Quantitation of Human Mammary Epithelial Stem Cells with in Vivo Regenerative Properties Using a Subrenal Capsule Xenotransplantation Assay

Methods to identify and enumerate primitive, and typically rare, undifferentiated cells in normal tissue using functional endpoints are powerful tools for acquiring insights into the mechanisms that regulate normal tissue stem cell turnover and differentiation. In this paper, we describe a xenotransplantation-based protocol that allows mammary stem cells with in vivo tissue regenerative properties to be specifically detected and quantified among the heterogeneous cell populations obtained from dissociated normal human mammary tissue. This methodology involves implanting a collagen gel containing the test cells in combination with supportive fibroblasts under the kidney capsule of highly immune-deficient, hormone-supplemented mice and then, 4 weeks later, searching for regenerated human cells with in vitro clonogenic activity. Quantification of the input human mammary stem cells is achieved using standard limiting dilution transplant approaches. This approach circumvents the need to modify the mouse mammary fat pad, and is objective, rapid (∼5 weeks) and economical to perform.