Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue

Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes.     

Expression of cell surface sialic acid and galactose by normal adult human melanocytes in culture

Normal adult human melanocytes grown either in the presence of phorbol ester or dialyzed hypothalamic extract were analyzed for their cell surface sialic acid and galactose content. In both cases, cells expressed large amounts of sialic acid, whereas they differed in their terminal nonreducing beta-D-galactosyl residues linked to N-acetyl galactosamine; such residues were accessible to peanut agglutinin and Bauhinia purpurea lectin on cells grown in phorbol ester and inaccessible on cells grown with dialyzed hypothalamic extract. In addition, striking differences in morphology and growth characteristics were observed between adult melanocytes grown with phorbol ester or with dialyzed hypothalamic extract. Thus, pure cultures of normal adult human melanocytes grown in the presence of dialyzed hypothalamic extract displayed cell surface properties different from those of melanocytes grown with phorbol ester. Cultures of melanocytes with dialyzed hypothalamic extract are likely to reflect known cell surface characteristics of human melanocytes in the skin. Such cultures could represent a useful model to study normal behavior and tumor progression of pigmented cells.     

Cellular and hormonal regulation of pigmentation in human ocular melanocytes

The purpose of this study was to examine some of the factors that may be relevant to regulating pigmentation in the human eye, specifically whether choroidal and iridial melanocytes are sensitive to regulation by epithelial and stromal cells and alpha-melanocyte stimulating hormone (alpha-MSH). Human choroidal and iridial melanocytes were established in culture and co-cultured with epithelial cells and stromal cells derived both from skin and from eye in order to determine their influence on choroidal and iridial melanocyte dopa oxidase activity. In all cases, co-culture of melanocytes with either epithelial cells or fibroblasts led to an increase in dopa oxidase activity during 5 days of co-culture. The extent of the increase ranged from 60% (non-significant) to as much as 185% when both fibroblasts and keratinocytes were present. The optimal ratio of fibroblasts to melanocytes was 1:10 (for dermal fibroblasts) or 1:2 (for iridial fibroblasts) and 1:1 for all epithelial cells to melanocytes. Both choroidal (three out of three cultures) and iridial (two out of three cultures) melanocytes showed increases in dopa oxidase activity to alpha-MSH when cultured in Green's media but the same cells cultured in MCDB153 were unresponsive to alpha-MSH. These in vitro studies suggest that ocular melanocytes have the capacity to be influenced by adjacent epithelial and stromal cells with respect to pigmentation.     

The Proteolytic Potential of Normal Human Melanocytes: Comparison With Other Skin Cells and Melanoma Cell Lines

To understand the contribution of epidermal melanocytes in the proteolytic potential of human skin, we have studied melanocytes grown in a low-serum medium deprived of phorbol esters, cholera toxin, and other non-physiological supplements. We focused on the plasminogen activation system and certain matrix metalloproteinases (gelatinases). Supposing that the proteolytic activity of cells can influence binding to collagen matrix and its reorganization, we have analyzed these parameters as well. We found that human melanocytes secreted tissue-type plasminogen activator and utilised it to generate cell-bound plasmin. No urokinase-type plasminogen activator was detected in the cultures but its receptor was found in cell extracts. Both the 72 kDa and 92 kDa gelatinases were secreted by the cells and in equal amounts. In addition, melanocytes secreted the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Melanocytes cast into collagen matrices retained a rounded morphology, did not extend processes, and were unable to contract collagen lattices. As a control, these parameters were investigated in parallel in cultures of human keratinocytes, dermal fibroblasts, and two melanoma cell lines. The obtained characteristics suggest that normal human melanocytes are proteolytically active cells. This function may pertain to skin physiology and pathophysiology.     

Transmission and scanning electron microscopy and freeze-fracture replication of normal human melanocytes and human melanoma cells in tissue culture

Basic LM, TEM, SEM, and FFR appearances of a pure line of normal human melanocytes derived from foreskin, and a human melanoma line, in cell culture are described. Normal melanocyte cultures exhibit side by side, cells of widely different melanogenic activities--possible clones--and melanosomes of bizarre shape and internal structure are frequent. Aggregates of melanosomes, with or without associated amorphous material, and with no discernible limiting membrane are present within many cells, and occasional simple specialised contacts occur between apposed cells. On replicas of plasma membrane of normal melanocytes, particle densities and diameters on P and E fracture faces were within the ranges for cells in general, and equivalent data for the melanoma cells were not significantly different. Similarly, there was no difference in density of distribution or diameter of nuclear pores between the normal and the tumoural cells.     

Responses of Cultured Melanocytes to Defined Growth Factors

To proliferate in vitro, normal melanocytes, unlike normal fibroblasts, require specific growth factors in addition to those supplied in serum. The substances that promote melanocyte proliferation, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and stimulators of cyclic adenosine monophosphate (cAMP), also promote pigmentation. Consequently, cell division and expression of at least some differentiated functions are not mutually exclusive for melanocytes. At present, the only known natural growth factor that can replace TPA in normal human melanocyte cultures is basic fibroblast growth factor (bFGF). Like TPA, bFGF is effective, most of the time, only in the presence of added cAMP. Some preparations of bFGF, however, may have a highly labile, intrinsinc cAMP stimulatory activity. It is thus possible that bFGF can assume two forms, dependent on and independent of cAMP stimulatory activity. Alternatively, a second factor may exist in pituitary glands that co-purifies with bFGF but deteriorates with storage. Abnormal melanocytes in culture, such as those derived from dysplastic nevi and primary melanomas, depend on the specific factors (bFGF and cAMP), whereas melanocytes from metastatic melanomas do not     

Selective growth and serial passage of mouse melanocytes from neonatal epidermis in a medium supplemented with bovine pituitary extract

Suspensions of disaggregated epidermal cells from skins of newborn C57BL/10JHir mice were plated in a growth medium that consisted of Ham's F-10 plus bovine pituitary extract (BPE), insulin, and transferrin. Fetal bovine serum (FBS) was added to the culture medium at a concentration of 4% at the time of plating. On the second day of culture, a small number of melanocytes was randomly distributed among large sheets of keratinocytes. From the third day onward, FBS was excluded from the culture medium to prevent the proliferation of keratinocytes and fibroblasts. The melanocytes began to grow preferentially, and after 12 days pure and enriched populations of melanocytes could be harvested. In the absence of the proliferation of keratinocytes and fibroblasts, melanocytes could be serially passaged in the growth medium supplemented with a conditioned medium (CM) prepared from keratinocyte-enriched cultures, namely, those at the early stages of the primary culture. FBS was added at a concentration of 1% for the first day. These results suggest that both BPE and keratinocyte CM contain growth factors required for proliferation of melanocytes.     

Purification of human melanocytes by monoclonal antibody combined with Percoll 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 R₂₄ 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 R₂₁ 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.

     

Hormonal effects of vitamin D3 on epidermal melanocytes

The role of cholecalciferol, 25(OH) D3, and 1,25(OH)2 D3, as modulators of melanocyte function and proliferation has been examined. Topical application of 100 micrograms cholecalciferol to the pinnal epidermis of DBA/2J mice for 5 or 10 days increased the number of L-dihydroxyphenylalanine-positive (DOPA-positive) melanocytes and had a synergistic effect with a low dose of ultraviolet B light (UVB). Application of 1 microgram 1,25(OH)2 D3 had a transient effect on epidermal melanocytes. Addition of cholecalciferol to pure cultures of human melanocytes did not alter their tyrosinase activity (therefore, melanin synthesis) or growth rate even after 72 hours of treatment. However, treatment of similar cultures with 1,25(OH)2 D3 at a concentration equal to or greater than 10(-8) M suppressed tyrosinase activity but did not affect proliferation. The effect of 25(OH) D3 was similar to, but lower in magnitude than, that of 1,25(OH)2 D3. We attempted to demonstrate the presence of specific receptors for 1,25(OH)2 D3 in normal human melanocytes using the monoclonal antibody (Mo Ab) 9A7 gamma raised against the receptor for 1,25(OH)2 D3. Melanocytes were exposed to 9A7 gamma and to a secondary biotinylated Ab and analyzed by the fluorescence activated cell sorter (FACS). An increase in the specific fluorescent signal was constantly observed. By using the immunoblotting technique, we observed a major immunoreactive species that migrated in the 53-kD region in normal melanocytes. The size of this major immunoreactive species was smaller in melanoma cells than in normal melanocytes. This correlates with the finding that the former cells were unresponsive to cholecalciferol, 25(OH) D3, or 1,25(OH)2 D3 treatment. These results predict a direct role for 1,25(OH)2 D3 as an effector of normal melanocyte function.     

Wnt inhibitory factor (WIF)‐1 promotes melanogenesis in normal human melanocytes

Wnt signaling plays a role in the differentiation as well as the development of melanocytes. Using a microarray analysis, hyperpigmentary skin of melasma expressed high levels of Wnt inhibitory factor‐1 (WIF‐1) compared with perilesional normal skin. In this study, the expression and functional roles of WIF‐1 on melanocytes were investigated. WIF‐1 was expressed both in the melanocytes of normal human skin and in cultured melanocytes. The upregulation of WIF‐1 on cultured normal human melanocytes significantly induced expressions of MITF and tyrosinase, which were associated with increased melanin content and tyrosinase activity. Consistent with the stimulatory effect of WIF‐1, WIF‐1 siRNA reduced melanogenesis in the cells. Moreover, WIF‐1 increases pigmentation in melanocytes co‐cultured with WIF‐1‐overexpressed fibroblasts and of organ‐cultured human skin. These findings suggest that melanocytes express WIF‐1 constitutively in vivo and in vitro and that WIF‐1 promotes melanogenesis in normal human melanocytes.     

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.     

In vivo and in vitro evidence of dermal fibroblasts influence on human epidermal pigmentation

Using chimeric human epidermal reconstructs, we previously demonstrated that epidermal pigmentation is dependent upon the phototype of melanocytes. We report here several lines of experimental evidence for dermal modulation of human epidermal pigmentation. First, phototype II-III epidermal reconstructs grafted on the back of immunotolerant Swiss nu/nu mice developed a patchy pigmentation dependent on the presence of colonizing human or mouse fibroblasts. Similarly, human white Caucasoid split-thickness skin xenografted on the same mouse strain became black within 3 months and histochemistry revealed a phototype VI pattern of melanin distribution. In vitro, human fibroblasts colonizing human dead de-epidermized dermis (DDD) induced a decrease in epidermal pigmentation whereas mouse (Swiss nu/nu) fibroblasts increased epidermal pigmentation. Conditioned medium from mice (Swiss nu/nu) fibroblasts also increased pigmentation whereas conditioned medium from human fibroblasts had no significant effect. Lastly, epidermal reconstructs made with normal or vitiligo keratinocytes and/or normal or vitiligo melanocytes from the same donor grown on DDD originating from several donors of the same clinical phototype did not pigment similarly and no specific dermal influence was noted for vitiligo. Thus, fibroblast secretion and acellular dermal connective tissue itself significantly influence melanocyte proliferation and melanin distribution/degradation. Our study suggests that murine fibroblasts are more potent than human fibroblasts in secreting soluble factors which can act directly on pigmentation, such as SCF, or activate keratinocytes to produce basement membrane proteins or melanogenic factors.     

Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes

Melanoma growth stimulatory activity (MGSA) was originally described as an endogenous growth factor for human melanoma cells. To test the hypothesis that an MGSA autocrine loop is responsible for the partial freedom from growth control observed in nevocytes and melanoma cells, MGSA growth response and MGSA mRNA/protein levels were examined in these cells compared with normal melanocytes. As a single agent, or in combination with other factors, MGSA stimulated the growth of normal human epidermal melanocytes as well as other growth promoters for melanocytes. Nevocytes were not as responsive to exogenous MGSA as melanocytes. MGSA mRNA was minimal or not detected in cultured normal melanocytes, although the protein was present when the cells were cultured in the presence of serum/growth factors and absent when serum/growth factors were omitted. In contrast, MGSA mRNA was constitutively expressed in the absence of exogenous growth factors in cultures established from benign intradermal and dysplastic nevi and melanoma lesions in different stages of tumor progression. Nevus cultures contained immunoreactive MGSA protein in the presence of serum but were negative or only faintly positive in the absence of serum. Melanoma cell lines were positive for MGSA protein in both the presence and the absence of serum. Thus, continued expression of both MGSA mRNA and MGSA protein in the absence of exogenous hormones or serum factors may correlate with partial freedom from growth control exhibited by malignant melanocytes.     

Biomarkers of human skin cells identified using DermArray DNA arrays and new bioinformatics methods

Biomarker genes of human skin-derived cells were identified by new simple bioinformatic methods and DNA microarray analysis utilizing in vitro cultures of normal neonatal human epidermal keratinocytes, melanocytes, and dermal fibroblasts. A survey of 4405 human cDNAs was performed using DermArray DNA microarrays. Biomarkers were rank ordered by "likelihood ratio" algorithms and stringent selection criteria that have general applicability for analyzing a minimum of three RNA samples. Signature biomarker genes (up-regulated in one cell type) and anti-signature biomarker genes (down-regulated in one cell type) were determined for the three major skin cell types. Many of the signature genes are known biomarkers for these cell types. In addition, 17 signature genes were identified as ESTs, and 22 anti-signature biomarkers were discovered. Quantitative RT-PCR was used to verify nine signature biomarker genes. A total of 158 biomarkers of normal human skin cells were identified, many of which may be valuable in diagnostic applications and as molecular targets for drug discovery and therapeutic intervention.     

Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells

The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs) present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes.     

pp60c-src in human melanocytes and melanoma cells exhibits elevated specific activity and reduced tyrosine 530 phosphorylation compared to human fibroblast pp60c-src.

Elevated levels of pp60c-src tyrosine kinase activity have been implicated in both tumorigenesis and cell differentiation. We have found a 2- to 4-fold elevation in pp60c-src specific activity in certain human melanoma cell lines compared to human foreskin fibroblasts. This activation of pp60c-src did not appear to be related to melanoma tumor progression, because when normal human epidermal melanocytes were examined, it was found that they contained pp60c-src having a 7-fold elevation in specific activity compared to pp60c-src from human fibroblasts. It was determined that pp60c-src from melanocytes was not the neuronal form, pp60c-src+. Melanocyte pp60c-src exhibited a reduced level of phosphorylation on its carboxyl-terminal regulatory site, tyrosine 530, which might be responsible for its elevated specific activity. These results suggest that, in melanocytes, regulation of tyrosine 530 phosphorylation-dephosphorylation favors activation of pp60c-src. This activation may be involved in the growth, differentiation, or function of human melanocytes.