Melanocyte stem cells in the skin: Origin, biological characteristics, homeostatic maintenance and therapeutic potential.

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.

Detecting normal and cancer skin cells via glycosylation and adhesion signatures: A path to enhanced microfluidic phenotyping.

Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive properties. Our study investigates the adhesion of skin cells to specific lectins, particularly focusing on advancements in lectin-based biosensors with diagnostic potential. We explore whether we can successfully capture normal skin (melanocytes and keratinocytes) and melanoma (WM35, WM115, WM266-4) cells in a low-shear flow environment by coating surfaces with lectins. Specifically, we coated surfaces with Dolichos biflorus (DBA) and Maackia Amurensis (MAL) lectins, which were used to detect and capture specific skin cells from the flow of cell mixture. Alterations in glycan expression (confirmed by fluorescent microscopy) demonstrated that DBA binds predominantly to normal skin cells, while MAL interacts strongly with melanoma cells. Assessing adhesion under static and dynamic low-shear stress conditions (up to 30 mPa) underscores the reliability of DBA and MAL as markers for discriminating specific cell type. Melanocytes and keratinocytes adhere to DBA-coated surfaces, while melanoma cells prefer MAL-coated surfaces. A comprehensive analysis encompassing cell shape, cytoskeleton, and focal adhesions shows the independence of our approach from the inherent characteristics of cells, thus demonstrating its robustness. Our results carry practical implications for lectin-biosensor designs, emphasizing the significance of glycan-based discrimination of pathologically altered cells. Combined with microfluidics, it demonstrates the value of cell adhesion as a discriminant of cancer-related changes, with potential applications spanning diagnostics, therapeutic interventions, and advanced biomedical technologies.

Molecular heterogeneity of quiescent melanocyte stem cells revealed by single-cell RNA-sequencing.

Melanocyte stem cells (McSCs) of the hair follicle are a rare cell population within the skin and are notably underrepresented in whole-skin, single-cell RNA sequencing (scRNA-seq) datasets. Using a cell enrichment strategy to isolate KIT+/CD45- cells from the telogen skin of adult female C57BL/6J mice, we evaluated the transcriptional landscape of quiescent McSCs (qMcSCs) at high resolution. Through this evaluation, we confirmed existing molecular signatures for qMcCS subpopulations (e.g., Kit+, Cd34+/-, Plp1+, Cd274+/-, Thy1+, Cdh3+/-) and identified novel qMcSC subpopulations, including two that differentially regulate their immune privilege status. Within qMcSC subpopulations, we also predicted melanocyte differentiation potential, neural crest potential, and quiescence depth. Taken together, the results demonstrate that the qMcSC population is heterogeneous and future studies focused on investigating changes in qMcSCs should consider changes in subpopulation composition.

Analysis of Proliferation, Apoptosis, and Motility in Mouse Embryonic Melanocytic Precursor Cells.

In this chapter, we provide a method to purify and culture embryonic melanocytic stem cells that express green fluorescent protein in a cell-type specific manner. Isolation of melanocytic lineage cell populations that are >98% pure is accomplished through the use of GFP-based fluorescence activated cell sorting. We also provide a method to culture the purified melanoblasts and to analyze their proliferation, apoptosis, and motility properties.

Human-Induced Pluripotent Stem Cell‒Derived Keratinocytes, as Therapeutic Option in Vitiligo.

Vitiligo is a skin condition affecting 1% of the global population, causing non-scaly, chalky-white macules on the skin and hair. It is caused by the pathologic destruction of melanocytes, which produce melanin. Research has focused on the abnormalities of melanocytes and their interaction with neighboring keratinocytes. Current treatments are mainly immunosuppressive drugs and UV radiation, which are scarce and ineffective. To treat vitiligo, regenerative medicine techniques, such as cell-based and cell-free methods, are recommended. Keratinocyte cell transplantation has shown promising results in treating vitiligo. Moreover, studies suggest individualized therapy for diseases can be provided by reprogramming somatic cells into induced pluripotent stem cells. On the other hand, differentiation into particular cell types is a key component of induced pluripotent stem cells-based treatment. In this chapter, the differentiation and validation of human induced pluripotent stem cells into a keratinocyte as a therapeutic option in vitiligo will be discussed.

Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Dedifferentiation maintains melanocyte stem cells in a dynamic niche.

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.

PRAME immunohistochemistry can distinguish melanocytic pseudonests of lichenoid reactions from melanoma in situ.

BACKGROUND: Distinguishing melanocytic pseudonests encountered in lichenoid dermatoses or lichenoid keratoses from melanoma in situ (MIS) with brisk lichenoid inflammation can prove challenging. METHODS: We designed a case-control study to evaluate the accuracy metrics of PRAME immunohistochemistry to distinguish melanocytic pseudonests in lichenoid dermatoses or keratoses from inflamed MIS. Immunostaining for PRAME was performed on paraffin-embedded formalin-fixed diagnostic tissue using a rabbit monoclonal antibody to PRAME (Abcam), with a 1:3200 dilution on a Leica Bond detection system. RESULTS: Our search identified 21 cases of melanocytic pseudonests (n = 21, 46%) encountered in lichenoid dermatoses and 24 cases of inflamed MIS (n = 24, 53%). Each method of evaluating PRAME immunohistochemistry (PRAME+ clusters, PRAME % of melanocytes by four categories and PRAME+ melanocyte counts per linear mm of epidermal basal layer) showed statistically significant differences between the MIS and the pseudonest cohorts (respectively, p < 0.001; p < 0.001; and p < 0.001). Receiver operating characteristics analysis for PRAME+ melanocyte counts per linear mm of epidermal basal layer revealed an area under the curve of 0.9 ± 0.05 (95% confidence interval 0.9-1.0). When determining an optimal cut-off point for the best Youden index [sensitivity (%) + specificity (%) - 100], the cut-off of 1.0 PRAME+ melanocytes per linear mm showed a sensitivity of 79.2% and specificity of 85.7% (Youden index 0.65) to distinguish MIS from pseudonests. CONCLUSION: PRAME immunohistochemistry may constitute an additional tool for this challenging differential diagnosis.

The Role of Oxidative Stress in the Pathogenesis of Vitiligo: A Culprit for Melanocyte Death.

Vitiligo is a common chronic acquired pigmentation disorder characterized by loss of pigmentation. Among various hypotheses proposed for the pathogenesis of vitiligo, oxidative stress-induced immune response that ultimately leads to melanocyte death remains most widely accepted. Oxidative stress which causes elevated levels of reactive oxygen species (ROS) can lead to dysfunction of molecules and organelles, triggering further immune response, and ultimately melanocyte death. In recent years, a variety of cell death modes have been studied, including apoptosis, autophagy and autophagic cell death, ferroptosis, and other novel modes of death, which will be discussed in this review in detail. Oxidative stress is also strongly linked to these modes of death. Under oxidative stress, ROS could induce autophagy by activating the Nrf2 antioxidant pathway of melanocytes. However, persistent stimulation of ROS might eventually lead to excessive activation of Nrf2 antioxidant pathway, which in turn will inactivate autophagy. Moreover, ferroptosis may be triggered by oxidative-related transcriptional production, including ARE, the positive feedback loop related to p62, and the reduced activity and expression of GPX4. Therefore, it is reasonable to infer that these modes of death are involved in the oxidative stress response, and that oxidative stress also acts as an initiator for various modes of death through some complex mechanisms. In this study, we aim to summarize the role of oxidative stress in vitiligo and discuss the corresponding mechanisms of interaction between various modes of cell death and oxidative stress. These findings may provide new ideas for exploring the pathogenesis and potential therapeutic targets of vitiligo.

Viscoelastic parameterization of human skin cells characterize material behavior at multiple timescales.

Countless biophysical studies have sought distinct markers in the cellular mechanical response that could be linked to morphogenesis, homeostasis, and disease. Here, an iterative-fitting methodology visualizes the time-dependent viscoelastic behavior of human skin cells under physiologically relevant conditions. Past investigations often involved parameterizing elastic relationships and assuming purely Hertzian contact mechanics, which fails to properly account for the rich temporal information available. We demonstrate the performance superiority of the proposed iterative viscoelastic characterization method over standard open-search approaches. Our viscoelastic measurements revealed that 2D adherent metastatic melanoma cells exhibit reduced elasticity compared to their normal counterparts-melanocytes and fibroblasts, and are significantly less viscous than fibroblasts over timescales spanning three orders of magnitude. The measured loss angle indicates clear differential viscoelastic responses across multiple timescales between the measured cells. This method provides insight into the complex viscoelastic behavior of metastatic melanoma cells relevant to better understanding cancer metastasis and aggression.

Stabilization of ß-catenin promotes melanocyte specification at the expense of the Schwann cell lineage.

The canonical Wnt/ß-catenin pathway governs a multitude of developmental processes in various cell lineages, including the melanocyte lineage. Indeed, ß-catenin regulates transcription of Mitf-M, the master regulator of this lineage. The first wave of melanocytes to colonize the skin is directly derived from neural crest cells, whereas the second wave of melanocytes is derived from Schwann cell precursors (SCPs). We investigated the influence of ß-catenin in the development of melanocytes of the first and second waves by generating mice expressing a constitutively active form of ß-catenin in cells expressing tyrosinase. Constitutive activation of ß-catenin did not affect the development of truncal melanoblasts but led to marked hyperpigmentation of the paws. By activating ß-catenin at various stages of development (E8.5-E11.5), we showed that the activation of ß-catenin in bipotent SCPs favored melanoblast specification at the expense of Schwann cells in the limbs within a specific temporal window. Furthermore, in vitro hyperactivation of the Wnt/ß-catenin pathway, which is required for melanocyte development, induces activation of Mitf-M, in turn repressing FoxD3 expression. In conclusion, ß-catenin overexpression promotes SCP cell fate decisions towards the melanocyte lineage.

Highly Effective Protocol for Differentiation of Induced Pluripotent Stem Cells (iPS) into Melanin-Producing Cells.

Melanin is a black/brown pigment present in abundance in human skin. Its main function is photo-protection of underlying tissues from harmful UV light. Natural sources of isolated human melanin are limited; thus, in vitro cultures of human cells may be a promising source of human melanin. Here, we present an innovative in vitro differentiation protocol of induced pluripotent stem cells (iPS) into melanin-producing cells, delivering highly pigmented cells in quantity and quality incomparably higher than any other methods previously described. Pigmented cells constitute over 90% of a terminally differentiated population and exhibit features characteristic for melanocytes, i.e., expression of specific markers such as MITF-M (microphthalmia-associated transcription factor isoform M), TRP-1 (tyrosinase-related protein 1), and TYR (tyrosinase) and accumulation of black pigment in organelles closely resembling melanosomes. Black pigment is unambiguously identified as melanin with features corresponding to those of melanin produced by typical melanocytes. The advantage of our method is that it does not require any sophisticated procedures and can be conducted in standard laboratory conditions. Moreover, our protocol is highly reproducible and optimized to generate high-purity melanin-producing cells from iPS cells; thus, it can serve as an unlimited source of human melanin for modeling human skin diseases. We speculate that FGF-8 might play an important role during differentiation processes toward pigmented cells.

Inhibition of Fam114A1 protects melanocytes from apoptosis through higher RACK1 expression.

Fam114A1 is a gene closely related to the development of nerve cells, melanocytes, and nerve cells that originate from the neural crest of the embryonic ectoderm. Recent studies showed that Fam114A1 has a role in the occurrence of ankylosing myelitis spondylitis and autoimmune enteritis; still, its cellular function remains poorly understood. In this study, we investigated the effect of Fam114A1 on the biological activity of melanocytes. We found that the expression of Fam114A1 in vitiligo melanocytes (MCV-L, MCV-N, PI3V) was higher than that in normal melanocytes, and the biological function of melanocytes was significantly affected when the Fam114A1 gene was silenced. Inhibition of Fam114A1 increased proliferation, migration, and melanin synthesis proteins, decreased apoptosis, while its overexpression reversed this process. Mechanistically, we discovered that RACK1 is a target protein of Fam114A1 and that RACK1 can be negatively regulated by Fam114A1. Further study showed that Fam114A1 inhibition could not protect melanocytes from apoptosis once the expression of RACK1 protein was silenced. In summary, Fam114A1 is an effective regulatory protein for regulating the function of melanocytes. Inhibition Fam114A1 protects melanocytes from apoptosis through increasing RACK1.

Kaempferol, the melanogenic component of Sanguisorba officinalis, enhances dendricity and melanosome maturation/transport in melanocytes.

Kaempferol, a representative flavonoid constituent of Sanguisorba officinalis, promotes melanogenesis, but the underlying mechanisms remain unknown. Here, we evaluated the effects of kaempferol on melanocytes morphology and behavior and determined the mechanisms regulating kaempferol-induced pigmentation. We observed that kaempferol increased melanin contents and dendritic length and stimulated melanocyte migration both in vitro and vivo. It significantly enhanced the expression of microphthalmia-associated transcription factor (MITF) and downstream enzymes of melanin biosynthesis-tyrosinase (TYR), tyrosinase-related protein (TRP-1), and dopachrome tautomerase (DCT). It also induced melanosome maturation (increased stage III and IV melanosomes) and melanin transfer to dendritic tips; this was evidenced as follows: kaempferol-treated melanocytes exhibited the perimembranous accumulation of HMB45-positive melanosomes and increased the expression of Rab27A, RhoA, and Cdc42, which improved melanosome transport to perimembranous actin filaments. These results jointly indicated that kaempferol promotes melanogenesis and melanocyte growth. Additionally, kaempferol stimulated the phosphorylation of P38/ERK MAPK and downregulated p-PI3K, p-AKT, and p-P70s6K expression. Pre-incubation with P38 (SB203580) and ERK (PD98059) signaling inhibitors reversed the melanogenic and dendritic effects and MITF expression. PI3K/AKT inhibitor augmented kaempferol-induced melanin content and dendrite length. In summary, kaempferol regulated melanocytes' dendritic growth and melanosome quantity, maturation, and transport via P38/ERK MAPK and PI3K/AKT signaling pathways.

Mitigating the pro-oxidant state and melanogenesis of Retinitis pigmentosa: by counteracting mitochondrial dysfunction.

Retinitis pigmentosa (RP) is a group of mitochondrial diseases characterized by progressive degeneration of rods and cones leading to retinal loss of light sensitivity and, consequently, to blindness. To date, no cure is available according to the clinical literature. As a disease associated with pigmentation-related, pro-oxidant state, and mitochondrial dysfunction, RP may be viewed at the crossroads of different pathogenetic pathways involved in adverse health outcomes, where mitochondria play a preeminent role. RP has been investigated in a number of experimental and clinical studies aimed at delaying retinal hyperpigmentation by means of a number of natural and synthetic antioxidants, as well as mitochondrial cofactors, also termed mitochondrial nutrients (MNs), such as alpha-lipoic acid, coenzyme Q10 and carnitine. One should consider that each MN plays distinct-and indispensable-roles in mitochondrial function. Thus, a logical choice would imply the administration of MN combinations, instead of individual MNs, as performed in previous studies, and with limited, if any, positive outcomes. A rational study design aimed at comparing the protective effects of MNs, separately or in combinations, and in association with other antioxidants, might foresee the utilization of animal RP models. The results should verify a comparative optimization in preventing or effectively contrasting retinal oxidative stress in mouse RP models and, in prospect, in human RP cases.

A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks.

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the ß tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.

MAPK and Notch-Mediated Effects of Meso-Xanthin F199 Compounds on Proliferative Activity and Apoptosis of Human Melanocytes in Three-Dimensional Culture.

Meso-Xanthin (Meso-Xanthin F199™) is a highly active antiaging injection drug of the latest generation. The main acting compound is fucoxanthin, supplemented with several growth factors, vitamins, and hyaluronic acid. Previous examination of fucoxanthin on melanocytes showed its ability to inhibit skin pigmentation through different signaling pathways focused on suppression of melanogenic-stimulating receptors. In turn, the anticancer property of fucoxanthin is realized through MAPK and PI3K pathways. We aimed to evaluate the effect of fucoxanthin and supplemented growth factors on melanocyte growth and transformation at a proteomic level. The effect of fucoxanthin on melanocytes cultivated in three-dimensional (3D) condition was examined using high-throughput proteomic and system biology approaches to disclose key molecular events of the targeted action. Our results demonstrated significant inhibition of cell differentiation and ubiquitination processes. We found that the negative regulation of PSME1 and PTGIS largely determines the inhibition of NF-κB and MAPK2. Besides, fucoxanthin selectively inhibits cell differentiation via negative regulation of Raf signaling and the upstream activation of IL-1 signaling. It is assumed that inhibition of Raf influences the Notch-4 signaling and switches off the MAPK/MAPK2 cascade. Blockage of MAPK/MAPK2 is feasible due to suppression of Ras and NF-κB by the addressed action of IKKB, IKK2, and TRAF6. Suggestively, Meso-Xanthin F199™ can manage processes of proliferative activity and inhibition of apoptosis due to composition of fucoxanthin and growth-stimulating factors, which may increase the risk of skin cancer development under certain condition.

Human embryonic stem cell-derived melanocytes exhibit limited immunogenicity.

Although surgical interventions have become optional for refractory vitiligo, grafting related injuries is inevitable. Embryonic stem cell (ESC) derivatives can be used in transplantation to address this issue, but the immune rejection due to allogeneic transplantation is of great concern. To investigate the immunogenicity of ESC derived melanocytes (ES-MC), we established a co-culture system of ES-MC and allogeneic PBMC. The results showed that ES-MC were similar to human primary melanocytes, with low expression of immune related molecules, and limited capability of stimulating allogeneic lymphocytes in vitro. Taken together, our findings confirm that ES-MC are of limited immunogenicity, providing new insights into the application of ES-MC in the regenerative medicine such as treating vitiligo.

Melanocyte progenitor cells reside in human subcutaneous adipose tissue.

Based on the assumption that some progenitor cells in an organ might reside in neighboring adipose tissue, we investigated whether melanocyte progenitor cells reside in human subcutaneous adipose tissue. First, we examined the expression of human melanoma black 45 (HMB45) and microphthalmia-associated transcription factor (MITF) in undifferentiated adipose-derived stem cells (ADSCs) by immunostaining, RT-PCR, and western blotting. These two markers were detected in undifferentiated ADSCs, and their expression levels were increased in differentiated ADSCs in melanocyte-specific culture medium. Other melanocytic markers (Melan A, MATP, Mel2, Mel EM, tyrosinase, KIT, and PAX3) were also detected at variable levels in undifferentiated ADSCs, and the expression of some markers was increased during differentiation into the melanocyte lineage. We further showed that ADSCs differentiated in melanocyte-specific culture medium localized in the basal layer and expressed tyrosinase and HMB45 in a 3D epidermal culture system. Melanin deposits were also induced by ultraviolet-light-B (UVB) irradiation. These results demonstrate that melanocyte progenitor cells reside in human subcutaneous adipose tissue and that these cells might have the potential to differentiate into mature melanocytes. Melanocyte and keratinocyte progenitors residing in human subcutaneous tissue can be used for the treatment of skin diseases and skin rejuvenation in the future.