Phenotypic characterization of mouse mammosphere-initiating cells (Ma-ICs) indicates they represent a novel progenitor population

Elucidation of the mechanisms regulating the development of the mammary gland would be facilitated by quantitative in vitro assays for mammary epithelial stem cells. Recent studies have shown that tight clusters of cells containing primitive clonogenic mammary cells are produced when dissociated human breast cells are cultured in serum-free liquid suspension cultures containing epidermal growth factor (EGF) and fibroblast growth factor (FGF). Single cell suspensions prepared from mouse breast tissue formed similar "mammospheres" under these conditions but cell aggregation was a significant factor in determining, their size and numbers. Addition of 1% methylcellulose to the culture medium prevented cell aggregation and resulted in the generation of smaller mammospheres over a 7-14-day period at a constant frequency of ~1 per 100 cells plated over a 100-fold range of cell concentrations. Interestingly, when subsets of antibody-stained cells in suspensions of freshly dissociated mouse mammary glands were first isolated by fluorescence activated cell sorting, the mammospheres they generated in semi-solid cultures were not from fractions containing highly purified cells with either mammary epithelial stem cell or progenitor activity (as identified by an ability to repopulate a cleared mammary fat pad, or generate colonies in standard 2D cultures containing feeder layers). These findings provide a quantitative assay for a previously uncharacterized clonogenic cell that is present in mouse breast tissue that may facilitate the survival or propagation of primititive mammary cells in vitro but whose developmental relationship to the mammary epithelial hierarchy has yet to be established.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat