We employed several complementary approaches to identify putative stem cell markers, to characterise an isolated stem cell population and to relate these to cells expressing ERα. ERα-positive cells were found to coexpress the putative stem cell markers p21CIP1 and Msi-1. Human breast epithelial cells with Hoechst dye-effluxing 'side population' (SP) properties characteristic of mammary stem cells in mice were demonstrated to be undifferentiated cells by lack of expression of myoepithelial and luminal epithelial membrane markers. These SP cells were sixfold enriched for ERα-positive cells and expressed several-fold higher levels of the ERα, p21CIP1 and Msi1 genes than non-SP cells. In contrast to non-SP cells, SP cells formed branching structures in matrigel which included cells of both luminal and myoepithelial lineages. The data suggest a model where scattered ERα-positive cells are stem cells that self-renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells.In recent studies we have been investigating breast cancers for the presence of a stem cell population. Using a nonadherent culture method analogous to neurosphere culture that enriches for neural stem cells, we have demonstrated that breast cancer cell lines and primary tumours contain a self-renewing population that is highly regulated by the Notch receptor signaling pathway. Inhibitors of this pathway could represent a new therapeutic modality in breast cancer, perhaps through combination with current treatments.In order to discover novel pathways that regulate stem cell self-renewal, we have applied functional genomics using an RNAi library targeting ~8,000 genes involved in cancer. This has revealed the importance of several pathways not previously associated with stem cell self-renewal. These pathways may represent novel targets for breast cancer therapy aimed at the breast cancer stem cells that survive conventional therapies.