Melanocyte Population Research Articles

Selective Clonal Regression After Interferon Therapy in Metastatic Melanoma.

Regression (total or partial) is a common phenomenon in melanoma. From a pathogenic perspective, it is highly complex and only partially understood, involving aspects of both the tumor and the individual. One of the determining factors is the clonal selection of the tumor, wherein some clones within the tumor survive while others perish. This clonal selection can sometimes occur as a selective mechanism after the initiation of a therapeutic intervention. In many of these cases, the effect is detrimental, because the surviving clone is resistant to the applied therapy. However, occasionally, the therapy can successfully select the less harmful clone. We present an example of the latter, where therapy with interferon induced regression of the metastatic-capable melanocytic population, with only the primary tumor melanocytic population persisting. To confirm this, we demonstrated BRAF mutational similarity between the 2 populations, and an additional NRAS mutation in the metastatic population, which was absent in the primary tumor.

The Treatment of Refractory Vitiligo With Autologous Cultured Epithelium Grafting: A Real-World Retrospective Cohort Study.

Surgical intervention is the main therapy for refractory vitiligo. We developed a modified autologous cultured epithelial grafting (ACEG) technique for vitiligo treatment. Between January 2015 and June 2019, a total of 726 patients with vitiligo underwent ACEG in China, with patient characteristics and clinical factors being meticulously documented. Using a generalized linear mixed model, we were able to assess the association between these characteristics and the repigmentation rate. ACEG demonstrated a total efficacy rate of 82.81% (1754/2118) in treating 726 patients, with a higher repigmentation rate of 64.87% compared to conventional surgery at 52.69%. Notably, ACEG showed a better response in treating segmental vitiligo, lesions on lower limbs, age ≤ 18, and stable period > 3 years. A keratinocyte:melanocyte ratio below 25 was found to be advantageous too. Single-cell RNA sequencing analysis revealed an increase in melanocyte count and 2 subclusters of keratinocytes after ACEG, which remained higher in repigmented sites even after 1 year. ACEG is a promising therapy for refractory vitiligo. Patient age, clinical type, lesion site, and stability before surgery influence repigmentation in ACEG. The mechanism of repigmentation after ACEG treatment is likely not confined to the restoration of melanocyte populations. It may also involve an increase in the number of keratinocytes that support melanocyte function within the affected area. These keratinocytes may aid the post-transplant survival and function of melanocytes by secreting cytokines and extracellular matrix components. registered with Chictr.org.cn (ChiCTR2100051405).

Vitiligo non-responding lesions to narrow band UVB have intriguing cellular and molecular abnormalities that may prevent epidermal repigmentation.

We have discovered that human vitiligo patients treated with narrow-band UVB (NBUVB) demonstrated localized resistance to repigmentation in skin sites characterized by distinct cellular and molecular pathways. Using immunostaining studies, discovery-stage RNA-Seq analysis, and confirmatory insitu hybridization, we analyzed paired biopsies collected from vitiligo lesions that did not repigment after 6 months of NBUVB treatment (non-responding) and compared them with repigmented (responding) lesions from the same patient. Non-responding lesions exhibited acanthotic epidermis, had low number of total, proliferative, and differentiated melanocyte (MC) populations, and increased number of senescent keratinocytes (KCs) and of cytotoxic CD8+ T cells as compared with responding lesions. The abnormal response in the non-responding lesions was driven by a dysregulated cAMP pathway and of upstream activator PDE4B, and of WNT/β-catenin repigmentation pathway. Vitiligo-responding lesions expressed high levels of WNT10B ligand, a molecule that may prevent epidermal senescence induced by NBUVB, and that in cultured melanoblasts prevented the pro-melanogenic effect of α-MSH. Understanding the pathways that govern lack of NBUVB-induced vitiligo repigmentation has a great promise in guiding the development of new therapeutic strategies for vitiligo.

Insights into human choroid melanocyte and stromal cells

Aims/Purpose: The human choroid is a complex highly vascular pigmented tissue between Bruch's membrane and the sclera. Its dynamic nature is important for eye homeostasis and for in pathology including macular degeneration and myopia. The stroma comprises heterogeneous cell populations including fibroblasts, melanocytes, immune cells, smooth muscle cells and neurons within a loose collagenous extracellular matrix. We used RNA transcriptome and multimarker immunolabelling of human choroidal melanocytes and tissue to further characterize adult choroidal melanocytes.Methods: Human choroidal melanocytes were cultured (n = 6 postmortem donor eyes, <18 h delay, UNSW HREC), and used between passage (P) 2 to 5 for RNA transcriptome and multimarker immunofluorescence labelling and confocal microscopy. Sections and flatmounts from central choroid were also compared. Immunolabelling included melanocyte (Tyrosinase, TRYP1, DCT, MelanA) and stromal cell (S1004A, vimentin, α‐smooth muscle actin, CD45) markers.Results: Cultured melanocytes displayed mostly bipolar morphology with heterogeneous eumelanin density related to donor eye iris colour. Whole transcriptome findings showed enriched melanogenesis genes (high level TYR, PMEL, MITF, TYRP1; MLANA; low level DCT1, MC1R) but not genes reported for RPE (e.g. RPE65), pericytes (e.g. ACTA2) or fibroblasts (e.g. IGF2) confirming melanocyte culture quality. We also found expression for proposed Schwann cell precursors PLP1 and SOX10. Immunolabelling confirmed overall expression of melanogenesis proteins, and subpopulations expressing PLP1 and Sox10, in culture and in choroidal tissue.Conclusions: Melanocytes and stromal fibroblast are among the most numerous and complex heterogeneous populations within the choroid and our study provides further details on characteristics markers and topography. A possible role for Schwann cell precursors in renewing adult melanocyte populations is intriguing and continues to be investigated.

Beyond the Epidermal-Melanin-Unit: The Human Scalp Anagen Hair Bulb Is Home to Multiple Melanocyte Subpopulations of Variable Melanogenic Capacity.

The visual appearance of humans is derived significantly from our skin and hair color. While melanin from epidermal melanocytes protects our skin from the damaging effects of ultraviolet radiation, the biological value of pigmentation in the hair follicle, particularly on the scalp, is less clear. In this study, we explore the heterogeneity of pigment cells in the human scalp anagen hair follicle bulb, a site conventionally viewed to be focused solely on pigment production for transfer to the hair shaft. Using c-KIT/CD117 microbeads, we isolated bulbar c-KIT-positive and c-KIT-negative melanocytes. While both subpopulations expressed MITF, only the c-KIT-positive fraction expressed SOX10. We further localized bulbar melanocyte subpopulations (expressing c-KIT, SOX10, MITF, and DCT) that exhibited distinct/variable expression of downstream differentiation-associated melanosome markers (e.g., gp100 and Melan-A). The localization of a second 'immature' SOX10 negative melanocyte population, which was c-KIT/MITF double-positive, was identified outside of the melanogenic zone in the most peripheral/proximal matrix. This study describes an approach to purifying human scalp anagen hair bulb melanocytes, allowing us to identify unexpected levels of melanocyte heterogeneity. The function of the more immature melanocytes in this part of the hair follicle remains to be elucidated. Could they be in-transit migratory cells ultimately destined to synthesize melanin, or could they contribute to the hair follicle in non-melanogenic ways?

Alhamd treatment of vitiligo, a study of needling+ excimer laser @308nm

Introduction: Vitiligo is a chronic autoimmune disorder where pigment producing cells in skin called melanocytes are attacked and destroyed causing milky white patches in skin. Our study to treat vitiligo is a combination of EXCIMER LASER @308nm,1,2,3 + NEEDLING.4 This study is based on a theme that the melanocytes in the normal skin around the vitiligo patch can be pushed into the Vitiligo patch and subsequent EXCIMER induced melanogenesis treats vitiligo.5 Methods: We use a 30G, 4mm stem needle. Push it through the normal skin into the vitiligo patch at the level of the D-E junction parallel to the skin. This needle push called NEEDLING drags epidermal cells including melanocytes as micro inoculation to produce multiple small populations of melanocytes in the vitiligo area,6 which is then exposed to EXCIMER to cause melanogenesis and hence repigmentation of the area.3,5,7 Multiple needle pushes are made through normal skin around the vitiligo patch, into the vitiligo patch. These needle pushes are done 1cm apart. All patients are having needling once weekly and EXCIMER 2-3 times per week.4,6 A study of this combination was done over 50 patients, in both sexes in different age groups from October 2021 to September 2022 and follow up still continued. A comparison was also done with EXCIMER alone (without needling) in some of these patients at some patches.8 Inclusion and exclusion criteria, a result criteria and a study Performa with follow-up details was set. Photos of all the patients were taken before treatment and then every 3 weeks. Results: Our combination treatment has proved very effective for vitiligo as compared to EXCIMER alone. Most patients had fast repigmentation. The best results are on the face and trunk4,6 with more then 90% repigmentation in all age groups. Repigmentation is good but slow towards peripheral parts.9,10 Repigmentation was specially noticed to start from the edge of the vitiligo patch (where needling is done) as tiny black dots and further needling through these dots gradually repigmented the central areas.4 (Figure 2, 3, 4, 6). Repigmentaion is also good over areas with grey hair with this technique as needling uses melanocytes of the skin surrounding the vitiligo patch rather than the follicular cells. This further strengthened the idea of needling as a useful combination with EXCIMER.

Generating Functional and Highly Proliferative Melanocytes Derived from Human Pluripotent Stem Cells: A Promising Tool for Biotherapeutic Approaches to Treat Skin Pigmentation Disorders

Melanocytes are essential for skin homeostasis and protection, and their loss or misfunction leads to a wide spectrum of diseases. Cell therapy utilizing autologous melanocytes has been used for years as an adjunct treatment for hypopigmentary disorders such as vitiligo. However, these approaches are hindered by the poor proliferative capacity of melanocytes obtained from skin biopsies. Recent advances in the field of human pluripotent stem cells have fueled the prospect of generating melanocytes. Here, we have developed a well-characterized method to produce a pure and homogenous population of functional and proliferative melanocytes. The genetic stability and potential transformation of melanocytes from pluripotent stem cells have been evaluated over time during the in vitro culture process. Thanks to transcriptomic analysis, the molecular signatures all along the differentiation protocol have been characterized, providing a solid basis for standardizing the protocol. Altogether, our results promise meaningful, broadly applicable, and longer-lasting advances for pigmentation disorders and open perspectives for innovative biotherapies for pigment disorders.

Chemical reprogramming of melanocytes to skeletal muscle cells.

Direct cell-fate conversion by chemical reprogramming is promising for regenerative cell therapies. However, this process requires the reactivation of a set of master transcription factors (TFs) of the target cell type, which has proven challenging using only small molecules. We developed a novel small-molecule cocktail permitting robust skin cell to muscle cell conversion. By single cell sequencing analysis, we identified a Pax3 (Paired box 3)-expressing melanocyte population holding a superior myogenic potential outperforming other seven types of skin cells. We further validated the single cell sequencing analysis results using immunofluorescence staining, in situ hybridization and FACS sorting and confirmed the myogenic potential of melanocytes during chemical reprogramming. We used single cell RNA-seq that detect the potential converted cell type, uncovering a unique role of Pax3 in facilitating chemical reprogramming from melanocytes to muscle cells. In this study, we demonstrated that the Pax3-expressing melanocytes to be a skin cell type for skeletal muscle cell fate conversion in chemical reprogramming. By developing a small-molecule cocktail, we showed an efficient melanocyte reprogramming to skeletal muscle cells (40%, P<0.001). The endogenous expression of specific TFs may circumvent the additional requirement for TF reactivation and form a shortcut for cell fate conversion, suggesting a basic principle that could ease cell fate conversion. Our study demonstrates the first report of melanocyte-to-muscle conversion by small molecules, suggesting a novel strategy for muscle regeneration. Furthermore, skin is one of the tissues closely located to skeletal muscle, and therefore, our results provide a promising foundation for therapeutic chemical reprogramming in vivo treating skeletal muscle degenerative diseases.

Induced pluripotent stem cells reprogramming overcomes technical limitations for highly pigmented adult melanocyte amplification and integration in 3D skin model.

Understanding pigmentation regulations taking into account the original skin color type is important to address pigmentary disorders. Biological models including adult melanocytes from different phenotypes allow to perform fine-tuned explorative studies and support discovery of treatments adapted to populations' skin color. However, technical challenges arise when trying to not only isolate but also amplify melanocytes from highly pigmented adult skin. To bypass the initial isolation and growth of cutaneous melanocytes, we harvested and expanded fibroblasts from light and dark skin donors and reprogrammed them into iPSC, which were then differentiated into melanocytes. The resulting melanocyte populations displayed high purity, genomic stability, and strong proliferative capacity, the latter being a critical parameter for dark skin cells. The iPSC-derived melanocyte strains expressed lineage-specific markers and could be successfully integrated into reconstructed skin equivalent models, revealing pigmentation status according to the native phenotype. In both monolayer cultures and 3D skin models, the induced melanocytes demonstrated responsiveness to promelanogenic stimuli. The data demonstrate that the iPSC-derived melanocytes with high proliferative capacity maintain their pigmentation genotype and phenotypic properties up to a proper integration into 3D skin equivalents, even for highly pigmented cells.

BAP1-Inactivated Melanoma Arising From BAP1-Inactivated Melanocytic Tumor in a Patient With BAP1 Germline Mutation: A Case Report and Review of the Literature.

BAP1-inactivated melanocytic tumors represent a subset of epithelioid melanocytic neoplasms resulting from biallelic inactivation of the BAP1 gene and by a driver mutation that activate the MAP kinase pathway, most commonly BRAFV600E. They occur sporadically or, less common, in the setting of BAP1 tumor predisposition syndrome caused by a BAP1 germline mutation that predisposes to several malignancies including cutaneous and uveal melanoma. To date, only few cases of BAP1-inactivated melanomas have been reported. We present a case of a 35-year-old woman presented with a melanocytic lesion microscopically composed of 3 distinct melanocytic populations, suggesting a stepwise progression model to melanoma from a conventional nevus through a melanocytoma stage. This progression was also supported from a molecular viewpoint given BRAFV600E, BAP1, and TERT-p hot spot mutations detected by targeted mutational analysis. Four atypical melanocytic lesions were removed from the patient's back, and the same A BAP1 c.856A>T, p.(Lys286Ter) mutation was detected on either tumoral or normal tissue samples. To the best of our knowledge, this is the first case of BAP1-inactivated melanoma with a documented TERT-p hot spot mutation manifesting as the first presentation of BAP1 tumor predisposition syndrome.

556 Functional and proliferative melanocyte-derived pluripotent stem cells: from developmental studies to clinical perspectives

Over the last decade, various protocols have been published to generate melanocytes from human pluripotent stem cells (CSP). However, there is limitations in their use specifically for developmental research studies and clinical applications in which a high purity of mel-CSP capable of high proliferative capacity is critical for a large-scale production of cells. To overcome the shortcomings associated to the previous protocols, we have successfully developed an efficient and robust stepwise method to generate a pure and homogenous melanocyte population with higher proliferate rate in culture.

PRAME is a useful marker for the differential diagnosis of melanocytic tumours and histological mimics.

Although the morphological assessment of melanoma is generally straightforward, diagnosis can be especially difficult when the significant morphological and immunohistochemical results overlap with those of benign and malignant melanocytic tumours and histological mimics. This study assessed the potential diagnostic utility of measuring PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemically in naevi, melanomas and clear cell sarcomas (CCSs) in Chinese patients. We examined the immunohistochemical expression of PRAME in 317 melanocytic naevi, 178 primary melanomas, 72 metastatic melanomas and 19 CCSs and compared the sensitivity and specificity of PRAME immunohistochemistry (IHC) in the differential diagnosis of melanocytic tumours and histological mimics. Of the 317 melanocytic naevi, 98.1%were completely negative for PRAME; six cases showed focal PRAME immunoreactivity in a minor population of lesional melanocytes. Diffuse nuclear immunoreactivity for PRAME was found in 89.9% of primary melanomas and 93.1% of metastatic melanomas. Regarding melanoma subtypes, PRAME was expressed in 100% of superficial spreading melanomas, 100% of melanomas arise in congenital naevus, 91.4% of nodular melanomas, 87.8% of acral lentigo melanomas, 80.0% of lentigo malignant melanomas, 60.0% of Spitz melanomas, 96.2% of mucosal melanomas and 80.0% of uveal melanomas. None of the two desmoplastic melanomas expressed PRAME. Of the 19 CCS cases, 89.5% were negative for PRAME and 10.5% showed focal weak PRAME immunoreactivity in a minor population of tumour cells. Our findings indicate that PRAME may be a useful marker to support a suspected diagnosis of melanoma. In addition, lack of PRAME expression is a valuable hint to CCS in a suspected case, and then molecular confirmation of the presence of EWSR1 rearrangement is necessary.

Elastin Peptides with Ferrous Ferric Chloride Activate Human Melanocytes and Elastin Fibers

Background: Elastin peptides stimulate the development of mouse melanocytes in neural crest culture. Ferrous ferric chloride (FFC) promotes mammalian melanocyte growth in culture. However, it is unclear whether elastin peptides in the presence of FFC can stimulate human melanocyte growth in situ. Objectives: This study aimed to investigate the mechanism of human melanocyte growth for skin and stem cell science since melanocytes control human skin color. Methods: In this clinical trial study, a lotion containing elastin peptides and/or FFC was applied to the normal skin of 6 volunteers twice a day for 1 to 3 months. Punch biopsies were taken from treated skin and surveyed by histochemical methods using the dopa reaction (detect melanocytes) and dopa-premelanin reaction (detect melanocytes and melanoblasts). Elastin fibers were detected by Victoria blue staining. Results: Only the combined treatment of elastin peptides and FFC increased melanocyte populations in addition to dopa reactivity, melanogenesis, dendritogenesis, and epidermal melanin pigmentation. Mitotic divisions of melanocytes were also observed. However, the melanoblast population failed to increase, and no mitotic melanoblasts were observed. In the dermis, elastin fibers became thicker and denser after the treatment. The data of statistical analyses were performed by tabulation, mean, and SD on Microsoft Excel for Macintosh OS Catalina 10 system. Conclusions: Our present study suggests that elastin peptides with FFC can promote melanocyte growth, melanin synthesis, skin pigmentation, and elastin fiber formation. Our study can be expected to contribute to advancing skin and stem cell science.

Renal organoid modeling of tuberous sclerosis complex reveals lesion features arise from diverse developmental processes.

Tuberous sclerosis complex (TSC) is a multisystem tumor-forming disorder caused by loss of TSC1 or TSC2. Renal manifestations predominately include cysts and angiomyolipomas. Despite a well-described monogenic etiology, the cellular pathogenesis remains elusive. We report a genetically engineered human renal organoid model that recapitulates pleiotropic features of TSC kidney disease invitro and upon orthotopic xenotransplantation. Time course single-cell RNA sequencing demonstrates that loss of TSC1 or TSC2 affects multiple developmental processes in the renal epithelial, stromal, and glial compartments. First, TSC1 or TSC2 ablation induces transitional upregulation of stromal-associated genes. Second, epithelial cells in the TSC1-/- and TSC2-/- organoids exhibit a rapamycin-insensitive epithelial-to-mesenchymal transition. Third, a melanocytic population forms exclusively in TSC1-/- and TSC2-/- organoids, branching from MITF+ Schwann cell precursors. Together, these results illustrate the pleiotropic developmental consequences of biallelic inactivation of TSC1 or TSC2 and offer insight into TSC kidney lesion pathogenesis.

Segmenting Skin Biopsy Images with Coarse and Sparse Annotations using U-Net.

The number of melanoma diagnoses has increased dramatically over the past three decades, outpacing almost all other cancers. Nearly 1 in 4 skin biopsies is of melanocytic lesions, highlighting the clinical and public health importance of correct diagnosis. Deep learning image analysis methods may improve and complement current diagnostic and prognostic capabilities. The histologic evaluation of melanocytic lesions, including melanoma and its precursors, involves determining whether the melanocytic population involves the epidermis, dermis, or both. Semantic segmentation of clinically important structures in skin biopsies is a crucial step towards an accurate diagnosis. While training a segmentation model requires ground-truth labels, annotation of large images is a labor-intensive task. This issue becomes especially pronounced in a medical image dataset in which expert annotation is the gold standard. In this paper, we propose a two-stage segmentation pipeline using coarse and sparse annotations on a small region of the whole slide image as the training set. Segmentation results on whole slide images show promising performance for the proposed pipeline.

Identification, Isolation, and Characterization of Melanocyte Precursor Cells in the Human Limbal Stroma.

Given their vital role in the homeostasis of the limbal stem cell niche, limbal melanocytes have emerged as promising candidates for tissue engineering applications. This study aimed to isolate and characterize a population of melanocyte precursors in the limbal stroma, compared with melanocytes originating from the limbal epithelium, using magnetic-activated cell sorting (MACS) with positive (CD117/c-Kit microbeads) or negative (CD326/EpCAM or anti-fibroblast microbeads) selection approaches. Both approaches enabled fast and easy isolation and cultivation of pure limbal epithelial and stromal melanocyte populations, which differed in phenotype and gene expression, but exhibited similar functional properties regarding proliferative potential, pigmentation, and support of clonal growth of limbal epithelial stem/progenitor cells (LEPCs). In both melanocyte populations, limbus-specific matrix (laminin 511-E8) and soluble factors (LEPC-derived conditioned medium) stimulated melanocyte adhesion, dendrite formation, melanogenesis, and expression of genes involved in UV protection and immune regulation. The findings provided not only a novel protocol for the enrichment of pure melanocyte populations from limbal tissue applying easy-to-use MACS technology, but also identified a population of stromal melanocyte precursors, which may serve as a reservoir for the replacement of damaged epithelial melanocytes and an alternative resource for tissue engineering applications.

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Single-cell RNA-sequencing analysis of the ciliary epithelium and contiguous tissues in the mouse eye

The ciliary epithelium plays a central role in ocular homeostasis but cells of the pigmented and non-pigmented layers are difficult to isolate physically and study. Here we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptional signatures of cells harvested from the ciliary body and contiguous tissues. Microdissected tissue was dissociated by collagenase digestion and the transcriptomes of individual cells were obtained using a droplet-based scRNA-seq approach. In situ hybridization was used to verify the expression patterns of selected differentially-expressed genes. High quality transcriptomes were obtained from 10,024 cells and unsupervised clustering distinguished 22 cell types. Although efforts were made to specifically isolate the ciliary body, approximately half of the sequenced cells were derived from the adjacent retina. Cluster identities were assigned using expression of canonical markers or cluster-specific genes. The transcriptional signature of cells in the PCE and NPCE were distinct from each other and from cells in contiguous tissues. PCE cell transcriptomes were characterized by genes involved in melanin synthesis and transport proteins such as Slc4a4. Among the most differentially expressed genes in NPCE cells were those encoding members of the Zic family of transcription factors (Zic1, 2, 4), collagen XVIII (Col18a1), and corticotrophin-releasing hormone-binding protein (Crhbp). The ocular melanocyte population was distinguished by expression of the gap junction genes Gjb2 and Gjb6. Two fibroblast signatures were detected in the ciliary body preparation and shown by in situ hybridization to correspond to uveal and scleral populations. This cell atlas for the ciliary body and contiguous layers represents a useful resource that may facilitate studies into the development of the ciliary epithelium, the production of the aqueous and vitreous humors, and the synthesis of the ciliary zonule.

Diagnostic utility of preferentially expressed antigen in melanoma immunohistochemistry in the evaluation of melanomas with a co-existent nevoid melanocytic population: A single-center retrospective cohort study

To the Editor: PRAME (PReferentially expressed Antigen in MElanoma) immunohistochemistry has emerged as a reliable tool for distinguishing benign and malignant melanocytic neoplasms.1-4 Diffuse nuclear immunoreactivity was found in ∼90% of conventional nonspindle cell primary cutaneous melanomas, compared to absent or focal staining in 86% and 13% of conventional nonspitzoid melanocytic nevi, respectively.2

What is your diagnosis? Fine-needle aspirate biopsy from a skin mass on the distal metatarsus of a dog.

What is your diagnosis? Fine-needle aspirate biopsy from a skin mass on the distal metatarsus of a dog.