Fibronectin combined with stem cell factor plays an important role in melanocyte proliferation,differentiation and migration in cultured mouse neural crest cells

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild-type mice, we previously demonstrated that KIT-positive and/or L-3, 4-dihydroxyphenylalanine (DOPA)-positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT-positive cells and DOPA-positive cells were detected in the presence of SCF on ECM-coated wells than on non-coated wells. A statistically significant increase in DOPA-positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA-positive cells and KIT-positive cells were detected on either the ECM-coated or non-coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA-positive cells decreased with RGDS concentration in a dose-dependent fashion. Immunohistochemical staining revealed the presence of integrin alpha5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.     

Establishment and characterization of a mouse neural crest derived cell line (NCCmelan5)

Stem cell factor (SCF) and endothelin 3 (EDN3) are both necessary for melanocyte development. We have established an immortal cell population of neural crest cells from C57BL/6 mice, cultivating them with SCF, EDN3 and 15% fetal calf serum without feeder cells, and have designated that line as C57NCC SE. C57NCC SE consists of a population of melanocytes in various stages of differentiation. We used a single-cell cloning method, in which only one cell is transferred to each new culture plate, and succeeded in establishing an immortal cell line named NCCmelan5. All NCCmelan5 cells were positive for KIT (SCF receptor), HMB45 (human melanosomal antigen), tyrosinase-related protein-1 (TYRP1), tyrosinase-related protein-2 (TYRP2), tyrosinase and endothelin receptor B (EDNRB) and all could oxidize 3,4-dihydroxyphenylalanine (DOPA) to form melanin. Measurement of their DNA content revealed that 88.6% of the cells were in the G0-G1 phase, suggesting that they retained normal DNA ploidy. Thus, NCCmelan5 cells have the characteristics of mature melanocytes except that they are immortal; these cells may prove useful to study factors that directly affect melanogenesis and melanocyte development without the influence of feeder cells. It is clear that our attempt to establish immortal cell lines from murine neural crest cells would have never been successful without the addition of SCF and EDN3, since C57NCC SE and NCCmelan5 cells require those factors to proliferate.     

EDNRB/EDN3 and Hirschsprung Disease Type II

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1–4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah‐Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type‐B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest‐derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest‐derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB‐mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3‐induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post‐coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3 – a receptor‐mediated pathway with pleiotropic effects.     

Generation of human melanocytes from induced pluripotent stem cells

Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.     

Establishment by an Original Single‐cell Cloning Method and Characterization of an Immortal Mouse Melanoblast Cell Line (NCCmelb4)

We devised a unique new single‐cell cloning method which uses microscope cover glasses and established a melanoblast cell line derived from mouse neural crest cells. A microscope cover glass was nicked and broken into small pieces and put on a dish. Culture medium and a suspension of 20–30 cells/ml were dropped in the dish. After 1–3 d, a piece of glass to which only one cell was adhered was picked up and transferred to another dish containing culture medium. The greatest advantage of this method is that the derivation of a colony from a single cell can be directly confirmed by microscopy and there is no risk of migratory cells being contaminated by other colonies. Using this single‐cell cloning method, in this study we established a cell line derived from a neural crest cell line (NCC‐S4.1) and designated it as NCCmelb4. When the culture medium was supplemented with stem cell factor (SCF) alone, NCCmelb4 cells were KIT‐positive and tyrosinase‐negative melanocyte precursors; they remained at an immature and undifferentiated stage. When the medium was supplemented with phorbol 12‐o‐tetradecanoyl‐13‐acetate (TPA) + cholera toxin (CT), the cell morphology changed and became l ‐3,4‐dihydroxyphenylalanine (DOPA)‐positive. This observation indicates that the NCCmelb4 cells are capable of further differentiation with suitable stimulation. NCCmelb4 cells derived from the mouse neural crest has characteristics of melanocyte precursors (melanoblasts), and is a cell line which can be utilized to study differentiation‐inducing factors and growth factors without the effects of feeder cells.     

Roles of endothelin signaling in melanocyte development and melanoma

Endothelin (Edn) signaling via the G-coupled, Edn receptor type B (Ednrb) is essential for the development of melanocytes from the neural crest (NC) and has been associated with melanoma progression. Edn3 plays varying roles during melanocyte development, promoting the proliferation and self-renewal of NC-derived multi- and bi-potential precursors as well as the survival, proliferation, differentiation and migration of committed melanocyte precursors. Melanocyte differentiation is achieved via the interaction of Ednrb and Kit signaling, with Ednrb being specifically required in the final differentiation step, rather than in the initial specification of melanocytic fate. Ednrb has also been implicated in the de-differentiation of mature melanocytes, a process that takes place during the malignant transformation of these cells. Ednrb was found to be upregulated in melanoma metastases and was shown to alter tumor–host interactions leading to melanoma progression. Antagonists to this receptor were shown to inhibit melanoma cell growth and increase the apoptotic rate of these cells, and to lead to disease stabilization in melanoma patients. Thus, Edn signaling inhibition may prove useful in the treatment of certain types of melanoma.     

Fibronectin Combined with Stem Cell Factor Plays an Important Role in Melanocyte Proliferation,Differentiation and Migration in Cultured Mouse Neural Crest Cells

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild‐type mice, we previously demonstrated that KIT‐positive and/or L ‐3, 4‐dihydroxyphenylalanine (DOPA)‐positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT‐positive cells and DOPA‐positive cells were detected in the presence of SCF on ECM‐coated wells than on non‐coated wells. A statistically significant increase in DOPA‐positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA‐positive cells and KIT‐positive cells were detected on either the ECM‐coated or non‐coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA‐positive cells decreased with RGDS concentration in a dose‐dependent fashion. Immunohistochemical staining revealed the presence of integrin a5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.     

Differentiation of avian neural crest cells in vitro: Absence of a developmental bias toward melanogenesis

Summary Neural crest cells from quail embryos grown in standard culture dishes differentiate almost entirely into melanocytes within 4 or 5 days when chick embryo extract (CEE) or occasional lots of fetal calf serum (FCS) are included in the medium. Gel fractionation showed that the pigment inducing factor(s) present in these media is of high molecular weight (> 400 K daltons). In the absence of CEE, the neural tube can also stimulate melanocyte differentiation. Culture medium supplemented by selected lots of FCS permits crest cell proliferation but little overt differentiation after up to 2 weeks in culture if the neural tube is removed within 18 h of explantation in vitro. Subsequent addition of CEE to such cultures promotes complete melanocyte differentiation. Crest cells from White leghorn chick embryos also differentiate into melanocytes in the presence of CEE, but do not survive well in its absence. Melanocyte differentiation of crest cells from both quail and chick embryos can by suppressed by culturing under a dialysis membrane, even in the presence of the neural tube and CEE, but neuronal differentiation appears greatly enhanced.     

Turtle Lung Cells Produce a Melanization-Stimulating Activity That Promotes Melanocytic Differentiation of Avian Neural Crest Cells

We found previously that neural crest cells in turtle embryos migrated into the lung buds and melanocytes were located in the lungs. The finding suggested to us that the lungs provide a stimulatory factor(s) to the differentiation of neural crest cells into melanocytes. We have established lung cell lines to facilitate analysis of the interactions of neural crest cells with the environment in melanocyte development. One cell line, TLC-2, was found to produce a putative melanization-stimulating activity (MSA), which promoted the melanocyte differentiation in vitro of avian neural crest cells. The TLC-2-derived MSA was different from that of basic fibroblast growth factor (bFGF), α-melanocyte stimulating hormone (α-MSH), and steel factor (SLF). Its molecular weight was estimated to be within the range of 150 kD. Our findings suggest that MSA may be a novel factor exercising a positive control over melanocyte differentiation.     

Steel factor is required for maintenance, but not differentiation, of melanocyte precursors in the neural crest.

Skin melanocytes are derived from neural crest cells that migrate into the dermis during embryogenesis. Two mouse mutants, Steel and White dominant-spotting, which have defects in melanocyte production, have recently been shown to have deletions in the genes that code for a new growth factor, steel factor (SLF), and its receptor, respectively. Here, we have investigated the role that SLF plays in melanogenesis using cultures of mouse neural crest and found that its primary action is the maintenance of melanocyte precursors. It has no effect on the final stage of melanocyte differentiation, the production of melanin, which appears to require an additional factor whose action is mimicked by the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate).     

Crosstalk in skin: melanocytes,keratinocytes, stem cells,and melanoma

In the vertebrate embryo, melanocytes arise from the neural crest, migrate to and colonize the basal layer within the skin and skin appendages. Post-migratory melanocytes are securely attached to the basement membrane, and their morphology, growth, adhesion, and migration are under control of neighboring keratinocytes. Melanoma is a malignant tumor originated from melanocytes or their progenitor cells. During melanocyte transformation and melanoma progression, melanocytes lose their interactions with keratinocytes, resulting in uncontrolled proliferation and invasion of the malignant cells. Melanoma cells at the advanced stages often lack melanocytic features and resemble multipotent progenitors, which are a potential melanocyte reservoir in human skin. In this mini-review, we will summarize findings on cell-cell interactions that are responsible for normal melanocyte homeostasis, stem cell self-renewal, and differentiation. Our ultimate goal is to define molecules and pathways, which are essential for normal cell-cell interactions but deregulated in melanoma formation and progression.     

Induction of Melanogenesis by Tetradecanoylphorbol‐13 Acetate and Endothelin 3 in Embryonic Avian Peripheral Nerve Cultures

In this study, we have analyzed the melanogenic potential of Schwann cells using in vitro cell cultures of embryonic quail peripheral nerves. It is shown that in Schwann cells, two factors, 12‐O‐tetradecanoylphorbol‐13 acetate (TPA) and endothelin 3, trigger a differentiation pathway toward melanocytes, and that Steel factor has no effect on these cells unless treated simultaneously with TPA. In these cultures, TPA induces the expression of c‐kit, whereas Steel factor enhances the development of melanocytes. In the assay system we employed, neither neuronal nor catecholaminergic phenotypes were obtained, regardless of various combinations of related factors added to the culture medium. These data support our previous observations indicating the existence of bipotent progenitors that are capable of differentiating into Schwann cells or into melanocytes, and the regulatory role of endothelin 3 on those precursors, as revealed by the clonal culture of neural crest cells.     

Wnt and BMP signaling govern lineage segregation of melanocytes in the avian embryo

Recent studies show that specification of some neural crest lineages occurs prior to or at the time of migration from the neural tube. We investigated what signaling events establish the melanocyte lineage, which has been shown to migrate from the trunk neural tube after the neuronal and glial lineages. Using in situ hybridization, we find that, although Wnts are expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, the Wnt inhibitor cfrzb-1 is expressed in the neuronal and glial precursors and not in melanoblasts. This expression pattern suggests that Wnt signaling may be involved in specifying the melanocyte lineage. We further report that Wnt-3a-conditioned medium dramatically increases the number of pigment cells in quail neural crest cultures while decreasing the number of neurons and glial cells, without affecting proliferation. Conversely, BMP-4 is expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, but is decreased coincident with the timing of melanoblast migration. This expression pattern suggests that BMP signaling may be involved in neural and glial cell differentiation or repression of melanogenesis. Purified BMP-4 reduces the number of pigment cells in culture while increasing the number of neurons and glial cells, also without affecting proliferation. Our data suggest that Wnt signaling specifies melanocytes at the expense of the neuronal and glial lineages, and further, that Wnt and BMP signaling have antagonistic functions in the specification of the trunk neural crest.