This study describes the in vitro propagation of human mammary epithelial cells (HMECs) in an undifferentiated state, using nonadherent mammospheres. These spheres are enriched in early progenitor/stem cells and can differentiate into all three mammary epithelial lineages. Gene expression analysis of mammosphere-derived cells revealed overlapping genetic programs with other stem and progenitor cells, identifying new markers for mammary stem cells. The isolation and characterization of these cells help elucidate the molecular pathways governing normal mammary development and carcinogenesis. The mammary gland has a lobulo-alveolar structure composed of three cell lineages: myoepithelial, ductal, and alveolar. These cells proliferate and differentiate during pregnancy and lactation, and undergo apoptosis during involution. Stem cells may generate the cellular repertoire of the mammary gland. Factors intrinsic to stem cells and their interactions with stromal cells and extracellular matrix are important in normal morphogenesis. Experimental and clinical data support the hypothesis that cancer may arise from mutations in stem cell populations. Transplantation studies in mice suggest the existence of self-renewing, multipotent mammary stem cells. Clonal dominant populations were generated, and serial transplantation recapitulated the functional repertoire of the gland. A recent study showed that mammary SCA-1+ cells are enriched in progenitor cells that can regenerate the gland in vivo. To study stem cell properties, one needs to identify and purify them, which is technically difficult due to the scarcity of stem cells. Stem cells are defined by their ability to self-renew and generate differentiated progeny. In adult organs, these properties are manifest by extensive self-renewal potential and the ability to generate the entire repertoire of cell types found in a tissue. A characteristic shared by many stem cells is their ability to exclude dyes like Hoechst or rhodamine, identified as the side population (SP). The SP fraction contains long-term repopulating hematopoietic stem cells, primitive myoblasts, and epidermal stem cells. In the mouse mammary gland, SP cells can regenerate the gland upon transplantation. Several studies have described bipotent human mammary epithelial progenitor cells based on immunosorting using ESA, MUC1, and α6 integrin. Using these markers, a 10-fold increase in bipotent progenitors was obtained compared with unsorted cells. An alternative approach is the isolation and propagation of normal human mammary progenitor cells from primary tissue. This approach has been limited by the lack of suitable systems for propagation in an undifferentiated state. When primary cultures of mammary epithelium are cultured on solid substrata, they undergo limited replication and differentiate in a process regulated by hormonal factors, extracellular matrix, and cell-cell interactions. A major advance in neural stem cell research was the discovery that undifferentiated multipotent neural cells can be grown in suspension asThis study describes the in vitro propagation of human mammary epithelial cells (HMECs) in an undifferentiated state, using nonadherent mammospheres. These spheres are enriched in early progenitor/stem cells and can differentiate into all three mammary epithelial lineages. Gene expression analysis of mammosphere-derived cells revealed overlapping genetic programs with other stem and progenitor cells, identifying new markers for mammary stem cells. The isolation and characterization of these cells help elucidate the molecular pathways governing normal mammary development and carcinogenesis. The mammary gland has a lobulo-alveolar structure composed of three cell lineages: myoepithelial, ductal, and alveolar. These cells proliferate and differentiate during pregnancy and lactation, and undergo apoptosis during involution. Stem cells may generate the cellular repertoire of the mammary gland. Factors intrinsic to stem cells and their interactions with stromal cells and extracellular matrix are important in normal morphogenesis. Experimental and clinical data support the hypothesis that cancer may arise from mutations in stem cell populations. Transplantation studies in mice suggest the existence of self-renewing, multipotent mammary stem cells. Clonal dominant populations were generated, and serial transplantation recapitulated the functional repertoire of the gland. A recent study showed that mammary SCA-1+ cells are enriched in progenitor cells that can regenerate the gland in vivo. To study stem cell properties, one needs to identify and purify them, which is technically difficult due to the scarcity of stem cells. Stem cells are defined by their ability to self-renew and generate differentiated progeny. In adult organs, these properties are manifest by extensive self-renewal potential and the ability to generate the entire repertoire of cell types found in a tissue. A characteristic shared by many stem cells is their ability to exclude dyes like Hoechst or rhodamine, identified as the side population (SP). The SP fraction contains long-term repopulating hematopoietic stem cells, primitive myoblasts, and epidermal stem cells. In the mouse mammary gland, SP cells can regenerate the gland upon transplantation. Several studies have described bipotent human mammary epithelial progenitor cells based on immunosorting using ESA, MUC1, and α6 integrin. Using these markers, a 10-fold increase in bipotent progenitors was obtained compared with unsorted cells. An alternative approach is the isolation and propagation of normal human mammary progenitor cells from primary tissue. This approach has been limited by the lack of suitable systems for propagation in an undifferentiated state. When primary cultures of mammary epithelium are cultured on solid substrata, they undergo limited replication and differentiate in a process regulated by hormonal factors, extracellular matrix, and cell-cell interactions. A major advance in neural stem cell research was the discovery that undifferentiated multipotent neural cells can be grown in suspension as