Phenotypic plasticity of melanocytes derived from human adult skin

We previously described a novel in vitro culture technique for dedifferentiated human adult skin melanocytes. Melanocytes cultured in a defined, cholera toxin and PMA free medium became bipolar, unpigmented, and highly proliferative. Furthermore, TRP-1 and c-Kit expression disappeared and EGFR receptor and nestin expression were induced in the cells. Here, we further characterized the phenotype of these dedifferentiated cells and by comparing them to mature pigmented melanocytes we detected crucial steps in their phenotype change. Our data suggest that normal adult melanocytes easily dedifferentiate into pluripotent stem cells given the right environment. This dedifferentiation process described here for normal melanocyte is very similar to what has been described for melanoma cells, indicating that phenotype switching driven by environmental factors is a general characteristic of melanocytes that can occur independent of malignant transformation.     

Cultured Human Melanocytes Respond to MSH Peptides and ACTH

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that α-MSH and its synthetic analogue Nle⁴DPhe⁷α-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.     

Keratinocytes suppress TRP-1 expression and reduce cell number of co-cultured melanocytes - implications for grafting of patients with vitiligo

A pilot study for grafting of patients with vitiligo using cultured epithelial autografts containing melanocytes gave disappointing clinical results, with pigmentation achieved in only one out of five patients. Irrespective of the fate of melanocytes grafted back onto the patients, we experienced problems in identifying melanocytes within these well-integrated keratinocyte sheets. This led us to explore the fate of these cells within these sheets in vitro and to seek to improve their number and function within the sheets. We report that the introduction of a fibroblast feeder layer can improve melanocyte number within melanocyte/keratinocyte co-cultures initially, but at very high keratinocyte density, there is a marked loss of melanocytes (as detected by staining for S100). Additionally, we found that keratinocytes not only down-regulate melanocyte number, but also pigmentary function; thus, it was possible to identify melanocytes that were S100 positive but tyrosinase-related protein-1 (TRP-1) negative in confluent well-integrated keratinocyte sheets. In summary, our data suggest that keratinocytes at high density initially suppress melanocyte pigmentation (as evidenced by a lack of TRP-1 expression) and then cause a physical loss of melanocytes. The introduction of a fibroblast feeder layer can help maintain melanocyte number while keratinocytes are subconfluent, but fails to oppose the inhibitory influence of the keratinocytes on melanocyte TRP-1 expression.     

Effect of keratinocytes on regulation of melanogenesis in culture of melanocytes

Melanocytes are the melanin-producing cells by melanogenesis, and the pigment melanin is primarily responsible for the color of skin. These cells contain dendrites that are in close contact with neighboring keratinocytes. Keratinocytes produce and secrete factors that regulate the proliferation and melanogenesis of melanocytes in vitro. Therefore, adopting only melanocyte pure culture may not clearly reflect the skin physiology in vivo. In this study, we applied a two-culture model using melanocytes and keratinocytes from human skin, such as melanocyte pure culture and melanocyte co-culture with keratinocyte. And then, there was compared the responses of melanocytes under different culture conditions (treatment with arbutin, MSH-α and UV-B irradiation). The results show that there was no significant difference in melanocyte proliferation and melanogenesis between arbutin and MSH-α treatment. However, the co-culture model was more stable than the pure culture model in terms of melanocyte proliferation and melanogenesis upon UV-B irradiation. Therefore, the co-culture model was superior to the pure culture as a useful method for the study of melanocytes and epidermal melanin unit.     

Primary culture of human face skin melanocytes for the study of hyperpigmentation

Facial epidermal pigmentation and skin tumors can be caused by UV exposure and other physical and chemical irritations. In this report we describe the primary culture of melanocytes from human face skin. The ability to culture these melanocytes will enable their morphological and biological properties to be investigated. Skin specimens were obtained from patients who had undergone lower blepharoplasty procedures. Digestion with neutral protease and trypsin was used to obtain single cell suspensions of epidermal cells. The cells were cultured in M254 medium supplemented with human melanocyte growth solution. Cell morphology was observed using inverted microscopy. Melanocytes were positively identified using both l-DOPA staining and S-100 protein immunohistochemical staining. Immunofluorescence was used to confirm the expression of tyrosinase-related protein-1, a melanocyte-specific protein. The cellular ultrastructure of the melanocytes was observed by transmission electron microscopy. The cultured human melanocytes from face skin were multi-dendritic, and many mature melanosomes were observed. Therefore, using a specific culture medium, melanocytes from face skin can be successfully cultured and made available for further investigations.     

DNA microarrays and likelihood ratio bioinformatic methods: discovery of human melanocyte biomarkers

In this article, some of the advantages and limitations of DNA microarray technologies for gene expression profiling are summarized. As a model experiment, DermArray DNA microarrays were utilized to identify potential biomarkers of cultured normal human melanocytes in two different experimental comparisons. In the first case, melanocyte RNA was compared with vastly dissimilar non-melanocytic RNA samples of normal skin keratinocytes and fibroblasts. In the second case, melanocyte RNA was compared with a primary cutaneous melanoma line (MS7) and a metastatic melanoma cell line (SKMel-28). The alternative approaches provide dramatically different lists of 'normal melanocyte' biomarkers. The most robust biomarkers were identified using principal component analysis bioinformatic methods related to likelihood ratios. Only three of 25 robust biomarkers in the melanocyte-proximal study (i.e. melanocytes vs. melanoma cells) were coincidentally identified in the melanocyte-distal study (i.e. melanocytes vs. non-melanocytic cells). Selected up-regulated biomarkers of melanocytes (i.e. TRP-1, melan-A/MART-1, silver/Pmel17, and nidogen-2) were validated by qRT-PCR. Some of the melanocytic biomarkers identified here may be useful in molecular diagnostics, as potential molecular targets for drug discovery, and for understanding the biochemistry of melanocytic cells.     

The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.     

Coordinated mRNA and Protein Expression of Human LAMP-1 in Induction of Melanogenesis After UV-B Exposure and Co-Transfection of Human Tyrosinase and TRP-1 cDNAs

In order to better understand the cascade of melanogenic events in melanocytes, this report has introduced our two recent approaches for the expression of melanogenesis/or melanosome-associated genes and encoded proteins in melanocytes (melanoma cells) after repeated exposure to UV -B and after cotransfection of two human genes, i.e., tyrosinase and tyrosinase-related protein-1 (TRP-1). Repeated exposure of UV B (2.5–5.0 mJ/cm²) caused not only upregulation of tyrosinase and TRP-1 genes but also coordinated increase in the gene and protein synthesis expression of Lamp-1 (lysosome-associated membrane protein-1). When COS-7 kidney cells and amelanotic melanoma (C32 and SKMEL-24) and melanotic melanoma (G361 and SK-MEL-23) cells were exposed to cotransfection of human tyrosinase and TRP-1 cDNAs, there was also an increased expression of Lamp-1 mRNA and protein along with tyrosinase activation and new melanin synthesis. Importantly, single transfectants of human tyrosinase cDNA revealed marked cellular degeneration, whereas this degeneration was not seen in single transfectants of TRP-1 cDNA or cotransfectants of human tyrosinase and TRP-1 cDNAs, indicating that TRP-1 prevented, along with Lamp-1, programmed death of melanocytes after transfection of tyrosinase gene. The coordinated expression of TRP-1 and Lamp-1 was further confirmed by antisense oligodeoxynucleotide hybridization experiment against Lamp-1 gene, showing the decreased expression of TRP-1 as identified by three different types of anti-TRP-1 monoclonal antibodies. We propose therefore that human tyrosinase and TRP-l, when activated or expressed together, will coordinate to upregulate the mRNA expression and protein synthesis of Lamp-1. The Lamp-1 molecules will, in turn, cover the inner surface of melanosomal membrane, together with TRP-1 molecules, thus protecting the melanosomal membrane from toxic melanin intermediates generated during melanogenesis in the presence of active tyrosinase. In contrast, the expression of other lysosome-related proteins, e.g., β-galactosidase and CD63 is not stimulated in new melanogenesis.     

Extracellular matrix modulates the function of human melanocytes but not melanoma cells

Normal human epidermal melanocytes are attached to a basement membrane, a specialized form of extracellular matrix (ECM), located between the epithelium and underlying dermal tissues. To determine whether ECM influences pigmented cell behavior in vitro, human epidermal melanocytes and melanoma cells were cultured on uncoated or ECM-coated plastic culture surfaces, and a comparison was made between growth and function in the presence or absence of ECM. Melanocytes cultured on ECM-coated surfaces developed flatter and larger cell bodies and produced more melanin than melanocytes cultured on uncoated surfaces. In the presence of phorbol-myristate-acetate and cholera toxin, the rate of melanocyte replication was increased by ECM. In the absence of these mitogens, ECM significantly enhanced the adhesiveness of nonproliferating melanocytes. ECM had little or no effect on these parameters (morphology, tyrosinase activity, replication) in a pigmented human malignant melanoma cell line. These findings indicate that normal human epidermal pigment cells have the ability to recognize and respond to matrix signals, whereas this capacity appears to be absent in melanoma cells.     

Effects of genic substitution at the pink-eyed dilution locus on the proliferation and differentiation of mouse epidermal melanocytes in vivo and in vitro

Cells positive to the dopa reaction (melanocytes) as well as to the combined dopa-premelanin reaction (melanoblasts and melanocytes) in the epidermis of C57BL/10JHir-p/p (pink-eyed dilution) mice were fewer and less reactive than in C57BL/10JHir (black, P/P) mice, suggesting that the proliferation and differentiation of p/p melanocytes are inhibited. To confirm the inhibitory effects of p gene on the proliferation and differentiation of epidermal melanocytes, we cultured epidermal cell suspensions of neonatal skins from P/P and p/p in a serum-free medium. The proliferation and differentiation of p/p melanoblasts/melanocytes in primary culture were greatly inhibited as compared to P/P melanoblasts/melanocytes. The morphology of p/p melanoblasts/melanocytes cultured in melanocyte growth medium, though non-pigmented, was similar to P/P melanocytes; namely, dendritic, polygonal, or epithelioid. About 8% of p/p cells cultured in melanocyte growth medium were positive to the dopa reaction, and about 25% were reactive to the combined dopa-premelanin reaction. Eumelanin content in p/p was extremely reduced compared to P/P. The immunocytochemical staining of p/p melanoblasts/melanocytes revealed that they are negative to tyrosinase, but reactive to tyrosinase-related protein (TRP)-1, TRP-2, and c-kit. However, the reactivities in p/p were lower than in P/P. Although the differentiation of p/p melanoblasts was not induced by endothelin (ET)-1, ET-2, and ET-3, the proliferation of p/p melanoblasts was stimulated by them. These results suggest for the first time that p gene exerts its influence on the proliferative activities of mouse epidermal melanoblasts by affecting the regulatory mechanisms dependent on the function of ETs.     

Human TRP-1 Has Tyrosine Hydroxylase but no DOPA Oxidase Activity

Human TRP-1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP-1 for the two substrates, L-tyrosine and L-DOPA. Immunopurified TRP-1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP-1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP-1 under conditions with low tyrosine concentration (≤0.8 μCi/ml of ³H-tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L-DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase-negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP-1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L-DOPA to the assay. These results suggested that TRP-1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP-1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.     

Melanocyte stimulating hormone induces the differentiation of mouse epidermal melanocytes in serum-free culture.

In serum-free primary culture of dissociated mouse epidermal cells, alpha-melanocyte stimulating hormone (alpha-MSH) and dibutyryl cyclic AMP (DBcAMP) induced the differentiation of melanocytes. Moreover, the proliferation of melanocytes was also induced in the dishes cultured with DBcAMP, but not with alpha-MSH. In order to clarify the role of keratinocytes in melanocyte proliferation and differentiation, pure cultures of keratinocytes were established in serum-free medium. Subconfluent primary keratinocytes were trypsinized and seeded into pure primary melanoblasts cultured with serum-free medium that did not contain alpha-MSH or DBcAMP. Melanoblasts were cultured with alpha-MSH or DBcAMP in the presence or absence of keratinocytes. alpha-MSH failed to induce melanocyte differentiation in the absence of keratinocytes. DBcAMP failed to induce melanocyte proliferation in the absence of keratinocytes, although it induced melanocyte differentiation even in the absence of keratinocytes. These results suggest that keratinocyte-derived factors are required not only for the induction of melanocyte differentiation by alpha-MSH but also for the induction of melanocyte proliferation by DBcAMP.