The mammary gland of adult female mice contains undifferentiated epithelial stem cells with in vivo regenerative and self-renewal properties. A biologically similar population likely exists in the human breast but a specific and quantitative methodology to identify and characterize these cells has been lacking. In this study I show that human mammary structures are reproducibly generated when dissociated suspensions of primary human mammary cells are propagated in collagen gels that have been implanted under the renal capsule of highly immunodeficient, hormone-treated mice. These structures contain differentiated cells of both mammary lineages in a spatial organization similar to normal mammary tissue, and display functional maturation into milk-producing glands when the hosts become pregnant. In vitro assays of single cell suspensions prepared from these regenerated glands revealed the consistent presence of mammary progenitor cells able to form adherent bi-lineage as well as pure luminal and myoepithelial colonies. In addition, when these cells are suspended in new gels and transplanted into secondary immunodeficient mice, similar progenitor-containing structures are demonstrable, indicative of a regenerative process that recreates the normal developmental hierarchy. This daughter progenitor production endpoint allows the frequency of these self-renewing human mammary stem cells to be derived from limiting dilution transplant assays as 1 per 10³–10⁴ cells in normal adult human reduction mammoplasty samples, and their phenotype to be established as CD49f⁺EpCAM⁻⁄low CD31⁻CD45⁻. I have also developed methodologies to isolate fractions of cells from mammoplasty tissue that are enriched in cells in different phases of the cell cycle (G0/G1/S/G2/M). Application of functional assays to these fractions indicates that a proportion of stem and progenitor cells in normal adult breast tissue exhibit phenotypes that are associated with actively proliferating cells. These studies support a model of mammary cell production that includes a significant rate of normal turnover of primitive cells, and sets the stage for further work to identify the factors and molecular interactions that regulate this process.Medicine, Faculty ofMedical Genetics, Department ofGraduat
