediated variability in melanocyte stem cell differentiation. Taking advantage of publicly-available mouse sequence and variant data, in silico protein prediction programs, and whole genome gene expression results we describe a short list of potential candidate genes that we anticipate to be involved in melanocyte stem cell biology in mice.

Mechanisms of repigmentation induced by photobiomodulation therapy in vitiligo.

Photobiomodulation (PBM) therapy is based on the exposure of biological tissues to low-level laser light (coherent light) or light-emitting diodes (LEDs; noncoherent light), leading to the modulation of cellular functions, such as proliferation and migration, which result in tissue regeneration. PBM therapy has important clinical applications in regenerative medicine. Vitiligo is an acquired depigmentary disorder resulting from disappearance of functional melanocytes in the involved skin. Vitiligo repigmentation depends on available melanocytes derived from (a) melanocyte stem cells located in the bulge area of hair follicles and (b) the epidermis at the lesional borders, which contains a pool of functional melanocytes. Since follicular melanoblasts (MBs) are derived from the melanocyte stem cells residing at the bulge area of hair follicle, the process of vitiligo repigmentation presents a research model for studying the regenerative effect of PBM therapy. Previous reports have shown favourable response for treatment of vitiligo with a low-energy helium-neon (He-Ne) laser. This review focuses on the molecular events that took place during the repigmentation process of vitiligo triggered by He-Ne laser (632.8nm, red light). Monochromatic radiation in the visible and infrared A (IRA) range sustains matrix metalloproteinase (MMP), improves mitochondrial function, and increases adenosine triphosphate (ATP) synthesis and O2 consumption, which lead to cellular regenerative pathways. Cytochrome c oxidase in the mitochondria was reported to be the photoacceptor upon which He-Ne laser exerts its effects. Mitochondrial retrograde signalling is responsible for the cellular events by red light. This review shows that He-Ne laser initiated mitochondrial retrograde signalling via a Ca2+ -dependent cascade. The impact on cytochrome c oxidase within the mitochondria, an event that results in activation of CREB (cyclic-AMP response element binding protein)-related cascade, is responsible for the He-Ne laser promoting functional development at different stages of MBs and boosting functional melanocytes. He-Ne laser irradiation induced (a) melanocyte stem cell differentiation; (b) immature outer root sheath MB migration; (c) differentiated outer root sheath MB melanogenesis and migration; and (d) perilesional melanocyte migration and proliferation. These photobiomodulation effects result in perifollocular and marginal repigmentation in vitiligo.

Medical and Maintenance Treatments for Vitiligo.

Medical treatments alone, or in combination with phototherapy, are key approaches for treating nonsegmental vitiligo and, to a lesser extent, segmental vitiligo. The treatments are useful for halting disease progression and have been proven effective for inducing repigmentation and decreasing risk of relapses. Although the treatments have side effects and limitations, vitiligo often induces a marked decrease in quality of life and in most cases the risk:benefit ratio is in favor of an active approach. Systemic and topical agents targeting the pathways involved in loss of melanocytes and in differentiation of melanocyte stem cells should provide more effective approaches in the near future.

Clinical and epidemiological analysis in 149 cases of rhododendrol-induced leukoderma.

Rhododendrol-induced leukoderma is an acquired depigmentation that develops mainly at the contact site after repeated use of skin-whitening cosmetics containing rhododendrol. In most cases, cessation of further depigmentation or occurrence of repigmentation is observed after discontinuing the use of cosmetics. However, some patients develop vitiligo vulgaris through the spread of depigmentation into the non-exposed areas. Our study aims to investigate the patient-specific factors that may affect the extent of depigmentation or repigmentation, as well as development of vitiligo vulgaris. The degree of depigmentation of the face, neck and hands where exposed to rhododendrol was scored using photographs over time. The relationships between depigmentation score at first visit/improvement rate of depigmentation score and patient demographics were evaluated and three important clinical observations were made. First, repigmentation of the face was superior compared with that of the hands and neck, suggesting a possible role for the migration and differentiation of melanocyte stem cells from hair follicles, as a mechanism of repigmentation. Second, the intensity of rhododendrol exposure did not contribute to differences in the severity of depigmentation. This suggested a possibility of underlying genetic susceptibility to melanocyte cytotoxicity or immune reaction. Third, depigmentation score at first visit and past history of atopic dermatitis were significantly high in patients who developed vitiligo vulgaris. This suggested that severe chemical damage of melanocytes by rhododendrol leads to a higher risk of developing vitiligo vulgaris through the possible involvement of an immune reaction. These clinical observations may help to further understand the pathogenesis of rhododendrol-induced leukoderma.

12-O-tetradecanoylphorbol-13-acetate activates hair follicle melanocytes for hair pigmentation via Wnt/β-catenin signaling

Melanocyte stem cells (McSCs) undergo cyclical activation and quiescence together with hair follicle stem cells (HFSCs). This process is strictly controlled by the autonomous and extrinsic signaling environment. However, the modulation of factors important for the activation of McSCs for hair pigmentation remains unclear. 12-O-tetradecanoylphorbol-13-acetate (TPA) mimics vital signaling pathways involved in melanocyte growth and melanogenesis in vitro. To investigate whether TPA regulates quiescent McSCs for hair pigmentation, we topically smeared TPA on 7-week-old mouse dorsal skin and found that TPA stimulated hair growth and hair matrix pigmentation. These changes were associated with a significant increase in the number of hair bulb melanocytes. Moreover, in the TPA-treated group, hair bulge McSCs and hair bulb melanoblasts actively proliferated. At the molecular level, nuclear β-catenin, a key factor of Wnt/β-catenin signaling, was highly synthesized in melanocytes and keratinocytes in TPA-induced hair bulbs. Inhibition of Wnt/β-catenin signaling by injecting Dickkopf1 plasmids into TPA-treated skin decreased hair matrix pigmentation and inhibited the proliferation and differentiation of McSCs. Our findings suggest that the topical application of TPA stimulates the proliferation and differentiation of McSCs and their progeny for hair matrix pigmentation by activating Wnt/β-catenin signaling. This might provide a useful experimental model for the study of signals controlling the activation of McSCs.

Inhibitory effect of Phalaenopsis orchid extract on WNT1-induced immature melanocyte precursor differentiation in a novel in vitro solar lentigo model

Recently, it has been reported that increased expression of WNT1 accelerates the differentiation of melanocyte stem cells (McSCs) in solar lentigines (SLs), hyperpigmented maculae commonly seen on sun-exposed areas of the skin. In this study, to establish an in vitro SL model, human epidermal squamous carcinoma cell line HSC-1, which expresses higher levels of WNT1 than normal human epidermal keratinocytes, was co-cultured with early passage normal human epidermal melanocytes (NHEMs) as an in vitro McSC model. As a result, mRNA expression levels of melanocyte differentiation-related genes MITF and TYR in NHEMs were significantly increased by co-culturing with HSC-1 cells. Furthermore, Phalaenopsis orchid extract (Phex) inhibited McSCs differentiation by suppressing WNT1 expression via down-regulation of DLX2, a transcriptional activator of WNT1, in HSC-1 cells. Therefore, our finding suggested that extracts such as Phex, which suppresses WNT1 expression, may be useful as a novel treatment of SLs.

EdnrB Governs Regenerative Response of Melanocyte Stem Cells by Crosstalk with Wnt Signaling.

Delineating the crosstalk between distinct signaling pathways is key to understanding the diverse and dynamic responses of adult stem cells during tissue regeneration. Here, we demonstrate that the Edn/EdnrB signaling pathway can interact with other signaling pathways to elicit distinct stem cell functions during tissue regeneration. EdnrB signaling promotes proliferation and differentiation of melanocyte stem cells (McSCs), dramatically enhancing the regeneration of hair and epidermal melanocytes. This effect is dependent upon active Wnt signaling that is initiated by Wnt ligand secretion from the hair follicle epithelial niche. Further, this Wnt-dependent EdnrB signaling can rescue the defects in melanocyte regeneration caused by Mc1R loss. This suggests that targeting Edn/EdnrB signaling in McSCs can be a therapeutic approach to promote photoprotective-melanocyte regeneration, which may be useful for those with increased risk of skin cancers due to Mc1R variants.

Understanding Melanocyte Stem Cells for Disease Modeling and Regenerative Medicine Applications.

Melanocytes in the skin play an indispensable role in the pigmentation of skin and its appendages. It is well known that the embryonic origin of melanocytes is neural crest cells. In adult skin, functional melanocytes are continuously repopulated by the differentiation of melanocyte stem cells (McSCs) residing in the epidermis of the skin. Many preceding studies have led to significant discoveries regarding the cellular and molecular characteristics of this unique stem cell population. The alteration of McSCs has been also implicated in several skin abnormalities and disease conditions. To date, our knowledge of McSCs largely comes from studying the stem cell niche of mouse hair follicles. Suggested by several anatomical differences between mouse and human skin, there could be distinct features associated with mouse and human McSCs as well as their niches in the skin. Recent advances in human pluripotent stem cell (hPSC) research have provided us with useful tools to potentially acquire a substantial amount of human McSCs and functional melanocytes for research and regenerative medicine applications. This review highlights recent studies and progress involved in understanding the development of cutaneous melanocytes and the regulation of McSCs.

Ectopic differentiation of melanocyte stem cells is influenced by genetic background.

Hair graying in mouse is attributed to the loss of melanocyte stem cell function and the progressive depletion of the follicular melanocyte population. Single-gene, hair graying mouse models have pointed to a number of critical pathways involved in melanocyte stem cell biology; however, the broad range of phenotypic variation observed in human hair graying suggests that additional genetic variants involved in this process may yet be discovered. Using a sensitized approach, we ask here whether natural genetic variation influences a predominant cellular mechanism of hair graying in mouse, melanocyte stem cell differentiation. We developed an innovative method to quantify melanocyte stem cell differentiation by measuring ectopically pigmented melanocyte stem cells in response to the melanocyte-specific transgene Tg(Dct-Sox10). We make the novel observation that the production of ectopically pigmented melanocyte stem cells varies considerably across strains. The success of sensitizing for melanocyte stem cell differentiation by Tg(Dct-Sox10) sets the stage for future investigations into the genetic basis of strain-specific contributions to melanocyte stem cell biology.

Accelerated differentiation of melanocyte stem cells contributes to the formation of hyperpigmented maculae.

It has been reported that the abnormal regulation of melanocyte stem cells (McSCs) causes hair greying; however, little is known about the role of McSCs in skin hyperpigmentation such as solar lentigines (SLs). To investigate the involvement of McSCs in SLs, the canonical Wnt signalling pathway that triggers the differentiation of McSCs was analysed in UVB-induced delayed hyperpigmented maculae in mice and human SL lesions. After inducing hyperpigmented maculae on dorsal skin of F1 mice of HR-1× HR/De, which was formed long after repeated UVB irradiation, the epidermal Wnt1 expression and the number of nuclear β-catenin-positive McSCs were increased as compared to non-irradiated control mice. Furthermore, the expression of dopachrome tautomerase (Dct), a downstream target of β-catenin, was significantly upregulated in McSCs of UVB-irradiated mice. The Wnt1 expression and the number of nuclear β-catenin-positive McSCs were also higher in human SL lesions than in normal skin. Recombinant Wnt1 protein induced melanocyte-related genes including Dct in early-passage normal human melanocytes (NHEMs), an in vitro McSC model. These results demonstrate that the canonical Wnt signalling pathway is activated in SL lesions and strongly suggest that the accelerated differentiation of McSCs is involved in SL pathogenesis.