Mature Melanocytes Research Articles

Hair regrowth in alopecia areata and re-pigmentation in vitiligo in response to treatment: Commonalities and differences.

Both alopecia areata (AA) and vitiligo share common pathogenesis involving, interferon-γ (IFN-γ) and interleukin-15 (IL-15) signalling pathways that activate cytotoxic CD8+ T lymphocytes. These shared mechanisms may explain why both diseases respond to currently available treatments (e.g. topical/systemic corticosteroid) and emerging treatment modalities. As compared with the speed of re-pigmentation in vitiligo lesions, the regeneration of pigmented terminal hair follicles in AA lesions appears fast in response to treatments targeting the inhibition of the Janus kinases (JAKs) and other kinases. We summarize the commonalities and differences between AA and vitiligo focusing on the treatment modalities, followed by recent findings associated with hair follicle stem cells (HFSC) in hair bulge (HBg) and melanocyte stem cells (McSC) in HBg and hair germ (HGm). We then discuss how HFSC and HGm-McSC are involved in the initiation of anagen phase, followed by pigmented terminal hair regrowth in the recovering AA lesions in association with immunology. We also discuss how HBg-McSC contribute to the migration of fully dendritic mature melanocytes into interfollicular epidermis and the equal distribution of melanin in recovering vitiligo lesions. Finally, we present four hypotheses to elucidate the delayed distribution of melanin by mature melanocytes in depigmented vitiligo lesions from the aspects of stem cell biology, as compared with quick hair recovery in AA: (1) McSC are less abundant than HFSC. (2) McSC require a long travel, whereas HFSC reside close to hair regeneration trigger point. (3) Keratinocyte scaffold to accept melanin is not well preserved, whereas scaffold for hair regrowth is well preserved. (4) Inhibitors targeting JAKs and other kinases have less direct effects on melanocyte proliferation and differentiation in vitiligo than hair regrowth in AA. Our review provides an overview of treatment modalities and bridges the gap between scientific advancement and clinical practice in AA and vitiligo management.

Canonical Wnt and TGF-β/BMP signaling enhance melanocyte regeneration but suppress invasiveness, migration, and proliferation of melanoma cells.

Melanoma is the deadliest form of skin cancer and develops from the melanocytes that are responsible for the pigmentation of the skin. The skin is also a highly regenerative organ, harboring a pool of undifferentiated melanocyte stem cells that proliferate and differentiate into mature melanocytes during regenerative processes in the adult. Melanoma and melanocyte regeneration share remarkable cellular features, including activation of cell proliferation and migration. Yet, melanoma considerably differs from the regenerating melanocytes with respect to abnormal proliferation, invasive growth, and metastasis. Thus, it is likely that at the cellular level, melanoma resembles early stages of melanocyte regeneration with increased proliferation but separates from the later melanocyte regeneration stages due to reduced proliferation and enhanced differentiation. Here, by exploiting the zebrafish melanocytes that can efficiently regenerate and be induced to undergo malignant melanoma, we unravel the transcriptome profiles of the regenerating melanocytes during early and late regeneration and the melanocytic nevi and malignant melanoma. Our global comparison of the gene expression profiles of melanocyte regeneration and nevi/melanoma uncovers the opposite regulation of a substantial number of genes related to Wnt signaling and transforming growth factor beta (TGF-β)/(bone morphogenetic protein) BMP signaling pathways between regeneration and cancer. Functional activation of canonical Wnt or TGF-β/BMP pathways during melanocyte regeneration promoted melanocyte regeneration but potently suppressed the invasiveness, migration, and proliferation of human melanoma cells in vitro and in vivo. Therefore, the opposite regulation of signaling mechanisms between melanocyte regeneration and melanoma can be exploited to stop tumor growth and develop new anti-cancer therapies.

The journey from melanocytes to melanoma.

Over the past decade, melanoma has led the field in new cancer treatments, with impressive gains in on-treatment survival but more modest improvements in overall survival. Melanoma presents heterogeneity and transcriptional plasticity that recapitulates distinct melanocyte developmental states and phenotypes, allowing it to adapt to and eventually escape even the most advanced treatments. Despite remarkable advances in our understanding of melanoma biology and genetics, the melanoma cell of origin is still fiercely debated because both melanocyte stem cells and mature melanocytes can be transformed. Animal models and high-throughput single-cell sequencing approaches have opened new opportunities to address this question. Here, we discuss the melanocytic journey from the neural crest, where they emerge as melanoblasts, to the fully mature pigmented melanocytes resident in several tissues. We describe a new understanding of melanocyte biology and the different melanocyte subpopulations and microenvironments they inhabit, and how this provides unique insights into melanoma initiation and progression. We highlight recent findings on melanoma heterogeneity and transcriptional plasticity and their implications for exciting new research areas and treatment opportunities. The lessons from melanocyte biology reveal how cells that are present to protect us from the damaging effects of ultraviolet radiation reach back to their origins to become a potentially deadly cancer.

Impact of KIT Editing on Coat Pigmentation and Fresh Meat Color in Yorkshire Pigs.

The white coat color of Yorkshire pigs is caused by the dominant white I allele, which has been associated with at least one copy of the 450-kb duplication encompassing the entire KIT gene and a splice mutation (G > A) at the first base of intron 17. The splice mutation in KIT has an adverse effect on pigmentation in mice. Therefore, removing the 450 kb duplications harboring the KIT copy with splice mutations is expected to affect Yorkshire pig pigmentation. In this study, we describe the use of a Yorkshire pig kidney cell strain with the I?/IBe-ed genotype, previously created by CRISPR-Cas9, as donor cells for somatic cell nuclear transfer to generate gene-edited Yorkshire pigs. The removal of the 450 kb duplications harboring the KIT copy with splice mutation did not alter the white coat color of Yorkshire pigs, which was confirmed by the absence of fully mature melanocytes and melanin accumulation in the hair follicles. Except for the improved transcription of tyrosinase, and slight increase in microphthalmia transcription factor and tyrosinase-related protein 1 protein expression, there was no significant impact of the removal of splice mutations on genes and signaling pathways (PI3K/AKT) involved in melanogenesis. However, the removal of the 450 kb duplications harboring the KIT copy with splice mutation substantially improved fresh meat color accompanied by significantly increased red blood cell number, which merits further investigation. Our study provides new insights into the role of structural mutations of the KIT gene in the formation of white coat color and erythropoiesis in Yorkshire pigs.

Lrig1- and Wnt-dependent niches dictate segregation of resident immune cells and melanocytes in murine tail epidermis.

ABSTRACTThe barrier-forming, self-renewing mammalian epidermis comprises keratinocytes, pigment-producing melanocytes and resident immune cells as first-line host defense. In murine tail skin, interfollicular epidermis patterns into pigmented ‘scale’ and hypopigmented ‘interscale’ epidermis. Why and how mature melanocytes accumulate in scale epidermis is unresolved. Here, we delineate a cellular hierarchy among epidermal cell types that determines skin patterning. Already during postnatal development, melanocytes co-segregate with newly forming scale compartments. Intriguingly, this process coincides with partitioning of both Langerhans cells and dendritic epidermal T cells to interscale epidermis, suggesting functional segregation of pigmentation and immune surveillance. Analysis of non-pigmented mice and of mice lacking melanocytes or resident immune cells revealed that immunocyte patterning is melanocyte and melanin independent and, vice versa, immune cells do not control melanocyte localization. Instead, genetically enforced progressive scale fusion upon Lrig1 deletion showed that melanocytes and immune cells dynamically follow epithelial scale:interscale patterns. Importantly, disrupting Wnt-Lef1 function in keratinocytes caused melanocyte mislocalization to interscale epidermis, implicating canonical Wnt signaling in organizing the pigmentation pattern. Together, this work uncovers cellular and molecular principles underlying the compartmentalization of tissue functions in skin.

Effect of An Atmospheric Plasma Jet on the Differentiation of Melanoblast Progenitor.

Melanoblasts are the cell source of regeneration for pigment restoration. The ability to differentiate into mature melanocytes is the essential feature of melanoblasts in depigmentation diseases. Cold atmospheric plasma is an ionized gas with near-room temperature and highly reactive species that has been shown to induce stem cell differentiation. The aim of the study was to explore the effect of cold atmospheric plasma on the differentiation of melanoblast progenitor cells. In this study, melanoblasts were exposed to the plasma jet and the cell morphology was observed. The cell cycle and cell proliferation were detected. Furthermore, the cell immunofluorescence and the detection of melanin particle and nitric oxide were carried out to investigate the differentiation of melanoblast progenitor cells. Cells that were treated with the plasma had longer and more synaptic structures, and the G1 phase of cell cycle was prolonged in the treated group. More melanin synthesis-related proteins and melanin particles were produced after plasma treatment. Nitric oxide was one of the active components generated by the plasma jet, and the nitric oxide content in the cell culture medium of the treated group increased. These results indicate that an increase in nitric oxide production caused by a plasma jet can promote cell differentiation. The application of plasma provides an innovative strategy for the treatment of depigmentation diseases.

Characterization of a melanocyte progenitor population in human interfollicular epidermis.

It is still unknown whether the human interfollicular epidermis harbors a reservoir of melanocyte precursor cells. Here, we clearly distinguish between three distinct types of melanocytes in human interfollicular epidermis: (1) cKit+CD90-, (2) cKit+CD90+, and (3) cKit-CD90+. Importantly, we identify the Kit tyrosine kinase receptor (cKit) as a marker expressed specifically in mature, melanin-producing melanocytes. Thus, both cKit+CD90- and cKit+CD90+ cells represent polydendritic, pigmented mature melanocytes, whereas cKit-CD90+ cells display bipolar, non-dendritic morphology with reduced melanin content. Additionally, using tissue-engineered pigmented dermo-epidermal skin substitutes (melDESSs), we reveal that the cKit expression also plays an important role during melanogenesis in melDESS invivo. Interestingly, cKit-CD90+ cells lack the expression of markers such as HMB45, TYR, and TRP1 invitro and invivo. However, they co-express neural-crest progenitor markers and demonstrate multilineage differentiation potential invitro. Hence, we propose that cKit-CD90+ cells constitute the precursor melanocyte reservoir in human interfollicular epidermis.

Targeting GPCRs and Their Signaling as a Therapeutic Option in Melanoma.

Simple SummarySixteen G-protein-coupled receptors (GPCRs) have been involved in melanogenesis or melanomagenesis. Here, we review these GPCRs, their associated signaling, and therapies.G-protein-coupled receptors (GPCRs) serve prominent roles in melanocyte lineage physiology, with an impact at all stages of development, as well as on mature melanocyte functions. GPCR ligands are present in the skin and regulate melanocyte homeostasis, including pigmentation. The role of GPCRs in the regulation of pigmentation and, consequently, protection against external aggression, such as ultraviolet radiation, has long been established. However, evidence of new functions of GPCRs directly in melanomagenesis has been highlighted in recent years. GPCRs are coupled, through their intracellular domains, to heterotrimeric G-proteins, which induce cellular signaling through various pathways. Such signaling modulates numerous essential cellular processes that occur during melanomagenesis, including proliferation and migration. GPCR-associated signaling in melanoma can be activated by the binding of paracrine factors to their receptors or directly by activating mutations. In this review, we present melanoma-associated alterations of GPCRs and their downstream signaling and discuss the various preclinical models used to evaluate new therapeutic approaches against GPCR activity in melanoma. Recent striking advances in our understanding of the structure, function, and regulation of GPCRs will undoubtedly broaden melanoma treatment options in the future.

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.

Bone Morphogenic Protein Signaling and Melanoma.

Malignant melanoma is a deadly form of skin cancer caused by neoplastic transformation of melanocytic cells. Despite recent progress in melanoma therapy, by inhibition of activated oncogenes or immunotherapy, survival rate for metastatic melanoma patients remains low. The remarkable phenotypic plasticity of melanoma cells allows for rapid development of invasive properties and metastatic tumors, the main cause of mortality in melanoma patients. Phenotypic and molecular analyses of developing tumors revealed that epithelial-mesenchymal transition (EMT), a cellular and molecular mechanism, controls transition from mature melanocyte to less differentiated melanocyte lineage progenitor cells forming melanoma tumors. This transition is facilitated by persistence of transcriptional regulatory circuit characteristic of embryonic stage in mature melanocytes. Switching of the developmental program of mature melanocyte to EMT is induced by accumulated mutations, especially targeting BRAF, N-RAS, or MEK1/2 signaling pathways, and further promoted by dynamic stimuli from local environment including hypoxia, interactions with extracellular matrix and growth factors or cytokines. Recent reports demonstrate that signaling mediated by transforming growth factor-β (TGF-β) and bone morphogenic proteins (BMPs) play critical roles in inducing EMT by controlling expression of critical transcription factors. BMPs are essential modulators of differentiation, proliferation, apoptosis, invasiveness, and metastases in developing melanoma tumors. They control transcription and epigenetic landscape of melanoma cells. Better understanding of the role of BMPs may lead to new strategies to control EMT processes in melanocyte cell lineage and to achieve clinical benefits for the patients.

Analysis of how obtaining melanocytes by magnetic cell separation contributes to autoepidermal transplantation technology in treating leucoderma.

Leucoderma, a depigmentation of the skin, is a common disease in humans, and has been observed in cattle, horses and buffalo as well. To analyze the correlation between melanin stem cells and the differentiation and proliferation of melanocytes (MCs). Magnetic cell separation was used to separate melan-A+ cells and PAX3+ cells, which were cultured in vitro. The L-DOPA staining was used to observe cell morphology; Cell Counting Kit-8 (CCK8) was used to determine the cell proliferation rate; and flow cytometry (FCM) was used to determine cell cycle changes. The relative mRNA levels of melanocyte-inducing transcription factor (MITF), dopachrome tautomerase (DCT) and melan-A in cells were determined with reverse-transcription polymerase chain reaction (RT-PCR). The number of MC dendrites increased and extended continually during in vitro culture following magnetic cell separation. The proportion of positive L-DOPA staining cells increased from a baseline 40.70% to 82.03%, and the cell proliferation rate increased from 335.0% at D3 to 1577.4% at D20. The results of FCM showed that the cell proportion at the G1 stage in the D20 group was significantly lower than the D3 group; the cell proportion at the G2/M stage also decreased significantly. The expression of MITF and melan-A increased as the culture time increased, while the expression of DCT decreased. The number of MC stem cells decreased and mature MCs increased gradually, indicating that MC stem cells can gradually differentiate into mature MCs during in vitro culture following magnetic cell separation.

The Danger of Being Too Sympathetic: Norepinephrine in Alzheimer's Disease and Graying of Hair.

Although alterations in the sympathetic nervous system (SNS) with age have been reported, and serious degenerative diseases of the autonomic nervous system such as multiple system atrophy are more likely to strike older people, connections between dysregulated adrenergic receptors and age-associated diseases and phenotypes have not been well studied. Two recent reports suggest that SNS may be more closely connected than previously appreciated. First, low nanomolar concentrations of Alzheimer's disease (AD)-associated Aβ42-amyloid oligomers alter signaling by SNS neurotransmitter norepinephrine (NE) to sufficiently activate kinase GSK3β to hyperphosphorylate tau, a key mediator of neurotoxicity in AD. Connecting beta-amyloid to tau in AD has been a key quest in understanding AD and developing therapeutics. The α2 adrenergic receptor inhibitory drug idazoxan reduces GSK3β activity and tau phosphorylation in AD mice with improved cognitive function, even in the presence of beta-amyloid deposits. In this study, SNS activation in the brain coupled with problematic Aβ42-amyloid oligomers result in serious consequences that can be ameliorated by reducing SNS signaling. A second example of the detrimental effects of increased SNS signaling is the premature graying of hair in response to stress. Secretion of NE resulting from stress causes differentiation of most hair pigment melanocyte stem cells (MeSCs) into melanocytes, rapidly depleting the hair follicle of pigment-producing cells as mature melanocytes undergo apoptosis and MeSCs are eventually eliminated. Blockade of NE SNS signaling preserves hair coloration in stressed animals. Increased SNS activation has serious apparently irreversible effects on homeostasis in both situations. Although neither report directly addresses aging, given that AD and the loss of hair pigmentation have strong age associations, it is of interest to better understand the role that SNS has in promoting age-associated phenotypes generally and determine if tuning the SNS through drug-mediated attenuation of SNS signaling may be of medical benefit.

14. Ancient melanocytic naevus (AN): A simulator of malignant melanoma

Background and aims: Ancient melanocytic naevus (AN) is an unusual but distinct melanocytic neoplasm within the spectrum of simulators of malignant melanoma. The name ‘ancient’ is used because it shares numerous features with ancient schwannoma. Ancient naevi are found most commonly on the face of older persons. The neoplasm is usually a dome-shaped, skin-coloured papule. Histopathologically, ancient naevi are exo-endophytic, mostly intradermal proliferations of two populations of melanocytes: one with large pleomorphic nuclei and the other with small monomorphic ones. A few mitotic figures may be present. We report a case of 73-year-old male who presented with an exo-endophytic pigmented lesion 5 mm in diameter on his right lateral thigh. It had been present for several years. An excision biopsy was done. Methods: Here we present a case of an ancient melanocytic naevus. Results: The histological examination of routine H&E stains showed a predominately dermal compound melanocytic lesion in the upper dermis composed of two populations of intermingled melanocytes. One with large pleomorphic round, oval, polygonal and spindled melanocytes with large hyperchromatic nuclei, some with prominent nucleoli and scant cytoplasm. The other with conventional, small, round to oval mature melanocytes arranged in a ‘congenital’ pattern. A few multinucleate pleomorphic melanocytes were also present. Ki-67 immunostain showed a proliferation index of less than 1%. Morphological features and immunohistochemistry were consistent with ancient melanocytic naevus. Conclusions: Ancient naevi may be misdiagnosed as melanoma arising in an intradermal naevus. Knowledge of the histopathologic characteristics of this benign melanocytic neoplasm should enable pathologists to avoid over diagnosis of melanoma arising in an intradermal naevus.

MiR-380-3p regulates melanogenesis by targeting SOX6 in melanocytes from alpacas (Vicugna pacos)

BackgroundMelanocytes are derived from neural crest stem cells in the embryonic stage. In mature melanocytes, a series of complex enzyme-catalyzed reactions leads to the production of melanins, which determine the hair and skin colors of animals. The process of melanogenesis is complex and can be regulated by mRNA, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) genes. MiRNAs are a type of endogenous noncoding RNA approximately 22 nt in size that predominantly regulate gene expression by inhibiting translation. miR-380-3p is a candidate miRNA potentially related to melanogenesis. To better understand the mechanism of miR-380-3p melanogenesis regulation, plasmids to overexpress or knockdown miR-380-3p were transfected into alpaca melanocytes, and their effects on melanogenesis were evaluated.ResultsIn situ hybridization identified a positive miR-380-3p signal in alpaca melanocyte cytoplasm. Luciferase activity assays confirmed that SOX6 is targeted by miR-380-3p. miR-380-3p overexpression and knockdown in alpaca melanocytes respectively downregulated and upregulated SOX6 expression at the mRNA and protein levels. Additionally, miR-380-3p overexpression and knockdown, respectively, in alpaca melanocytes decreased and increased the mRNA levels of melanin transfer-related genes, including microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosine-related protein-1 (TYRP1), and dopachrome tautomerase (DCT). In contrast, miR-380-3p overexpression and knockdown respectively increased and decreased the mRNA levels of β-catenin. Additionally, the effect of miR-380-3p on melanogenesis was assessed by Masson-Fontana melanin staining.ConclusionsThe results demonstrated that miR-380-3p targeted SOX6 to regulate melanogenesis by influencing β-catenin and MITF transcription and translation, which reduced the expression of downstream genes, including TYR, TYRP1, and DCT. These results provide insights into the mechanisms through which miR-380-3p controls melanogenesis.

Skin transcriptome profiles associated with black- and white-coated regions in Boer and Macheng black crossbred goats

To increase the current understanding of the gene-expression profiles in different skin regions associated with different coat colors and identify key genes for the regulation of color patterns in goats, we used the Illumina RNA-Seq method to compare the skin transcriptomes of the black- and white-coated regions containing hair follicles from the Boer and Macheng Black crossbred goat, which has a black head and a white body. Six cDNA libraries derived from skin samples of the white-coated region (n = 3) and black-coated region (n = 3) were constructed from three full-sib goats. On average, we obtained approximately 76.5 and 73.5 million reads for skin samples from black- and white-coated regions, respectively, of which 75.39% and 76.05% were covered in the genome database. A total of 165 differentially expressed genes (DEGs) were detected between these two color regions, among which 110 were upregulated and 55 were downregulated in the skin samples of white- vs. black-coated regions. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that some of these DEGs may play an important role in controlling the pigmentation of skin or hair follicles. We identified three key DEGs, i.e., Agouti, DCT, and TYRP1, in the pathway related to melanogenesis in the different skin regions of the crossbred goat. DCT and TYRP1 were downregulated and Agouti was upregulated in the skin of the white-coated region, suggesting a lack of mature melanocytes in this region and that Agouti might play a key developmental role in color-pattern formation. All data sets (Gene Expression Omnibus) are available via public repositories. In addition, MC1R was genotyped in 200 crossbred goats with a black head and neck. Loss-of-function mutations in MC1R as well as homozygosity for the mutant alleles were widely found in this population. The MC1R gene did not seem to play a major role in determining the black head and neck in our crossbred goats. Our study provides insights into the transcriptional regulation of two distinct coat colors, which might serve as a key resource for understanding coat color pigmentation in goats. The region-specific expression of Agouti may be associated with the distribution of pigments across the body in Boer and Macheng Black crossbred goats.

Essential Role for Integrin-Linked Kinase in Melanoblast Colonization of the Skin

Melanocytes are pigment-producing cells found in the skin and other tissues. Alterations in the melanocyte lineage give rise to a plethora of human diseases, from neurocristopathies and pigmentation disorders to melanoma. During embryogenesis, neural crest cell subsets give rise to two waves of melanoblasts, which migrate dorsolaterally, hone to the skin, and differentiate into melanocytes. However, the mechanisms that govern colonization of the skin by the first wave of melanoblasts are poorly understood. Here we report that targeted inactivation of the integrin-linked kinase gene in first wave melanoblasts causes defects in the ability of these cells to form long pseudopods, to migrate, and to proliferate invivo. As a result, integrin-linked kinase-deficient melanoblasts fail to populate normally the developing epidermis and hair follicles. We also show that defects in motility and dendricity occur upon integrin-linked kinase gene inactivation in mature melanocytes, causing abnormalities in cell responses to the extracellular matrix substrates collagen I and laminin 332. Significantly, the ability to form long protrusions in mutant cells in response to collagen is restored in the presence of constitutively active Rac1, suggesting that an integrin-linked kinase-Rac1 nexus is likely implicated in melanocytic cell establishment, dendricity, and functions in the skin.

Stem Cell-Derived Models of Neural Crest Are Essential to Understand Melanoma Progression and Therapy Resistance.

During development, neural crest (NC) cells are early precursors of several lineages including melanocytes. Along their differentiation from multipotent cells to mature melanocytes, NC cells will go through successive steps which require either proliferative or motile capacities. For example, they will undergo Epithelial to Mesenchymal Transition (EMT) in order the separate from the neural tube and migrate to their final location in the epidermis (Larribere and Utikal, 2013; Skrypek et al., 2017). The differentiated melanocytes are the cells of origin of melanoma tumors which progress through several stages such as radial growth phase, vertical growth phase, metastasis formation, and often resistance to current therapies. Interestingly, depending on the stage of the disease, melanoma tumor cells share phenotypes with NC cells (proliferative, motile, EMT). These phenotypes are tightly controlled by specific signaling pathways and transcription factors (TFs) which tend to be reactivated during the onset of melanoma. In this review, we summarize first the main TFs which control these common phenotypes. Then, we focus on the existing strategies used to generate human NCs. Finally we discuss how identification and regulation of NC-associated genes provide an additional approach to improving current melanoma targeted therapies.

835 Role of ILK in melanoblasts

Melanocytes are pigment-producing cells found in the skin and other tissues. Alterations in the melanocyte lineage contribute to various human diseases, including neurocristopathies and pigmentation disorders. During embryogenesis, neural crest cell subsets give rise to the first wave of melanoblasts, which migrate dorsolaterally, homing to the skin and differentiating into melanocytes. Targeted inactivation of Ilk in these melanoblasts causes defects in their ability to form long pseudopods, to migrate and to proliferate. As a result, integrin-linked kinase (ILK)-deficient melanoblasts fail to normally populate the developing epidermis and hair follicles. Similar defects occur upon Ilk gene inactivation in mature melanocytes, causing abnormalities in integrin-associated cell responses to the extracellular matrix substrates collagen and laminin-332. Significantly, the ability to form long protrusions in these cells is restored in the presence of constitutively active Rac1, suggesting that an integrin-ILK-Rac1 nexus is critically important for melanocytic cell establishment and functions in the skin.

Therapeutic Potential of Patient iPSC-Derived iMelanocytes in Autologous Transplantation

Induced pluripotent stem cells (iPSCs) are a promising melanocyte source as they propagate indefinitely and can be established from patients. However, the invivo functions of human iPSC-derived melanocytes (hiMels)remain unknown. Here, we generated hiMels from vitiligo patients using a three-dimensional system with enhanced differentiation efficiency, which showed characteristics of human epidermal melanocytes with high sequence similarity and involved inmultiple vitiligo-associated signaling pathways. A modified hair follicle reconstitution assay invivo showed that MITF+PAX3+TYRP1+ hiMels were localized in the mouse hair bulb and epidermis and produced melanin up to 7weeks after transplantation, whereas MITF+PAX3+TYRP1- hiMelanocyte stem cells integrated into the bulge-subbulge regions. Overall, these data demonstrate the long-term functions of hiMels invivo to reconstitute pigmented hair follicles and to integrate into normal regions for both mature melanocytes and melanocyte stem cells, providing an alternative source of personalized cellular therapy for depigmentation.

Novel Rest functions revealed by conditional gene ablation.

Rest is a regulator of neuronal development and has been suggested to function in maintaining the pluripotent state of embryonic stem cells (ESCs); however, this remains controversial. Since Rest null mice show embryonic lethality, we herein generated conditional Rest knockout (CKO) models to investigate Rest functions in more detail. Our results revealed that Rest was not necessary for maintaining the pluripotency of ESCs and instead promoted primitive endoderm differentiation. In contrast to the repressive role of Rest in vitro, including ESCs, neural stem cells, and fibroblasts, on the expression of target neural genes, Rest CKO did not affect the in vivo development of brain tissue. However, the same Rest CKO mice showed an abnormal lens morphology after birth with augmented Notch signaling and down-regulated lens fiber regulator gene expression. The ablation of Rest during neural crest cell (NCC) development caused neonatal lethality due to swelling of the digestive tract with reductions in acetylcholinesterase activity in the myenteric plexus derived from NCCs. Furthermore, a reduced number of melanocyte precursors also derived from NCCs resulted in white spotted coat color phenotypes lacking mature melanocytes. Rest controls thousands of target genes and may have many unknown functions related to diseases.