HISTORY AND PERSPECTIVES
Sandrine Bessou & Alain Taïeb, Laboratoire de Dermatologie,
Université de Bordeaux II
146, rue Léo Saignat - F - 33076 Bordeaux Cédex
Tel: 57571702, Fax: 56795987
Human skin is the first organ for which attempts of ex-vivo
reconstruction have been made over the last two decades.
A landmark was the discovery by Rheinwald & Green that irradiated 3T3 murine fibroblasts could be efficiently used to grow keratinocytes from epidermal cell suspensions (1). Epidermal sheets were obtained with incomplete differentiation and fibroblast contamination was frequent, but variants of this technique remained as a reference for human therapy especially in the burn patient, because of the selection of cells of high clonogenic potential.
A better differentiation of the reconstructed of the epidermis was noted by Prunieras et al (2) when keratinocytes were cultured on a dead deepidermized dermis and lifted at the air-liquid interface (3-4). The presence of living fibroblasts in a dermal equivalent, like in Bell's model (5), has been considered by some as an important factor to reconstruct an epidermis with better differentiation.
A further advance in epidermal culture was the development of a serum-free medium in Ham's laboratory (6). The first generation media were based on a mixture of aminoacids, vitamins and minerals completed by fetal calf serum, EGF and cholera toxin (CT). Boyce & Ham adjusted by a careful stepwise approach the basic medium composition to the specific metabolic needs of keratinocytes. In this low calcium medium feeder layers, the cAMP agonist cholera toxin and serum were no more required. The addition of bovine pituitary extract (BPE) and aminoacid supplementation allowed to grow primary cultures to confluency and to passage epidermal monolayers.
The switch to high calcium generated multilayers much similar to those obtained in Green's system (1). Since the early eighties, melanocytes had been grown in a few laboratories using Eisinger and Marko's (7) or Halaban's (8) techniques with serum, cAMP agonists (cholera toxin or IBMX) and phorbol esters (PKC agonists). Gilchrest's system using a pituitary extract(BPE), cholera toxin and a basic medium without serum showed the way towards more physiological culture condition of melanocytes, but cell growth and differentiation were poor (9).
In these techniques phorbol esters increased the mitotic activity of melanocytes and their morphology, and cAMP agonists like CT increased dendricity and melanogenesis.
Using Boyce & Ham's technique for keratinocyte culture (MCDB 153), preliminary observations were made in several laboratories about "melanocyte contamination" of cultures, fibroblasts being eliminated by the low calcium concentration and absence of serum.
Pittelkow et al introduced in the system the phorbol ester TPA and were able to grow pure melanocyte cultures (10). Modifications to this system have been made at several institution and this technique is now used with variants worldwide.
In our lab, Donatien has successfully grown well-differentiated melanocytes without CT or phorbol esters using MCBB153 supplemented with BPE, insulin, hydrocortisone, 3% serum and calcium (11). This physiological modified medium allowed to answer the controversial question of a-MSH receptors on human melanocytes (12).
The two dimensional culture systems so far described are however poor models for studying melanocyte biology ex-vivo. The in-vivo epidermal melanization unit is a well-organized three dimensional structure in which melanocytes are polarized to the basal layers and maintain close contacts with both the basal lamina and neighbouring keratinocytes. The influence of keratinocyte factors to maintain the growth and differentiation of melanocytes ex-vivo either by contact or by diffusible factors, is now well-established (13-14-15).
The reconstruction of the epidermal melanization unit ex vivo has been achieved recently.
Bertaux et al have obtained epidermal explants and stimulated melanogenesis with UVB ex vivo (16).
De Luca et al have obtained a coculture of keratinocytes and melanocytes resulting in a moderately differentiated epidermis with melanocytes polarized in basal position when epidermal keratinocytes were used but only in basal and suprabasal position when non keratinizing oral epithelial cells were used (17-18). Similarly, the polarisation of neonatal and fetal melanocytes and the ratio keratinocytes/melanocytes was studied by Haake & Scott in a epidermis reconstructed on a collagen lattice. The role of keratinocytes in stereologic and ratio regulation was confirmed (19-20).
All these experiments show that a mature epidermis is necessary to regulate the epidermal melanization unit.
Other experiments in cocultures of keratinocytes have been conducted at various institutions [Topol (21), Stainao (22), Valyi (23)].
More recently, Todd et al (24) have stimulated with UVB an epidermis reconstructed ex vivo from pure cultures of melanocytes and keratinocytes on a dead deepidermized dermis with at least 10% melanocytes. The cells came from non caucasian donors and melanosome transfer was not demonstrated.
Our experience with keratinocyte and melanocyte culture led us to set up experiments of epidermal reconstruction with melanocytes ex vivo which have been reported at the last ESPCR meeting in Vienna (25). Using pure cultures of keratinocytes and melanocytes of the same donors, we have been successful in obtaining a well differentiated epidermis with basal functional melanocytes. The system was basically a modification of the Pruniras model using 5% melanocytes in the seeding suspension and a novel culture medium.
UVB irradiation induced a stimulation of melanogenesis macroscopically, microscopically and increased melanin concentrations. These results have been reproduced with skin of various phototypes and melanosome transfer to keratinocytes was demonstrated by electron microscopy.
Despite all these exciting new developments, the complete reconstruction of the human skin remains a challenge, since other epidermal components like Langerhans cells are not yet amenable to long-term cultivation, and that dermal reconstruction has not made real breakthroughs over the last decade.
However, the European Center for the Validation of Alternative Methods (ECVAM) setup by the European Union in Ispra, Italy, has considered skin reconstruction among the most advanced projets for the development of in vitro pharmacotoxicology and research programs in this field should be stimulated.
The future of the models of epidermal reconstruction with melanocytes looks promising. First, with better standardization, assays for pharmacologic and cosmetic agents suspected to interfere with pigmentation may be developped to screen interesting molecules, with a better relevance than monolayers of melanocytes. Second, pathophysiologic and therapeutic studies in human pigmentary disorders may be approached directly ex-vivo or after grafting the reconstructed epidermis on the nude mouse.
1. Rheinwald JG, Green HG. Serial cultivation of human epidermal
keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331-344,
2. Pruniéras M, Régnier M, Schlotterer M. Nouveau procédé de culture des cellules épidermiques humaines sur derme homologue ou hétérologue: préparation de greffons recombinés. Ann Chir Plast 24:28s-33s, 1983.
3. Pruniéras M, Régnier M, Woodley D. Methods for cultivation of keratinocytes with an air-liquid interface. J Invest dermatol 91:28s-33s, 1983.
4. Asselineau D, Pruniéras M. Reconstructed of "simplified" skin: control of fabrication. Br J Dermatol Suppl 27:219-222, 1984.
5. Bell E, Ehrlich P, Sher S, Merrill C, Sarber R, Hull B, Nakatsuji T, Church D, Buttle DJ. Development and use of a living skin equivalent. Plast Reconstr Surg 67:386-392, 1981.
6. Boyce ST, Ham R. Calcium regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum free serial culture. J Invest Dermatol 81:33s-40s, 1983.
7. Eisinger M, Marko L. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol esthers and cholera toxin. Proc Natl Acad Sci USA 79:2018-2022, 1982.
8. Halaban R, Pomerantz SH, Marshall S, Lambert DT, Lerner AB. Regulation of tyrosinase in human melanocytes grown in culture. J Cell Biol 97:480-488, 1983.
9. Gilchrest BA, Vrabel MA, Flynn E, Szabo G. Selective cultivation of human melanocytes from newborn and adult epidermis. J Invest Dermatol 83:370-376, 1984.
10. Pittelkow MR, Shipley GD. Serum-free culture of normal human adult melanocytes: growth kinetics and growth factor requirements. J Cell Physiol 140:565-576, 1989.
11. Donatien PD, Surlève-Bazeille JE, Thody AJ, Taïeb A. Growth and differentiation of normal human melanocytes in a TPA-free, Cholera Toxin-free, low-serum medium and influence of keratinocytes. Arch Dermatol Res 285:385-392, 1993.
12. Donatien PD, Hunt G, Pieron C, Lunec J, Taïeb A, Thody AJ. The expression of functional MSH-receptors on cultured human melanocytes. Arch Dermatol Res 284:424-426, 1992.
13. Halaban R, Ghosh S, Baird A. bFGF is the putative natural growth factor for human melanocytes. In Vitro Cell Dev Biol 23:47-52, 1987.
14. Halaban R, Langdon R, Birchall N, Cuono C, Baird A, Scott G, Moellman G, Guire JMc. Basic fibroblast growth factor from human keratinocytes is a natural mitogens for melanocytes. J Cell Biol 107:1611-1619, 1988.
15. Gordon PR, Mansur CP, Gilchrest BA. Regulation of human melanocytes growth, dendricity, and melanization by keratinocyte derived factors. J Invest Dermatol 92:565-572, 1989.
16. Bertaux B, Morliere P, Moreno G, Courtelon A, Massé JM, Dubertret L. Growth of melanocytes in a skin equivalent model in vitro. Br J Dermatol 119:503-512, 1988.
17. De Luca M, Franzi AT, D'Anna F, Zicca A, Albanese E, Bondanza S, Cancedda R. Coculture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organised in the basal layer of the cultured epithelium. Eur J Cell Biol 46:176-180, 1988.
18. De Luca M, D'Anna F, Bodanza S, Franzi AT, Cancedda R. Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocyte regulates its proper differentiation in the absence of dermis. J Cell Biol 107:1919-1926, 1988.
19. Haake AR, Scott GA. Physiologic distribution and differentiation of melanocytes in human fetal and neonatal skin equivalents. J Invest Dermatol 96:71-77, 1991.
20. Scott GA, Haake AR. Keratinocytes regulate melanocytes number in human fetal and neonatal skin equivalents. J Invest Dermatol 97:776-781, 1991.
21. Topol BM, Hainmes HB, Dubertret L, Bell E. Transfer of melanosomes in a skin equivalent model in vitro. J Invest Dermatol 87:642-647, 1986.
22. Staiano-Coico L, Hefton JM, Amadeo C, Pagan-Charry I, Madden MR, Cardon-Cardo C. Growth of melanocytes in human epidermal cell cultures. J Trauma 30:1037-1043, 1990.
23. Valyi-Nagy IT, Murphy GF, Mancianti ML, Witaker D, Herlyn M. Phenotypes and interactions of human melanocytes and keratinocytes in an epidermal reconstruction model. Lab Invest 62:314-324, 1990.
24. Todd C, Hewitt SD, Kempenaar J, Noz K, Thody AJ, Ponec M. Coculture of human melanocytes and keratinocytes in a skin equivalent model: effect of ultraviolet radiation. Arch Dermatol Res 285:455, 1993.
25. Bessou S, Surlève-Bazeille JE, Sorbier E, Taïeb A. Ex-vivo reconstruction of epidermis with melanocytes and uvb influence (in press).