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The mammary gland of normal adult virgin female mice contains cells that regenerate a complete new gland containing both ductal and lobular elements when transplanted at limiting dilutions into surgically cleared mammary fat pads of 3-week-old syngeneic female hosts. We have used this powerful quantitative in vivo assay in combination with immunomagnetic and FACS methods to characterize adult mammary stem cells phenotypically and biologically and to develop a method that allows their purification to near homogeneity. The frequency of these cells in the adult mammary gland is approximately 10-4; i.e., 250-fold lower than the frequency of co-isolated mammary epithelial progenitor cells that form adherent colonies of differentiated luminal and/or myoepithelial cells in vitro. Functional analysis of adult mammary cells stained with Hoechst 33342 and Pyronin Y showed all of the transplantable stem cells to be in either G1 or S/G2/M. A Go fraction of mammary stem cells could not be detected and all of the stem cells retained the dye, Rhodamine-123. Hoechst 33342 staining also revealed the presence of a side population (SP) of Hoechst-effluxing cells but these contained less than 10% of all the mammary stem cells. Adult mouse mammary stem cells could be isolated at a purity of 1 in 19 cells based on their high expression of CD49f and CD24, low but positive expression of Sca-1, and lack of expression of CD45, Ter119 and CD31 (antigens used to remove blood cells and endothelial cells). Assays of glands regenerated in primary recipients of 12 such purified mammary cells produced similar complete glands in secondary mice, indicating the input stem cells had undergone at least 10 symmetric self-renewal divisions and many more self-renewal divisions assuming some were asymmetric. These results demonstrate that the mammary stem cells of normal adult virgin female mice differ from other described stem cell populations in their lack of a quiescent fraction and inability to efflux Hoechst 33342 or Rhodamine-123. These features of mammary stem cells are consistent with an intrinsic susceptibility to mutagenic toxins and a physiology that allows their continuous accumulation of genetic perturbations.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]