Abstract
In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation.
Highlights
IntroductionMelanocytes arise from the neural crest [1]. Melanoblasts are unpigmented cells containing only immature melanosomes that lack functional tyrosinase, the critical enzyme of melanin synthesis [2]
In vertebrates, melanocytes arise from the neural crest [1]
Melanocytes cultured in media that contains phorbol 12-myristate 13-acetate (PMA), cholera toxin (CT) or in some cases 3isobutyl-1-methylxanthine (IBMX) show the phenotype of fully differentiated melanocytes
Summary
Melanocytes arise from the neural crest [1]. Melanoblasts are unpigmented cells containing only immature melanosomes that lack functional tyrosinase, the critical enzyme of melanin synthesis [2]. Examination of melanoblasts is important to analyze basic mechanisms of cell differentiation, and to study the pathomechanisms of melanoma and genetic disorders of melanocyte development [4]. Melanocyte differentiation is under the control of microphthalmia transcription factor (MITF), a basic helix-loop-helix leucine zipper transcription factor, that activates genes involved in pigment production, such as TYR, TRP-1 and TRP-2[5] and melanocyte survival, e.g. Bcl-2 [5,6]. Towards the completion of hair follicle morphogenesis, several distinct follicular melanocytic cell populations were defined: undifferentiated, nonpigmented c-Kit-negative melanoblasts in the outer root sheath and bulge and highly differentiated melanocytes adjacent to the hair follicle dermal papilla above the Auber’s line [12]. Autocrine SCF stimulation of Kit receptor seems to be an important step in melanoma genesis in its early phases, but it is down-regulated in later stages [13,14]
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