Melanocyte dysfunctions: future and promise of stem cells.

Skin is the major tissue where melanocytes develop, and skin keratinocytes provide the necessary micro-environment for melanocyte survival, proliferation, differentiation, and migration. In this paper, we will discuss the ligands for receptor tyrosine kinases produced as environmental cues to support melanocyte development in the skin.

Mysteries of salt and pepper

Melanocyte stem cells (MSCs), melanocyte lineage-specific skin stem cells derived from the neural crest, are observed in the mammalian hair follicle, the epidermis or the sweat gland. MSCs differentiate into mature melanin-producing melanocytes, which confer skin and hair pigmentation and uphold vital skin functions. In controlling and coordinating the homeostasis, repair and regeneration of skin tissue, MSCs play a vital role. Decreased numbers or impaired functions of MSCs are closely associated with the development and therapy of many skin conditions, such as hair graying, vitiligo, wound healing and melanoma. With the advancement of stem cell technology, the relevant features of MSCs have been further elaborated. In this review, we provide an exhaustive overview of cutaneous MSCs and highlight the latest advances in MSC research. A better understanding of the biological characteristics and micro-environmental regulatory mechanisms of MSCs will help to improve clinical applications in regenerative medicine, skin pigmentation disorders and cancer therapy. KEY POINTS: This review provides a concise summary of the origin, biological characteristics, homeostatic maintenance and therapeutic potential of cutaneous MSCs. The role and potential application value of MSCs in skin pigmentation disorders are discussed. The significance of single-cell RNA sequencing, CRISPR-Cas9 technology and practical models in MSCs research is highlighted.

Hair graying is one of the prototypical signs of human aging, but its mechanism is largely unknown. To elucidate the mechanism of hair graying, we investigated gene expression related to melanogenesis in human hair. The key molecules in melanogenesis, microphthalmia-associated transcription factor-M (MITF-M), Sox10, Pax3, tyrosine related protein-1 (TRP-1), and tyrosinase, were absent or greatly reduced in the bulbs of white hair compared to black hair. Melanocyte stem cells (MSCs) or melanocytes express markers for neural crest cells, Sox10, Pax3, and MITF-M. Taken together, our data suggest that hair graying is caused by defective migration of MSCs into the bulb area of hair.

One of the wonders of development is the long migration undertaken by cells of the neural crest to their final destinations where they undergo differentiation. For cells of the melanocyte lineage, migration is driven by the ability of melanoblasts to receive and interpret extracellular cues that control their rate of proliferation and give directional signals so that they take the correct route. This intrinsic capacity of neural crest cells for long-distance movement may be one reason why melanomas are so aggressive; oncogene-mediated deregulation of key developmental signal transduction pathways coupled with microenvironmental stress may be sufficient to reactivate the embryonic migratory potential without the need for acquisition of pro-metastasis mutations. The processes underpinning development and those implicated in melanoma genesis and progression are intimately related, and the lessons from one system are likely to be applicable to the other. One key concern is the origin of melanoma: do the initial mutational events that lead to malignant transformation occur in differentiated melanocytes or their stem cells? Given that differentiated cells most likely have heterochromatinized the genes required for proliferation, the melanocyte stem cell might represent an attractive candidate. Moreover, there is increasing evidence that the heterogeneity observed within a melanoma may in part reflect the presence of ‘melanoma stem cells’ that would provide a reservoir of potentially drug-resistant cells with a capacity to form new tumours. Understanding how such a population of melanoma stem cells might be generated, and how the microenvironment might control the switch from proliferation to quiescence and vice versa is a key issue in the field. Presumably the tumour microenvironment will provide a ‘niche’ that is permissive for the generation and maintenance of the melanoma stem cell population. However, the dynamic nature of melanoma provides major challenges to understanding the nature of the putative melanoma stem cell niche. By contrast, understanding the molecular mechanisms that lead to the generation, maintenance and re-activation of normal melanocyte stem cells is tractable. Advances made over recent years, particularly by the Nishikawa laboratory, first identified melanocyte stem cells in the hair follicle bulge region and subsequently led to the molecular characterization of the hair follicle melanocyte stem cells in terms of their gene expression characteristics. The results obtained provide the basis for addressing a key question in stem cell biology: what determines a stem cell niche? The review from Masatake Osawa and Shin-Ichi Nishikawa in this issue summarizes the progress made to date on the characterization of melanocyte stem cells and proposes a model to describe the events that lead to the generation of a melanocyte stem cell. Although there is yet a long way to go, it is clear that unlike most other stem cell systems, the melanocyte lineage provides an excellent model for defining what is required for a stem cell niche. Importantly, the answers obtained to date suggest that melanocyte and melanoma stem cells may have a great deal in common. While melanoma represents a potentially fatal disease of the melanocyte lineage, it is relatively well characterized at the molecular level compared with vitiligo, a disease in which there is loss of melanocytes in the skin. While not life threatening, vitiligo nevertheless represents a major challenge to our understanding of pigment cell biology and as pointed out by Alain Taieb in his News and Views article, there is no obvious angle of attack. Although there is evidence that one component of the disease is stress sensitivity, one aspect that is undoubted is that vitiligo is characterized by a cell-based immune response against differentiated melanocytes and that this is underpinned by a genetic component. The current status of the genetics of vitiligo is reviewed by Rich Spritz, who not only highlights specific candidate genes for the disease, but also points out several candidates that appear to have fallen by the wayside. Progress in understanding vitiligo is likely to require a very broad approach in multiple disciplines, but rigorous application of statistics to genetic analysis will be critical if any progress in identifying true vitiligo-associated genes is to be made. Moreover, while understanding the molecular basis of vitiligo is clearly important, there is an urgent need for more effective treatment now. In that respect, while the immune response may eliminate differentiated melanocytes, the melanocyte stem cells in their niche do not express melanocyte markers and therefore will escape destruction. Understanding the mechanisms that lead to reactivation of the stem cell population and consequent repopulation of de-pigmented skin with new melanocytes represents the most viable approach for an effective anti-vitiligo therapy. Again, the review from Osawa and Nishikawa in this issue provides a sound basis for studies directed towards stem cell-based vitiligo therapy.

OBJECTIVE:To identify global research trends of follicle and melanocyte stem cells, and their application in neuroscience.DATA RETRIEVAL:We performed a bibliometric analysis of studies from 2002 to 2011 on follicle and melanocyte stem cells, and their application in neuroscience, which were retrieved from the Web of Science, using the key words follicle stem cell or melanocyte stem cell, and neural, neuro or nerve.SELECTION CRITERIA:Inclusion criteria: (a) peer-reviewed published articles on follicle and melanocyte stem cells, and their application in neuroscience, which were indexed in the Web of Science; (b) original research articles, reviews, meeting abstracts, proceedings papers, book chapters, editorial material, and news items. Exclusion criteria: (a) articles that required manual searching or telephone access; (b) documents that were not published in the public domain; and (c) a number of corrected papers from the total number of articles.MAIN OUTCOME MEASURES:(1) Distribution of publications on follicle and melanocyte stem cells by years, journals, countries, institutions, institutions in China, and most cited papers. (2) Distribution of publications on the application of follicle and melanocyte stem cells in neuroscience by years, journals, countries, institutions, and most cited papers.RESULTS:Of the 348 publications from 2002 to 2011 on follicle and melanocyte stem cells, which were retrieved from the Web of Science, more than half were from American authors and institutes. The most prolific institutions in China for publication of papers on follicle and melanocyte stem cells were the Fourth Military Medical University and Third Military Medical University. The most prolific journals for publication of papers on follicle and melanocyte stem cells were the Journal of Investigative Dermatology, Pigment Cell & Melanoma Research. Of the 63 publications from 2002 to 2011 on the application of follicle and melanocyte stem cells in neuroscience, which were retrieved from the Web of Science, more than half were from American authors and institutes, and no papers were from Chinese authors and institutes. The most prolific journals for publication of papers on the application of follicle and melanocyte stem cells in neuroscience were the Journal of Investigative Dermatology, Pigment Cell & Melanoma Research.CONCLUSION:Based on our analysis of the literature and research trends, we found that follicle stem cells might offer further benefits in neural regenerative medicine.

Tfap2b specifies an embryonic melanocyte stem cell that retains adult multifate potential

Melanocytes, our pigment producing cells, are replenished from multiple stem cell niches in adult tissues. Although pigmentation traits are known risk-factors for melanoma, we know little about melanocyte stem cell (MSC) populations other than hair follicle MSCs, and lack key lineage markers with which to identify MSCs and study their function. Here, we discover that Tfap2b, and a select set of its target genes, specifies an MSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and instead is essential for MSC-dependent melanocyte regeneration. Fate-mapping data reveal that tfap2b-expressing MSCs have multi-fate potential, and are the cell-of-origin for large patches of adult melanocytes, and two other pigment cell types, iridophores and xanthophores. Hence, Tfap2b confers MSC identity in early development, thereby distinguishing MSCs from other neural crest and pigment cell lineages, and retains multi-fate potential in the adult zebrafish.

Conditional Immortalization Establishes a Repertoire of Mouse Melanocyte Progenitors with Distinct Melanogenic Differentiation Potential

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.

During embryogenesis, the transcription factor, Sox10, drives the survival and differentiation of the melanocyte lineage. However, the role that Sox10 plays in postnatal melanocytes is not established. We show in vivo that melanocyte stem cells (McSCs) and more differentiated melanocytes express SOX10 but that McSCs remain undifferentiated. Sox10 knockout (Sox10fl; Tg(Tyr::CreER)) results in loss of both McSCs and differentiated melanocytes, while overexpression of Sox10 (Tg(DctSox10)) causes premature differentiation and loss of McSCs, leading to hair graying. This suggests that levels of SOX10 are key to normal McSC function and Sox10 must be downregulated for McSC establishment and maintenance. We examined whether the mechanism of Tg(DctSox10) hair graying is through increased expression of Mitf, a target of SOX10, by asking if haploinsufficiency for Mitf (Mitfvga9) can rescue hair graying in Tg(DctSox10) animals. Surprisingly, Mitfvga9 does not mitigate but exacerbates Tg(DctSox10) hair graying suggesting that MITF participates in the negative regulation of Sox10 in McSCs. These observations demonstrate that while SOX10 is necessary to maintain the postnatal melanocyte lineage it is simultaneously prevented from driving differentiation in the McSCs. This data illustrates how tissue-specific stem cells can arise from lineage-specified precursors through the regulation of the very transcription factors important in defining that lineage.

Animal pigment pattern: An integrative model system for studying the development, evolution, and regeneration of form

Transplantation of Melanocytes Into Iris: Method for Iris Repigmentation

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.

For unknow reasons, the melanocyte stem cell (McSC) system fails earlier than other adult stem cell populations1, which leads to hair greying in most humans and mice2,3. Current dogma states that McSCs are reserved in an undifferentiated state in the hair follicle niche, physically segregated from differentiated progeny that migrate away following cues of regenerative stimuli4–8. Here we show that most McSCs toggle between transit-amplifying and stem cell states for both self-renewal and generation of mature progeny, a mechanism fundamentally distinct from those of other self-renewing systems. Live imaging and single-cell RNA sequencing revealed that McSCs are mobile, translocating between hair follicle stem cell and transit-amplifying compartments where they reversibly enter distinct differentiation states governed by local microenvironmental cues (for example, WNT). Long-term lineage tracing demonstrated that the McSC system is maintained by reverted McSCs rather than by reserved stem cells inherently exempt from reversible changes. During ageing, there is accumulation of stranded McSCs that do not contribute to the regeneration of melanocyte progeny. These results identify a new model whereby dedifferentiation is integral to homeostatic stem cell maintenance and suggest that modulating McSC mobility may represent a new approach for the prevention of hair greying.