Mixed parenchymal-stromal populations of rat adrenocortical cells support the proliferation and differentiation of steroidogenic cells.

The mechanisms which regulate adrenocortical steroidogenesis in differentiated parenchymal cells have been studied in great detail. However, the stem cells that are responsible for regeneration of the adult cortex have never been identified or isolated, and their characteristics are unknown. We have developed a tissue culture system that supports the simultaneous proliferation and differentiation of steroidogenic adrenocortical cells. Utilizing density gradient separation, a cell population composed of a mixture of stromal, endothelial and parenchymal cells (MIX) was isolated from the adult rat adrenal cortex. In primary culture, MIX populations formed high saturation density multilayers from which rounded cells emerged. These cells proliferated, contained lipid, and expressed the steroidogenic enzymes delta 5,3 beta-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage cytochrome P-450scc. After selective passaging, rounded MIX-derived cells retained their steroidogenic potential, even in the absence of trophic hormone treatment. In contrast, parenchymal cells obtained from the zonae fasciculata (FASC) and glomerulosa (GLOM) respectively, formed homogeneous monolayers in primary culture, gradually de-differentiated, and no longer responded to trophic hormone treatment after passaging. Thus, primary MIX cultures provided a microenvironment that resulted in the production of adrenocortical cells with stem cell-like qualities. These cultures provide for the first time, a system for the identification of specific inducers that are responsible for both adrenocortical cytogenesis and its associated proliferation and steroidogenic differentiation.