A high proportion of myoepithelial cells have mammary stem cell potential. Mus musculus

The basal cell lineage in the mouse mammary gland is perceived to be composed mostly of differentiated myoepithelial cells intermixed with a rare subpopulation of mammary stem cells. Here, we show that a high proportion (~30%) of basal cells have colony forming capacity when actin-myosin contraction is prevented. A basal cell subpopulation enriched for mammary repopulating units (MRUs) is shown to be enriched for dividing cells. Inducing basal cells to proliferate in vitro increases MRU frequency 10-fold and permits a ~750-fold expansion of MRUs. Transplantation of single-cell-derived basal colonies into cleared fat pads demonstrates that the majority (~80%) of these colonies can repopulate a mammary gland. This suggests that a high proportion of basal cells, most of which were previously perceived to be terminally differentiated myoepithelial cells, have latent mammary stem cell potential. Mammary glands from 10-14 week old, female, virgin C57BL/6J mice were dissociated into single cells and basal cells were sorted by flow cytometry at a purity of ~95%. Inducing basal cell proliferation in vitro causes mammary stem cell expansion over 7 days in culture. Basal cells were cultured on Matrigel-coated cell culture plastic in a keratinocyte stem cell media supplemented with the Rho kinase inhibitor Y-27632 and were co-seeded with irradiated NIH 3T3 fibroblast feeder cells. In order to determine the molecular changes that occur during culture that may contribute to mammary stem cell expansion, gene expression profiling was conducted on basal cells pre-culture and after 1- or 7-day-culture. Overall design: Mammary glands from 6 C57BL/6J mice were pooled for each experiment. Purified basal cells sorted from the same pooled sample were split into 3 groups: pre-culture for immediate RNA extraction, 1-day-culture and 7-day-culture. Cultured basal cells were sorted to exclude fibroblast feeder cells. Total RNA was extracted for each group in 4 independent experiments