Figure 3.

Experimental design for assessing the differentiation and self-renewal potential of cells grown as mammospheres. (A) Differentiation is tested by plating cells at clonogenic densities. (i) Differentiation into ductal and myoepithelial cells is assessed on cells cultivated on collagen in the presence of serum. (ii) Differentiation into ductal, myoepithelial, and alveolar cells is tested in the same conditions as in i with the addition of prolactin and Matrigel as an overlayer. (iii) The ability to generate complex ductal-alveolar structures is tested in 3D Matrigel culture. (B) Self-renewal is tested by evaluating the ability of mammosphere-derived cells to form new spheres, containing multipotent cells.