Mammary epithelial differentiation in vitro: minimum requirements for a functional response to hormonal stimulation

Mammary epithelial differentiation is the culmination of responses to a complex sequence of hormonal stimuli. An in vitro model for this process should retain the basic features of in vivo epithelial differentiation. The IM-2 mouse mammary cell line responds to lactogenic hormone stimulation by synthesizing the milk protein beta-casein. Epithelial and fibroblastic clones derived from IM-2 lack this ability, but cocultures of these clones regain responsiveness to lactogenic hormone stimulation. Studies of the epithelial cell clone 31E under various culture conditions reveal that the role of fibroblastic cells in supporting synthesis and secretion of beta-casein can be supplanted by culture in filter chambers without addition of exogenous extracellular matrix components. Electron microscopic and immunofluorescence studies show that, under these conditions, 31E epithelial cells exhibit the morphology and intercellular organization characteristic of mammary epithelium. Transepithelial electrical resistance measurements indicate that the cells are well polarized. Analysis of glucose metabolism is consistent with this polarization; glucose is utilized from the basal chamber, and lactate is excreted into the basal chamber. Immunoblot analysis demonstrates the vectorial protein secretion expected of polarized mammary epithelium: laminin is secreted into the basal chamber, whereas beta-casein is secreted into the apical chamber in response to lactogenic hormone stimulation from the lower chamber. Thus, the maintenance of a polarized intercellular organization that permits access of the basolateral cell surface to nutrients is sufficient for a pure culture of an established mammary epithelial cell clone to retain differentiated epithelial function in vitro.