Substrate ifluences human epidermal melanocyte attachment and sperading in vitro

Summary Previous culture systems for melanocytes have employed serum-supplemented medium and uncoated plastic dishes, prohibiting examination of possible substrate influences on cellular morphology and function. We now report, using a sensitive serum-free system and a quantitative procedure for evaluating cellular morphology, that modification of the plating surface affects human epidermal melanocyte attachment rate and subsequent morphology in vitro. Melanocytes attach and spread more rapidly on surfaces coated with fibronectin or Type I/III collagen or on surfaces previously conditioned by human keratinocytes, dermal fibroblasts, melanocytes, or melanoma cells than do melanocytes on untreated control surfaces. Type IV collagen and laminin, although minimally beneficial for cell attachment, do support a characteristics melanocyte morphology that differs from that seen either on the other coated surfaces or on uncoated plastic controls. Addition of fetal bovine serum at the time of inoculation has no appreciable effect on attachment but markedly improves cell spreading on untreated surfaces, while addition of nerve growth factor with or without serum to this system fails to affect cell attachment or spreading. Our data establish that human epidermal melanocytes are indeed capable of responding morphologically to substrate signals. The ability of several biochemically unrelated surfaces to enhance melanocyte attachment rate and spreading suggests that melanocytes have surface receptors with a variety of specificities. This work is relevant to the development of improved culture systems for melanocytes in vitro and to understanding melanocyte behavior in vivo. This work was supported by the USDA Agricultural Research Service, by a grant from Cheesebrough-Ponds, Inc., and by a Dermatology Foundation Fellowship (Dr. Yaar).     

Regulatory factors that determine growth and phenotype of normal human melanocytes

Normal human melanocytes, unlike malignant melanoma cells, required at least three growth-promoting agents, i.e., phorbol ester for protein kinase C activation and the growth factors basic fibroblast growth factor (bFGF) and insulin, for growth in chemically defined W489 medium. Cell growth was further stimulated by addition of agents that increase intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP) to the medium. Among these agents, the pituitary hormones alpha-melanocyte-stimulating hormone (alpha-MSH) and follicle-stimulating hormone were the most potent, whereas bacterial toxins, including cholera, tetanus, and pertussis toxin and their subunits either were less mitogenic or gave variable results depending on the culture tested. Medium containing phorbol ester PMA, growth factors bFGF and insulin (or insulin-like growth factor-I), and synthetic alpha-MSH supported melanocyte growth for more than 5 months with doubling times between 5 and 8 days. Two copper-binding proteins, ceruloplasmin and tyrosinase, were mitogenic when added to medium and ceruloplasmic induced a long bi- to tripolar-shape of cells. Addition of 1 mM dibutyryl cAMP to the medium led to the formation of dendrites in all cells, with an average of 28 extensions per cell. Although cell growth was inhibited by dibutyryl cAMP, cells were not terminally differentiated and continued to proliferate. Dendritic melanocytes showed a 2.2-fold increase in activity of the tyrosine kinase pp60c-src. The induction of dendritic processes in melanocytes by dibutyryl cAMP or sodium butyrate was reversible and appears to reflect the expression of the mature melanocytic phenotype in situ.     

New procedure for epidermal cell isolation using kiwi fruit actinidin,and improved culture of melanocytes in the presence of leukaemia inhibitory factor and forskolin

Objectives

Conventional isolation of epidermis from the dermis and disruption of epidermal sheets to liberate the cells, are performed using proteolytic enzymes such as thermolysin or collagenase. Selective population expansion of melanocytes is achieved by suppressing proliferation of keratinocytes and fibroblasts in epidermal cell suspensions, using phorbol esters and cholera toxin. Here, we introduce a new procedure for isolation of epidermal cells, using proteolytic activity of kiwi fruit actinidin, and also an improved growth medium for melanocytes in the presence of leukaemia inhibitory factor (LIF) and forskolin.

Materials and methods

Dermo‐epidermal separation and epidermal sheet cell dispersion were performed using actinidin compared to conventional proteases including collagenase, thermolysin or trypsin. Thereafter, melanocyte culture was performed in two common media and one modified medium to discover optimization for these cells.

Results

We found that dermo‐epidermal separation and epidermal sheet cell dispersion using kiwi fruit actinidin were considerably better than previously used methods, both from the aspect of less fibroblast and keratinocyte contamination, and of more viable native cells. Also, melanocytes proliferated better in phorbol ester‐ and cholera toxin‐free proliferation medium supplemented with LIF and forskolin.

Conclusion

Less contamination and higher numbers of viable cells were actinidin preferential for separation of epidermis and isolation of epidermal cells. Supplementation of LIF and forskolin to new medium increased proliferation potential of melanocytes in comparison to exogenous mitogens.

     

Modulation of Normal Human Melanocyte Dendricity by Growth-Promoting Agents

Dendrite formation and extension, which comprise a characteristic morphology of human normal melanocytes in the skin, represent one of the functional activities of melanocytes, the ability to transfer melanosomes into neighboring keratinocytes. However, the morphology of the melanocyte in vitro is usually quite different from that observed in vivo. it is probably due to the hyperproliferative condition of the melanocytes in culture. No studies have ever compared the effects of a single factor on both dendricity and proliferation at the same time. Therefore, we have compared the effects of six growth-promoting agents commonly used for melanocyte cultures on dendrite formation and proliferation. The addition of agents that increase the intracellular levels of cyclic adenosine monophosphate (cAMP)—dibutyryl cyclic adenosine monophosphate (db cAMP; 1 mM) or isobutylmethyl xanthine (IBMX; 0.1 mM)—had a strong effect on dendrite formation and a negative effect on proliferation. This was especially true with db cAMP. In the presence of 2% or 5% of heat-inactivated fetal bovine serum (FBS), dendrite formation was significantly increased as was proliferation. The number of dendrites was decreased in the culture with 12-o-tetradecanoylphorbol-13-acetate (TPA), but cell growth was slightly increased. With human recombinant basic fibroblast growth factor (bFGF) (0.5, 1.0 ng/ml) in the presence of bovine pituitary extract (BPE) (60 μg/ml), cell growth was increased. With 2 ng/ml of bFGF, however, a strong inhibitory effect on proliferation was observed. However, dendrite formation was constant at all concentrations of bFGF tested (0.5, 1.0 or 2.0 ng/ml) with BPE (30 or 60 μg/ml). In this study, we have demonstrated that dendrite formation was suppressed by the reagents that stimulate melanocyte proliferation, and vice versa, with the only exception being heat-inactivated FBS. Both dendrite formation and proliferation were induced by the heat-inactivated FBS. This approach is crucial to the development of an adequate culture system for proliferation and/or dendrite formation of normal human melanocytes. It is necessary to keep these aspects in mind as we further investigate the biology of melanocytes, especially the cell-to-cell interactions between melanocytes and keratinocytes, involved in melanogenesis and melanin pigmentation in vivo. This study also provides practical and important information for a future reconstitutive skin system composed of melanocytes, keratinocytes, and fibroblasts in a single culture medium.     

Isolation and culture of amelanotic melanocytes from human hair follicles

We report a method to establish amelanotic melanocytes (AMMC) in culture and we investigate the effects of various components in the culture medium. Normal human scalp from cadaver donors was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. The individual hair follicles were washed exhaustively and suspensions of hair follicle cells were prepared and cultured in Eagle's minimum essential medium supplemented with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), cholera toxin and keratinocyte serum-free medium (K-SFM). Geneticin was used to eliminate contaminating fibroblasts. Proliferation of AMMC was observed after addition of TPA and K-SFM including bovine pituitary extract (BPE) into the culture medium. Cell type was determined by staining with monoclonal antibodies, NKI/beteb and HMB-45, which recognize premelanosomal and melanosomal antigens, respectively. The AMMC were also examined using transmission electron microscopy. Treatment with geneticin eliminates the majority of fibroblasts and does not impair the growth of keratinocytes or AMMC. After contaminating fibroblasts and keratinocytes were removed, two distinct cell morphologies remained: (1) large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and (2) small bipolar cells, which initially were unpigmented and proliferated very rapidly. We observed that TPA at various concentrations stimulated the proliferation of the cells, and at high concentrations could induce the formation of multiple dendrites. K-SFM including BPE accelerated the proliferation of the cells in a dose-dependent manner. After passage 3, almost all cells expressed premelanosomal and melanosomal antigens, recognized by NKI/beteb and HMB-45, respectively. Active mitochondria, abundant rough endoplasmic reticulum, Golgi complexes, ribosomes and melannosomes (predominantly in stages I, II or III with some at stage IV in some AMMC) were observed ultrastructurally in the cytoplasm of the cultured cells.     

Basic FGF and TGF-beta 1 influence commitment to melanogenesis in neural crest-derived cells of avian embryos

In previous studies, we showed that neural crest (NC)-derived cells from embryonic quail dorsal root ganglia (DRG) and peripheral nerve (PN), which do not normally give rise to melanocytes, become committed to melanogenesis following treatment in culture with the phorbol ester drug 12-O-tetradecanoyl phorbol-13-acetate (TPA). These and other observations support the notion that melanocytes and Schwann cells are derived from a common bipotent intermediate in the neural crest lineage--the melanocyte/Schwann cell progenitor. In this study, we test the possibility that peptide growth factors found in the embryonic environment might act similarly to TPA to influence the fates of these cells. DRG and PN explants were cultured in medium supplemented with a variety of growth factors, and then the cultures were examined for the presence of pigment cells. We found that basic fibroblast growth factor (bFGF), but not various other growth factors, induced pigmentation in about 20% of these cultures. When low concentrations of TPA were included in the culture medium, bFGF augmented the TPA-induced pigmentation, significantly increasing the proportion of pigmented cultures. These effects of bFGF were age-dependent, and could be blocked by addition of a bFGF-neutralizing antibody to the culture medium. In contrast to these stimulatory effects of bFGF, transforming growth factor-beta 1 (TGF-beta 1) was found to inhibit the TPA- or bFGF-induced pigmentation of DRG cultures. These data suggest, therefore, that at least some NC-derived cells are responsive to bFGF and TGF-beta 1, and that these growth factors may play an important role in the control of NC cell fate.     

Increased expression of integrins and decreased apoptosis correlate with increased melanocyte retention in cultured skin substitutes

Losses of human melanocytes (HM) in transplantation of cultured skin substitutes (CSS) may result from poor cellular attachments. To test this hypothesis, HM integrin expression was measured in four culture media: (a) melanocyte growth medium (MGM), an HM proliferation medium; (b) UCMC 160, a CSS maturation medium; (c) mMGM, modified MGM with 1.8 mM calcium; and (d) modified UCMC 160 with HM supplements (mUCMC 160). HM grew well in all media except UCMC 160. Increased expression of beta1, beta4, alpha3beta1 and alpha5 integrins on HM cultured in MGM and mMGM versus UCMC 160 was found by flow cytometry. Annexin V-allophycocyanin (APC) labeled HM in apoptosis and increased significantly in UCMC 160 (31.1%) compared with MGM (11.9%) or mMGM (13.9%). CSS were incubated in UCMC 160, mMGM or mUCMC 160 media, and grafted to athymic mice. In the mMGM group, grafts were darker as measured with a chromameter through 6 weeks and the average number of basal HM per field was greater at 12 weeks post-grafting. Increased graft loss was observed in the mMGM group which corresponded with the poor epidermal morphology in vitro. Although HM retention improved in vivo using mMGM to culture the CSS, the stability of the epidermis decreased. These results indicate that expression of integrins on HM in vitro correlates with HM retention in CSS and short-term survival after transplantation, but that long-term survival depends also on stable epithelium.     

Stimulation of the proliferation and differentiation of mouse pink-eyed dilution epidermal melanocytes by excess tyrosine in serum-free primary culture

The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink-eyed dilution (p) locus (black, C57BL/10JHir-P/P) and their congenic mutant mouse (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with a serum-free melanocyte growth medium supplemented with additional L-tyrosine (Tyr) from initiation of the primary culture. L-Tyr inhibited the proliferation of P/Pmelanocytes in a dose-dependent manner, whereas L-Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose. On the other hand, L-Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose-dependent manner. In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L-Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed. The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L-Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased. On the contrary, in p/p melanoblasts and melanocytes L-Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes. Contents of PTCA and eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of AHP and pheomelanin precursor, 5-S-cysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/Pmelanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L-Tyr, but difficult to accumulate them in melanosomes.     

Stimulation of Cultured Melanocytes in Medium Containing a Serum Substitute: Ultroser-G

Melanocyte cultures were established and maintained routinely in Ham's F-10 medium containing 12-O-tetradecanoyl-phorbol-13-acetate (TPA), isobutylmethylxanthine (IBMX), cholera toxin (CT) and fetal calf serum (FCS). Three serum substitutes (Ultroser-G, Nutridoma-Hu and Nutricyte-H) were tested in order to obtain a medium without FCS having a more constant composition. Melanocyte proliferation was examined in long-term culture experiments by in situ cell counts at different periods of time. Only with Ultroser-G (1-2%) was the proliferation of melanocytes maintained without both FCS and CT, whereas the addition of the other two serum substitutes resulted in stabilization of melanocyte densities in the cultures up to 28 days. In the medium containing 1% Ultroser-G and IBMX without TPA minimal or no increases in melanocyte density were found. Addition of basic fibroblast growth factor (bFGF, 1 ng/ml) to the medium without TPA resulted in a partial restimulation of growth in different experiments. In this system with 1% Ultroser-G and 1 ng/ml bFGF, IBMX could also be replaced by other factors (dbcAMP LTC4 and a purified form of α-melanocyte stimulating hormone). The culture medium with 1% Ultroser-G containing TPA and IBMX is now used for routine melanocyte culture. In this medium TPN/IBMX can easily be replaced by bFGF/dbcAMP with optimal growth stimulation. The combination bFGF/α-MSH and other more physiological stimulators offers an alternative to study responses of melanocytes in culture with respect to proliferation, metabolism, and phenotype.     

Ca2+ and UVB Radiation Have No Effect on E-Cadherin-Mediated Melanocyte-Keratinocyte Adhesion

Direct cell-cell contact between melanocytes and keratinocytes has been shown to play an important role in the regulation of human melanocyte function and skin pigmentation. An important role for the calcium-dependent epithelium-specific cell adhesion molecule, E-cadherin, in melanocyte-keratinocyte adhesion was suggested previously. To further clarify regulation of E-cadherin-mediated melanocyte-keratinocyte interactions, we investigated the effects of physiological (Ca²⁺) and environmental (ultraviolet B [UVB] radiation) stimuli on the expression and functional activity of E-cadherin in melanocyte-keratinocyte adhesion. Expression of E-cadherin mRNA was detected by Northern blot analysis in cultured normal human melanocytes at levels similar to those in keratinocytes. Flow cytometry analysis with anti-human and anti-mouse-E-cadherin antibodies (anti-uvomorulin and ECCD-2) showed that cultured normal human keratinocytes, melanocytes, and two metastatic melanoma cell lines express E-cadherin strongly on the cell surfaces. Melanocyte adhesion, particularly to differentiating keratinocytes (cultured in 1.2 mM calcium) but not to proliferating keratinocytes or to fibroblasts, was decreased by 41.7 ± 4.5% in the absence of 1 mM Ca²⁺ during the binding assay. Addition of anti-mouse-E-cadherin antibody (ECCD-1) to the binding assay inhibited the adhesion of melanocytes to differentiating keratinocytes by 88.2 ± 1.1%, while addition of anti-P-cadherin antibody (PCD-1) had no effect. The levels of E-cadherin expression in melanocytes were not changed by the presence of calcium (1 mM) in the medium or by UVB irradiation (20 mJ/cm²) for one day before flow cytometry analysis. Moreover, these treatments had no effect on melanocyte-keratinocyte adhesion. These results demonstrate that E-cadherin is strongly involved in melanocyte adhesion to keratinocytes and suggest the implication of E-cadherin in the overall regulation of the skin pigmentary system.     

The effects of phorbol ester on anabolism of lipids of cultured human skin fibroblasts

We have investigated the independent effects of phorbol ester (phorbol 12-myristate 13-acetate) on anabolism of the major lipid components in cultured diploid human skin fibroblasts. When we incubated these cells with [3H]acetate in serum-free medium for 18 h in the presence of 16 nM phorbol ester, [3H]acetate incorporation and the cellular content of cholesterol ester increased, and free cholesterol decreased. Enhancement of [3H]acetate incorporation into cholesterol ester was also observed when the cells were incubated with phorbol ester for 5 h in medium containing lipoprotein-deficient serum. Incorporation of [3H]galactose into glycosphingolipids increased many fold upon exposure of the cells either to fetal calf serum or separately to phorbol ester. Therefore, phorbol ester independently affects cholesterol and glycosphingolipid metabolism in a way that may be similar to that reported for serum low-density lipoproteins and unknown other factors in fetal calf serum. We have observed these effects of phorbol ester in the intact living cell. These findings should provide useful means for the study of metabolism of the plasma membrane lipid components in fibroblasts.     

Alterations in Ca<Superscript>2+</Superscript>-dependent and cAMP-dependent signaling pathways affect neurogenesis and melanogenesis of quail neural crest cells in vitro

Trunk neural crest cells primarily form neurons, nerve supportive cells of the peripheral nervous system and melanocytes. We are interested in signal transduction pathways that affect the production of peripheral neurons or melanocytes. Quail neural crest cell cultures were treated with a variety of drugs that affect components of protein kinase A- (PKA-), protein kinase C- (PKC-) and inositol-3-phosphate- (I3P-) dependent pathways. Forskolin, a drug that increases cAMP levels, augmented melanocyte populations and reduced neuronal populations in our cultures. H8 and H89, two drugs that inhibit PKA, reduced melanocyte populations well below control levels. Down regulation of PKC with a phorbol ester, PMA, or with calphostin C inhibited neurogenesis. PMA also enhanced melanogenesis. Increasing intracellular calcium levels (with A23187 or thapsigargin) resulted in cultures with few melanocytes but many neurons, compared to untreated controls. An antagonist of the I3P pathway, wortmannin, prevented the appearance of neurons but did not affect melanocyte populations. In summary, molecules that altered the PKA-dependent pathway affected melanogenesis. Manipulations of the I3P and/or PKC-dependent pathways influenced neurogenesis. Stimulation of one pathway often inhibited appearance of cells associated with the alternative pathway.Edited by D.A. Weisblat     

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.     

Calcium-independent growth of human ovarian carcinoma cells

Evidence in the literature suggests that cancer cell growth in vitro is generally not sensitive to external calcium. A human ovarian carcinoma cell line (SKOV3) retained 60% of its normal growth in Dulbecco modified Eagle's medium (DME) when the calcium concentration was reduced from 3 mM to 10 microM. Chinese hamster ovary cells (CHO) were growth-arrested in media containing less than 500 microM calcium. In low-calcium (10 microM) DME, 10 microM of a calmodulin antagonist W7 inhibited the growth of SKOV3 cells by more than 90%, while 100 microM of its inactive analog W5 was mildly inhibitory (20%). The growth inhibition by W7 was antagonized by increasing calcium concentrations in the culture media, while the inhibition by W5 was calcium-independent. The phorbol ester TPA was also effective in antagonizing W7's growth inhibition in low-calcium DME, suggesting that the W7 effect is mediated via protein kinase C inhibition. SKOV3 total cellular protein kinase C activity was 1.6 times higher than CHO cells when incubated in normal DME. When incubated in low-calcium DME, a large drop in protein kinase C activity in the CHO cells was observed while the enzyme activity was unchanged in the SKOV3 cells. Our data suggest that these human ovarian tumor cells have altered cellular calcium regulatory processes associated with the defective down-regulation of protein kinase C. This defect may confer these cells the ability to proliferate independently of the external calcium concentration. Targeting the cellular signal transduction components may be useful in cancer chemotherapy.     

Investigations into the nature of growth-related proteolysis in human fibroblasts

Previous experiments have shown that cultured human fibroblasts possess a cell-surface proteinase (the growth-related proteinase; GRP) which is essential to cell proliferation. In the present work, proteinase inhibition in defined and complex serum-free media and in pre-conditioned normal medium, still resulted in a corresponding inhibition of cell proliferation. Proteinase inhibition also blocked the action of a range of peptide growth factors and of a phorbol ester. Elevated extracellular calcium concentrations were still mitogenic in the presence of proteinase inhibitors. Proteinase inhibition did not affect the mobilisation of intracellular calcium, nor the metabolism of inositol phosphate derivatives in response to a mitogenic stimulus.