Identification of Stem Cells in a Novel Human Mammary Epithelial Culture (HMEC) System that Reproducibly Demonstrates Ductal Organotypic Architecture in 3 Weeks

Our laboratory has published a novel culture system for Human Mammary Epithelial Cells HMEC, both normal and malignant. This system allows for unusually long-term 3 months or longer establishment of normal primary cultures that begin as three-dimensional mammospheres, which are structures made up of 40-100 epithelial cells. These mammospheres subsequently differentiate into complex organotypic branching ducts and lobules that demonstrate Epithelial Specific Antibody ESA staining, lumen, polarized nuclei, desmosomes along the lateral surfaces of the cells, and microvilli on the apical surfaces We hypothesize that since we have demonstrated de novo formation of multicellular organotypic epithelial ductal and lobular structures, that our cultures must contain multipotent stem cells, possibly because of the use of our novel tissue culture medium and the use of matrigel. We will identify the stem cell component of our HME cultures and determine the percentage of cells that have this profile. We will isolate these putative stem cells and reintroduce them after labeling, into our culture system to determine their ability to contribute to multiple lineages under the juxtacrine and paracrine influences of the other cells present. We will dentify and isolate putative mammary stem cells using Hoechst exclusion from our primary cultures before they have actually differentiated ductal structures pre-14 days in culture. Determine whether these flow sorted cells match