Experimental design for assessing self-renewal and differentiation potential of cells grown as mammospheres. (a) Self-renewal is assessed by evaluating the ability of mammosphere-derived cells to form new spheres, containing multipotent cells. (b) Differentiation into all the three mammary lineage types on collagen in the presence of serum [immunostained with lineage-specific markers: brown, ductal epithelial (ESA); purple, myoepithelial (CD10); red, alveolar (β-casein)]. (c) Generate complex ductal-alveolar structures in three-dimensional Matrigel culture. (d) Differentiation and self-renewal in vivo are tested by implanting human mammary epithelial cells into the cleared mammary fat pads of immunodeficient mice (NOD/SCID mice). EGF, epidermal growth factor.