Mammospheres contain multipotent cells, capable of differentiating along all three lineages in the mammary gland. (A) Monolineage ductal epithelial colony immunostained for ESA. (B) Monolineage myoepithelial colony immunostained for CD10. (C) Bilineage colony (ESA, red; CD10, purple). The percentage of cells with bilineage differentiation potential is considerably higher in primary mammospheres compared with dissociated tissue. (D) Secondary mammospheres contain virtually only bipotent progenitors. (E) Triple-lineage colony (ESA, red; CD10, purple). (F) The same colony immunostained for β-casein (FITC, green) to identify the third lineage, alveolar cells. (G) Triple-lineage colony (ESA, brown; CD10, purple; β-casein, red). (H) Distribution of colony types generated by mammospheres derived cells in conditions that allow differentiation along all three lineages. (I) Ductal-acinar structure generated by a single cell in 3D Matrigel culture. (J) Acinar structure generated by a single cell in 3D Matrigel culture. (K) Acinar structure, immunostaining for myoepithelial lineage (CD10, FITC, green) and ductal epithelial lineage (ESA, Texas red). (L) Acinar structure, immunostaining for β-casein (red).