Pure Melanocyte Cultures Research Articles

Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin by Eisinger and Marko

Melanocytes are pigment producing cells that arise from the neural crest and migrate to the skin early in fetal development. The pigment that melanocytes synthesize, melanin, plays a critical role in protecting the skin from mutagenic ultraviolet irradiation. Melanocytes are also precursor cells for melanoma, a deadly form of skin cancer. Because melanocytes make up a minority population of cells in the epidermis they have been difficult to propagate in culture. The landmark paper by Eisinger and Marko, described below, was the first successful report of large scale propagation of pure cultures of melanocytes. This paper set the stage for an explosive growth in knowledge in the biology of human melanocytes and allowed scientists to begin dissecting the different oncogenic events involved in the transition of melanocytes to melanoma.

Melanocytes—A novel tool to study mitochondrial dysfunction in Duchenne muscular dystrophy

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal–epidermal junction. In addition the full-length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments. J. Cell. Physiol. 228: 1323–1331, 2013. © 2012 Wiley Periodicals, Inc.

Pax3GFP, a new reporter for the melanocyte lineage, highlights novel aspects of PAX3 expression in the skin

The paired box gene 3 (Pax3) is expressed during pigment cell development. We tested whether the targeted allele Pax3(GFP) can be used as a reporter gene for pigment cells in the mouse. We found that enhanced green fluorescent protein (GFP) can be seen readily in every melanoblast and melanocyte in the epidermis and hair follicles of Pax3(GFP/+) heterozygotes. The GFP was detected at all differentiation stages, including melanocyte stem cells. In the dermis, Schwann cells and nestin-positive cells of the piloneural collars resembling the nestin-positive hair follicle multipotent stem cells exhibited a weaker GFP signal. Pigment cells could be purified by fluorescent activated cell sorting and grown in vitro without feeder cells, giving pure cultures of melanocytes. The Schwann cells and nestin-positive cells of the piloneural collars were FACS-isolated based on their weak expression of GFP. Thus Pax3(GFP) can discriminate distinct populations of cells in the skin.

In Vitro Dedifferentiation of Melanocytes from Adult Epidermis

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.

Human adult epidermal melanocytes cultured without chemical mitogens express the EGF receptor and respond to EGF

We describe a novel chemical mitogen-free in vitro culture technique for obtaining pure melanocyte cultures using normal human adult epidermis as a source. The culture medium consists equal parts of the commercially available Keratinocyte Basal and AIM-V media (both from Gibco), as basal medium, which is supplemented with fetal bovine serum, bovine pituitary extract and recombinant human epidermal growth factor (EGF). Melanocytes harvested from human adult skin proliferate extensively and can be passaged serially up to 10-15 times using this medium. We have verified the identity of the cultured cells by tyrosinase mRNA expression and TRP-1 protein staining. Moreover, we showed that autologous human serum alone, without additional supplements is able to provide sufficient growth support for the cultured cells in the basal medium, making this culture technique suitable for autologous melanocyte transplantation. In this culture system normal human adult melanocytes expressed both EGF receptor (EGFR) mRNA and protein and EGF showed a dose dependent mitogenic effect on the cells. EGF itself had no significant influence on EGFR mRNA expression.

Hypopigmenting agents: an updated review on biological, chemical and clinical aspects

An overview of agents causing hypopigmentation in human skin is presented. The review is organized to put forward groups of biological and chemical agents. Their mechanisms of action cover (i) tyrosinase inhibition, maturation and enhancement of its degradation; (ii) Mitf inhibition; (iii) downregulation of MC1R activity; (iv) interference with melanosome maturation and transfer; (v) melanocyte loss, desquamation and chemical peeling. Tyrosinase inhibition is the most common approach to achieve skin hypopigmentation as this enzyme catalyses the rate-limiting step of pigmentation. Despite the large number of tyrosinase inhibitors in vitro, only a few are able to induce effects in clinical trials. The gap between in-vitro and in-vivo studies suggests that innovative strategies are needed for validating their efficacy and safety. Successful treatments need the combination of two or more agents acting on different mechanisms to achieve a synergistic effect. In addition to tyrosinase inhibition, other parameters related to cytotoxicity, solubility, cutaneous absorption, penetration and stability of the agents should be considered. The screening test system is also very important as keratinocytes play an active role in modulating melanogenesis within melanocytes. Mammalian skin or at least keratinocytes/melanocytes co-cultures should be preferred rather than pure melanocyte cultures or soluble tyrosinase.

Culture of normal adult human melanocytes.

Melanocytes isolated from normal adult human skin were cultured in vitro. Separation of the epidermis from the dermis by trypsin flotation proved better than collagenase treatment for providing viable cultures of melanocytes with a minimum of fibroblast contamination. Centrifugation on a discontinuous, rather than a continuous Percoll gradient, was more efficient in separating the epidermal cell types. Most of the melanocytes were usually found in one particular layer, and most of the viable keratinocytes were in the sediment. None of the layers produced a uniformly high percentage of melanocytes on routine culture, but enriched melanocyte cultures could be obtained by seeding the epidermal cells in magnesium- and calcium-free medium for 24 to 48 hours, and then transferring them to fibroblast-conditioned medium containing horse serum and polyamines. Melanocytes were identified by their dendritic morphology, ultrastructure, reaction to cholera toxin and pigment production after treatment with melanocyte stimulating hormone. Pure cultures of melanocytes have been cultivated by this method for more than 43 weeks (ten passages).

Keratinocytes and dermal factors activate CRABP-I in melanocytes.

Recognition that cellular retinoic acid binding protein (CRABP)-I and CRABP-II are found in different cell types has provided additional support for the presumably divergent roles of these two proteins in mediating retinoic acid (RA) effects in human skin. CRABP-II is expressed in fibroblasts and keratinocytes, and CRABP-I in as yet unidentified cells, possibly epidermal melanocytes. Recently, we demonstrated that each of these RA-binding proteins in human skin possesses two classes of binding sites, possibly related to the state of phosphorylation of the proteins. We now characterize the cutaneous origin of CRABP-I further using an anion-exchange HPLC assay that allows effective separation of the two proteins in human skin, and a fluorescent in situ hybridization technique. We report that CRABP-I is expressed in isolated melanocytes at the mRNA level, although under these circumstances the protein has minimal RA-binding activity, and that keratinocytic and dermal influences are required for CRABP-I activity in melanocytes. This melanocyte origin for CRABP-I and the improvement by RA of the irregular hyperpigmentation associated with photoaging led us to examine the effects of RA using various cellular associations, from conventional pure cultures of melanocytes grown on plastic dishes to a pigmented skin equivalent consisting of melanocytes and keratinocytes grown on a dermal equivalent. We established that the inhibitory effects of RA on melanogenesis do not result from a direct effect on melanocytes alone but also involve keratinocytes and dermal influence. These data expand our understanding of cell-to-cell signaling in cutaneous pigmentation, and strongly suggest a role for CRABP-I in mediating RA effects on melanogenesis.

Isolation of enriched human melanocyte cultures from fetal, newborn and adult skin

Since melanocytes replicate slowly compared to other skin cells, it has been common practice to isolate pure melanocytes early, prior to overgrowth by fibroblasts and keratinocytes. Contact with other skin cells in culture such as keratinocytes and fibroblasts, however, can enhance melanocyte viability and promote melanocyte replication in the presence of mitogens. We routinely observed that melanocytes appeared earlier and were cultured more efficiently using primary cultures of ‘mixed’ skin cells in mitogen-containing growth medium. A method incorporating dermo-epidermal separation with thermolysin prior to epidermal digestion with trypsin proved most reliable in isolating melanocytes in mixed culture from neonatal skin samples. Pure melanocyte cultures were subsequently obtained from the primary co-culture by applying traditional separation techniques with minor modifications.

Successful culture of adult human melanocytes obtained from normal and vitiligo donors.

The development of growth conditions for human melanocytes from uninvolved skin from vitiligo patients and age-matched normal adults is a prerequisite to understanding the etiology of this pigmentary disorder. By using new growth conditions, pure melanocyte cultures were prepared from normal adults and the pigmented skin of vitiligo donors. Both cell types grew without a lag period and were maintained for more than six months (4-8 passages). The cultures could be expanded from several thousand melanocytes in the original cell suspension to several million cells (2-7 x 10(6] in pure culture. To obtain these results, the current standard conditions for the culture of fetal melanocytes were substantially modified. MEM-S medium was less satisfactory than MCDB-153. Adult normal and vitiligo cells also required the presence of exogenous catalase (20 micrograms/ml) during isolation and primary seeding. Thereafter, this enzyme was not necessary. Melanocytes grew best and gave the highest yields if the concentrations of both calcium (200 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) were low (4-8 nM). Other growth factors included in the MCDB-153 media were bFGF, crude bovine pituitary extract, insulin, transferrin, hydrocortisone, 5% FCS, and the antioxidant alpha-tocopherol. Cholera toxin and isobutylmethylxanthine (IBMX) were omitted from the MCDB-153 growth medium because they slowed down growth even at very low concentrations. The results indicate adult and vitiligo melanocytes can be cultured. Preliminary studies of the normal and vitiligo cells indicate that vitiligo melanocytes retain some of the ultrastructural abnormalities observed in skin even when grown in culture.

Growth characteristics of human epidermal melanocytes in pure culture with special reference to genetic differences

Human melanocyte cultures were established using disaggregated epidermal cell suspensions derived from foreskins and plated onto culture dishes in medium containing 2% fetal bovine serum, growth factors, hormones, and melanocyte growth factor (MGF) extracted from bovine hypothalamus (Wilkins et al., J.Cell. Physiol., 122:350, 1985). After 2 days in culture the cells were transferred to serum-free medium to eliminate keratinocyte and fibroblast growth. Melanocytes grew preferentially and pure melanocyte populations could be harvested after 12-16 days in vitro. Melanocytes were later subcultured in the presence of 1% FBS. Pure melanocyte cultures were characterized by light and electron microscopic criteria, as well as by cytochemical demonstration of the melanocyte-specific enzyme, tyrosinase. At the ultrastructural level, cultured melanocytes derived from black (negroid) neonatal skin (B-M) had numerous mature rod-shaped stage IV melanosomes, while white (caucasoid) skin-derived melanocytes (W-M) in culture contained no mature melanosomes. Growth rate, cell yield, and in vitro lifespan for B-M were more than twice that for W-M in pure melanocyte cultures in the presence of MGF. Our results suggest that MGF-dependent growth of B-M differs from that of W-M.

Efficient culturing of human melanocytes from suction blisters.

The culturing of normal human melanocytes from the roof of suction blisters of adult oriental volunteers was carried out. Since the epidermal roofs of blisters did not contain any fibroblasts, and since keratinocytes did not attach to the culture dishes in the presence of PMA (phorbol 12-myristate 13-acetate), many melanocytes were obtained which grew well in the presence of PMA. This method is a very simple and easy way to establish pure melanocyte cultures.

Purification of human melanocytes by monoclonal antibody combined with Percoll TM gradients

Cultures of human melanocytes obtained by differential plating of human epidermal cells in 12-myristate 13-acetate (PMA) were purified using a monoclonal antibody R 24 which detects a restricted glycolipid antigen present on melanoma cells and melanocytes. Melanocytes were rosetted using protein A-conjugated human red blood cells and separated from non-rosetted ftbroblasts on discontinuous Percoll ™ gradients. 100% pure cultures of melanocytes obtained in this fashion were then successfully grown in tissue culture in the presence of PMA and cholera toxin for at least thirty passages (corresponding to approx. sixty cell doublings). We conclude that: 1. 1. Pure cultures of melanocytes are a prerequisite for the establishment of long-term cultures. 2. 2. Since most human melanomas express substantial levels of R 21 antigen, this method can be applied to purification of melanomas and can be easily adapted to separation of subpopulations of melanocytes and melanoma cells recognized by specific monoclonal antibodies.

Keratinocyte substrate adhesion is magnesium-dependent and calcium-independent

Primary cell cultures were prepared from guinea pig epidermis and six days old mice kidneys and hearts. Along with subcultures of Hep cells, plating experiments were performed using media devoid of bivalent cations containing dialzyed fetal calf serum. Upon stepwise supplementation with either calcium ++ or magnesium ++, keratinocytes displayed a unique dependency on magnesium ++ for cell attachment, whereas all other cell types could make use of either of these ions. Melanocytes, being a minority component of the epidermal cell population, do not conform to the predilection of keratinocytes for magnesium ++. Epidermal cell cultures prepared in media containing only calcium ++, therefore, result in pure melanocyte cultures.