Figure 3.

Effect of modulators of Notch signaling on self-renewal of mammary stem cells and on lineage commitment of mammary progenitor cells. (a)–(g) Effect of Notch agonist and antagonist treatment on primary and secondary mammosphere formation. Primary and secondary mammospheres were grown in standard conditions (a, d), in the presence of DSL peptide (b, e) and in the presence of N4 blocking antibody (N4Ab) (c, f). Primary mammospheres grown in the presence of N4 antibody (c) are smaller than the control (a). Secondary mammosphere formation is completely blocked by treatment with Notch4 blocking antibody (d, f). The addition of DSL peptide increases primary, secondary and tertiary mammosphere formation (a, b, d, e, g). Data are presented as the mean ± standard error of the mean. The calculated number of multilineage progenitor cells (number of spheres × % multilineage progenitors) shows a sevenfold increase after one passage, and a 100-fold increase after two passages, in mammospheres cultured in the presence of DSL compared with mammospheres cultured without added DSL. (h)–(k) Effect of Notch activation on lineage specification of human mammary progenitor cells. DSL treatment of mammospheres in suspension culture (DSL1) and on mammosphere-derived cells on the collagen substratum (DSL2) increases the number of myoepithelial progenitors, as shown by the clonogenic assay (h), and increases the rate of proliferation of bipotent and myoepithelial progenitors, as reflected by the colony size (i). DSL treatment in suspension culture increases the percentage of myoepithelial cells, as shown by flow-cytometry analysis (j). Cells were stained red (phycoerythrin [PE]) with myoepithelial marker CD10, and green (FITC) with the ductal epithelial marker epithelial-specific antigen (ESA). Treatment with DSL peptide does not have a lineage selective effect on differentiated cells, as shown by flow-cytometry analysis of human mammary epithelial cells passaged twice on collagen (k). Data are presented as the mean ± standard error of the mean.