Differentiation of Separated Mouse Mammary Luminal Epithelial and Myoepithelial Cells Cultured on EHS Matrix Analyzed by Indirect Immunofluorescence of Cytoskeletal Antigens

Abstract

SUMMARYWe have previously demonstrated that purified virgin mouse mammary luminal epithelial and myoepithelial cells promiscuously express cell type-specific cytokeratins when they are cloned in vitro. Changes in cytokeratin expression may be indicators of the loss or change of the differentiated identity of a cell. To investigate the factors that may be responsible for the maintenance of differentiated cellular identity, specifically cell-cell and cell-matrix interactions, we cloned flow-sorted mouse mammary epithelial cells on the extracellular matrix (ECM) derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Changes in cell differentiation on EHS, compared with culture on glass, were analyzed by comparing patterns of cytokeratin expression. The results indicate that ECM is responsible for maintenance of the differentiated identity of basal/myoepithelial cells and prevents the inappropriate expression of luminal antigens seen on glass or plastic. Luminal cell identity in the form of retention of luminal markers and absence of basal/myoepithelial antigens, on the contrary, appears to depend on homotypic cell-cell contacts and interactions. The results also show that luminal cells (or a subpopulation of them) can generate a cell layer that expresses only basal cytokeratin markers (and no luminal cytokeratin markers) and may form a pluripotent compartment. To fully understand how the development and differentiation of the mammary gland parenchyma and of the different cell types within it are regulated, it is necessary to be able to study separately the behavior of the individual cell types within the gland. The mature rodent mammary epithelium consists of two cell populations, the luminal epithelial cells and the basal/ myoepithelial cells. The luminal epithelial cells are cuboidal or low columnar cells that line the ducts and alveoli and are responsible for milk secretion. Primary and secondary ducts may be composed of several layers of luminal epithelial cells, which become fewer with higher orders of branching. Most terminal ducts have only one layer of luminal cells (Sekhri et al. 1967). The elongated myoepithelial cells form a monolayer over the ductal luminal epithelial cells. They make a continuous sheath in which the cells are orientated parallel to the duct. In alveoli, the myoepithelial cells form a basket-like network and in the spaces between their cell processes the luminal epithelial cells are in contact with basement membrane (Emerman and Vogl 1986).We have previously described (Smalley et al. 1998) a system for the separation of virgin mouse mammary epithelium by flow cytometry and methods for the clonal culture of such separated cells on tissue culture plastic and glass. We have characterized mouse mammary luminal epithelial and myoepithelial cell-derived clones on the basis of phase-contrast morphology and

Functional and molecular characterisation of mammary side population cells

“…The morphology and ratios of the clone types were consistent with the type A-D classification found when unsorted primary mammary epithelial cells were cloned [8,15]. Identical clone types and ratios of types A-D were observed following growth of SP and non-SP preparations.…”

“…Double immunofluorescence staining of clones, using the antibodies LE61 and LP2K (anticytokeratin 18 and anticytokeratin 19, respectively; in vivo markers of mammary luminal epithelial cells) and LLOO2 (anticytokeratin 14; a marker of mammary myoepithelial cells) [8,15], demonstrated that SP-derived and non-SP-derived clones had identical staining patterns. All were uniformly strongly positive for cytokeratin 18, and most cells within clones also double stained for cytokeratin 14.…”

Myoepithelial Cells: Their Origin and Function in Breast Morphogenesis and Neoplasia

“…Smalley et al demonstrated that sorted mouse myoepithelial cells give rise only to a single type of clones, whereas sorted luminal epithelial cells give rise to three morphologically distinct clonal types which show progressive acquisition of myoepithelial markers (34,37). In another study, albeit not using cell separation protocols, Kao et al defined two breast epithelial cell types by clonal dilution culture.…”

Function of phosphoenolpyruvate carboxykinase in mammary gland epithelial cells

“…Isolation of adipocytes and epithelial cells was performed as previously described ( 17,18 ). Briefl y, thoracic mammary glands were dissected from virgin female mice (three glands for each preparation).…”