Hydrocortisone is involved in regulating the proliferation and differentiation of mouse epidermal melanoblasts in serum-free culture in the presence of keratinocytes

Abstract

Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in serum-free medium (MPM) supplemented with dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). After 12 days, almost all keratinocytes died, and pure and enriched cultures of melanoblasts (approximately 90%) and melanocytes (approximately 10%) could be obtained. In order to clarify the role of hydrocortisone (HC) which is thought to be important for the regulation of melanocyte proliferation and differentiation, the hormone was added to MPM from the initiation of primary culture. The proliferation of melanoblasts was inhibited by HC in a dose-dependent manner. Instead, most of the proliferating melanoblasts were induced to differentiate into melanocytes by HC in a dose-dependent manner. A small number of pure melanoblasts derived from primary cultures at 12 days with MPM depleted of DBcAMP and bFGF were cultured with MPM with or without HC in the presence of secondary keratinocytes that were subcultured from a pure population of primary keratinocytes in a serum-free medium. The inhibition of the proliferation of melanoblasts by HC as well as the stimulation of the differentiation of melanoblasts into melanocytes by HC was observed in the presence of keratinocytes, but not in the absence of keratinocytes. Conditioned media or extracts prepared from pure keratinocytes in serum-free primary culture failed to replace the role of keratinocytes. These results suggest that HC plays an important role in the regulation of the proliferation and differentiation of mouse epidermal melanoblasts in culture in cooperation with factors supplied by keratinocytes.