Stem/progenitor cells in murine mammary gland: isolation and functional characterization.

The presence of adult functional mammary epithelial stem/progenitor cells in mammary gland and recent identification of enriched fraction of transplantable mammary stem cells (MaSCs) through specific cell surface markers have revolutionized the study to delineate the role of mammary stem/progenitor functions in both mammary gland development and mammary tumorigenesis. In this chapter, we have described detailed methods of isolation of mammary epithelial cells from murine mammary glands, enrichment of stem/progenitor fractions by fluorescence-activated cell sorting (FACS), in vitro mammosphere culture, and differentiation assays in two- and three-dimensional culture. We have also described the detailed protocol of in vivo cleared mammary fat pad transplantation assay for the assessment of the MaSC repopulating activity, which indicates their multilineage differentiation and self-renewal potential in vivo and is considered the "gold standard" assay of functional stem cells.