Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis

Human keratinocytes isolated from a skin biopsy and cultured in vitro reconstitute a stratified squamous epithelium suitable for grafting on burned patients. Melanocytes coisolated from the same skin biopsy also proliferate under these culture conditions and maintain differentiated functions (i.e., synthesize melanin granules, regularly intersperse in the basal layer of the cultured epidermis, and transfer melanosomes in the cytoplasm of contiguous keratinocytes) (De Luca, M., A. T. Franzi, F. D'Anna, A. Zicca, E. Albanese, S. Bondanza, and R. Cancedda. 1988. Eur. J. Cell Biol. 46:176-180). Isolated melanocytes in culture grow in the presence of specific growth factors with a mean population doubling time of 4-10 d. In this paper we show that (a) human keratinocytes and oral epithelial cells possess strong and specific melanocyte growth stimulating activity (doubling time, 24 h); (b) melanocyte growth is not autonomous but requires close keratinocyte contact and is regulated to maintain a physiological melanocytes/keratinocytes ratiol and (c) pure skin keratinocytes, but not oral epithelial cells, have all the information required for the proper physiological location and differentiation of melanocytes in the epidermis.     

Melanosome capping of keratinocytes in pigmented reconstructed epidermis--effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis

Reconstructed pigmented epidermis was established by co-seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air-liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3-isobutyl-1-methyl-xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.     

Melanosome Capping of Keratinocytes in Pigmented Reconstructed Epidermis – Effect of Ultraviolet Radiation and 3‐Isobutyl‐1‐Methyl‐Xanthine on Melanogenesis

Reconstructed pigmented epidermis was established by co‐seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air–liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3‐isobutyl‐1‐methyl‐xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.     

Distinct Melanogenic Response of Human Melanocytes in Mono‐culture,in Co‐Culture with Keratinocytes and in Reconstructed Epidermis,to UV Exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm²), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm² had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.     

Vitiligo: Where Do We Stand?

Vitiligo is a puzzling disorder characterized by a disappearance of epidermal and/or follicular melanocytes by unknown mechanisms. This very common disorder involving 1–4% of the world population is thus of great importance for the practicing dermatologist. The cellular and molecular mechanisms leading to the destruction of melanocytes in this disorder have not yet been elucidated, making it of major interest for the cell biologist involved in melanocyte research. Recent advances in this field, due largely to the availability of techniques for culturing normal human melanocytes, opened new perspectives in the understanding of vitiligo. Although vitiligo has long been considered a disorder confined to the skin, there is now good evidence that it also involves the extracutaneous compartment of the “melanocyte organ.” It is also clear that vitiligo is not only a melanocyte disorder, but that it also involves cells, such as keratinocytes and Langerhans cells, found in the epidermis and follicular epithelium. The three prevailing theories of the pathogenesis of vitiligo are the immune hypothesis, the neural hypothesis, and the self-destruct hypothesis. New hypotheses suggest that vitiligo may be due to (1) a deficiency in an unidentified melanocyte growth factor, (2) an intrinsic defect of the structure and function of the rough endoplasmic reticulum in vitiligo melanocytes, (3) abnormalities in a putative melatonin receptor on melanocytes and (4) a breakdown in free radical defense in the epidermis. None of these hypotheses has been demonstrated, and according to the available data, it is likely that the loss of epidermal and follicular melanocytes in vitiligo may be the result of several different pathogenetic mechanisms.     

CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.     

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.     

The melanocytorrhagic hypothesis of vitiligo tested on pigmented, stressed, reconstructed epidermis

Common generalized vitiligo is an acquired depigmenting disorder characterized by a chronic and progressive loss of melanocytes from the epidermis and hair follicles. We previously proposed a new theory that vitiligo involves the chronic detachment and transepidermal loss of melanocytes caused by autoimmune, neural and impaired redox mechanisms associated with mechanical trauma. In this study, we reconstructed epidermis on dead de-epidermized dermis with normal and/or non-segmental non-lesional vitiligo (NSV) cells and tested catecholamines or sera or hydrogen peroxide. Under unstressed conditions, the number of melanocytes located in the basal layer was significantly lower in reconstructs made with melanocytes from non-lesional NSV skin and normal keratinocytes compared with controls made with autologous normal melanocytes. The number of non-lesional NSV melanocytes was even lower in reconstructs made with keratinocytes from non-lesional NSV skin. Epinephrine and H(2)O(2) could trigger the transepidermal loss of normal and vitiligo melanocytes. Some sera induced melanocyte detachment but without any clear correlation with disease activity in the donors. In conclusion, our results are the first step to obtaining a reproducible melanocytorrhagic model in vitro with some of the stressors investigated. They support the hypothesis that NSV melanocytes have an intrinsic defect, which limits their adhesion in a reconstructed epidermis, with an enhancer effect of the vitiligo keratinocyte milieu.     

Ex Vivo Reconstruction of the Epidermis With Melanocytes and the Influence of UVB

To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm²× 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.     

Organotypic Culture of Human Skin to Study Melanocyte Migration

An ex vivo model system was developed to investigate melanocyte migration. Within this model system, melanocytes migrate among other epidermal cells in the epibolic outgrowth of skin explants. This process is initiated by loss of contact inhibition of epidermal cells at the rim of the explants and by locally produced chemotactic factors. Punch biopsies provided explants of reproducible diameter. Optimal culture conditions include medium consisting of Dulbecco's Minimal Essential Medium containing 10% inactivated normal human serum and placement of explants epidermal side up at the air-liquid interphase. Within 7 days, epidermal cells completely surround the explant. Approximately 3 days after the onset of keratinocyte migration, melanocytes distribute themselves within the newly formed epidermis. Throughout the 7-day culture period, melanocytes and keratinocytes show maintenance of subcellular morphology, and the dermo-epidermal junction remains intact. Melanocyte migration was quantified using immunoperoxidase staining in combination with light microscopy and computer-aided image analysis. Preliminary results using the model system to compare migration in control and nonlesional vitiligo skin indicate that no inherent migration defect is responsible for impaired repigmentation of vitiligo lesions. The organotypic culture model system allows for investigations on melanocytes within their environment of autologous epidermal and dermal components, closely resembling in vivo circumstances in human skin.     

Distinct melanogenic response of human melanocytes in mono-culture, in co-culture with keratinocytes and in reconstructed epidermis, to UV exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co-cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm2), an increase in melanin synthesis whereas in melanocyte mono-cultures, UVB doses up to 50 mJ/cm2 had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono- and co-cultures. Removing certain keratinocyte growth factors from the co-culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte-melanocyte co-cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB-induced pigmentation, (ii) UVA-induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV-induced pigmentation in vitro.     

Crosstalk in skin: melanocytes,keratinocytes, stem cells,and melanoma

In the vertebrate embryo, melanocytes arise from the neural crest, migrate to and colonize the basal layer within the skin and skin appendages. Post-migratory melanocytes are securely attached to the basement membrane, and their morphology, growth, adhesion, and migration are under control of neighboring keratinocytes. Melanoma is a malignant tumor originated from melanocytes or their progenitor cells. During melanocyte transformation and melanoma progression, melanocytes lose their interactions with keratinocytes, resulting in uncontrolled proliferation and invasion of the malignant cells. Melanoma cells at the advanced stages often lack melanocytic features and resemble multipotent progenitors, which are a potential melanocyte reservoir in human skin. In this mini-review, we will summarize findings on cell-cell interactions that are responsible for normal melanocyte homeostasis, stem cell self-renewal, and differentiation. Our ultimate goal is to define molecules and pathways, which are essential for normal cell-cell interactions but deregulated in melanoma formation and progression.     

Corticotropin Expression by Human Melanocytes in the Skin

Highly dendritic melanocytes have been observed in rapidly proliferating seborrheic keratosis, epidermis overlying melanomas, and in melanomas. On staining for the presence of POMC with monoclonal antibody against human ACTH, the melanocytes show cytoplasmic positivity. Short term organ cultures of whole skin from the marginal zone of vitiligo patients show that 22.7% of controls and 45.5% on dark incubation in adriamycin and 87.5% exposed to a pulse of UV on adriamycin treatment show melanocytes positive for ACTH. The surrounding keratinocytes in the epidermis and in the seborrheic keratosis are negative, whereas in melanomas, isolated groups of melanocytes are positive for ACTH. These findings indicate that ACTH is expressed by the melanocytes in the G₂-phase, the activity being enhanced on UV exposure. Thus UV dependent pigmentation is associated with POMC production in human skin. From this work it is evident that the melanocyte network varies the MSH/ACTH levels in correlation with repigmentation and depigmentation in the marginal zone in vitiligo by expressing POMC locally and is related to the UV-sensitivity of the melanocytes.     

The mathematics of tanning

Background  

The pigment melanin is produced by specialized cells, called melanocytes. In healthy skin, melanocytes are sparsely spread among the other cell types in the basal layer of the epidermis. Sun tanning results from an UV-induced increase in the release of melanin to neighbouring keratinocytes, the major cell type component of the epidermis as well as redistribution of melanin among these cells. Here we provide a mathematical conceptualization of our current knowledge of the tanning response, in terms of a dynamic model. The resolution level of the model is tuned to available data, and its primary focus is to describe the tanning response following UV exposure.     

Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes

In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte–keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.     

Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes

In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte-keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.     

Pigmented Human Skin Equivalent: New Method of Reconstitution by Grafting an Epithelial Sheet Onto a Non-Contractile Dermal Equivalent

We have established a new protocol for reconstituting a pigmented human skin equivalent (PSE) and have evaluated its functional responses to environmental stimulus, UVB. The PSE is reconstituted by grafting an epithelial sheet consisting of keratinocytes and melanocytes onto a porous non-contractile dermal equivalent populated with mitotically and metabolically active fibroblasts. i) The PSE has a multilayered, well-differentiated epidermis with cuboidal basal cells and highly organised dermis with newly synthesised extracellular matrix components. ii) Ki67-positive proliferating keratinocytes (18.1 ± 7.4%) were detected on the basal layer of the epidermis. iii) Melanocytes located exclusively within the basal layer were detected by monoclonal antibody against tyrosinase-related protein (TRP-1). iv) After exposure to UVB (100 mJ/cm² per day) for 7 consecutive days, the intensity of TRP-1 staining was increased in the PSE, showing their functional state, whereas the number of melanocytes was not changed. This non-contractile and functioning new PSE is potentially useful as a model for studying the role of melanocyte-keratinocyte-fibroblast interactions in photoprotection of the skin in more complex cutaneous microenvironment than monolayer culture, and for developing in vitro disease models and therapeutic protocols with genetically altered cells both in epidermis and dermis.     

A variation of pigmentation in the glabrous skin of dogs

The usual pigmentation pattern in mammalian skin consists of fixed melanocytes in the basal layer of the epidermis, supplying keratinocytes with melanosomes. We observed that the glabrous skin (rhinaria and footpads) of dogs deviates from this pattern. In dogs, melanocytes are found in both the dermis and epidermis. The epidermal melanocytes are situated in the intercellular spaces of the basal and spinous layers. They are characterized by a quantity of cytoplasm containing a centriole, also developing melanosomes, and in some cases annulate lamellae. There is a high frequency of closely apposed melanocytes in the epidermis. Melanosomes in different stages of formation are also abundant. The morphology of the glabrous skin of dogs suggests transport of melanocytes from the dermis into the epidermis and formation of melanosomes in the epidermis. A distributed and intense pigment formation may be necessary to achieve the black noses of many dog breeds and wild canids, as well as dark footpads despite heavy abrasion and rapid skin renewal.     

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.