Evidence for a multipotent mammary progenitor with pregnancy-specific activity

Introduction: The mouse mammary gland provides a powerful model system for studying processes involved in epithelial tissue development. Although markers that enrich for mammary stem cells and progenitors have been identified, our understanding of the mammary developmental hierarchy remains incomplete. Methods: We used the MMTV promoter linked to the reverse tetracycline transactivator to induce H2BGFP expression in the mouse mammary gland. Mammary epithelial cells (MECs) from virgin mice were sorted by flow cytometry for expression of the mammary stem cell/progenitor markers CD24 and CD29, and H2BGFP. Sorted populations were analyzed for in vivo repopulation ability, expression of mammary lineage markers, and differential gene expression. Results: The reconstituting activity of CD24(+)/CD29(+) cells in cleared fat pad transplantation assays was not distinguished in GFP(+) compared to GFP-subpopulations. However, within the CD24(+)/CD29(lo) luminal progenitor-enriched population, H2BGFP(+), but not H2BGFP(-), MECs formed mammary structures in transplantation assays; moreover, this activity was dramatically enhanced in pregnant recipients. These outgrowths contained luminal and myoepithelial mammary lineages and produced milk, but lacked the capacity for serial transplantation. Transcriptional microarray analysis revealed that H2BGFP(+)/CD24(+)/CD29(lo) MECs are distinct from H2BGFP(-)/CD24(+)/CD29(lo) MECs and enriched for gene expression signatures with both the stem cell (CD24(+)/CD29(+)) and luminal progenitor (CD24(+)/CD29(lo)/CD61(+)) compartments. Conclusions: We have identified a population of MECs containing pregnancy-activated multipotent progenitors that are present in the virgin mammary gland and contribute to the expansion of the mammary gland during pregnancy.