Characterization in vitro of luminal and myoepithelial cells isolated from the human mammary gland by cell sorting

Abstract

Abstract Luminal and myoepithelial cells have been separated from normal adult human breast epithelium using fluorescence activated cell sorting. Their isolation was based on the exclusive expression of two surface antigens, epithelial membrane antigen (EMA) and the common acute lymphoblaslic leukaemia antigen (CALLA/ CD10/neutral endopeptidase 24.11). Sorted luminal and myoepithelial cells displayed distinctively different morphologies when maintained in monolayer culture, differences which were enhanced by the addition of hydrocortisone. insulin and cholera toxin to the culture medium. The EMA-positive cells formed an attenuated monolayer with indistinct cell boundaries while CALLA-positivc cells, by contrast, formed tightly packed arrays of refractile cells. The distribution of the cell type-specific markers cytokeratin 18 (luminal cells) and smooth muscle α-actin (myoepithelial cells) indicated that the sorted populations were approximately 98% pure. However, a significant minority (-15%) of sorted luminal cells consistently expressed the basal-cell marker cylokeratin 14 in culture. A marked difference was noted in the proliferativc behaviour of the two types of sorted cells, with myoepithelial cells dividing rapidly in response to the humoural additives, in contrast to the luminal cells which proliferated slowly. Both types of sorted cells could be cloned in the presence of feeder layers of mouse fibroblasts. Clones of luminal and myoepilhelial cells were also distinctive; all “spread” luminal clones were similar in appearance to each other, although some cellular heterogeneity, including squamous metaplasia, was observed in “compact” myoepithelial clones. Both types were shown to have retained their original surface markers and to exhibit different cytoskelelal anligenic phenotypcs when they were re-analysed after a 3-week growth period. Both spread and compact phenolypes were obtained when separately isolated duels and alveoli were cloned. This detailed characterization of cells isolated from the human breast epithelium by flow cytometry provides the basis for further studies of luminal-myoepithelial interactions and growth responses of purified cell types in vitro.