AbstractThe myoepithelial (MEP) lineage of human breast comprises bipotent and multipotent progenitors in ducts and terminal duct lobular units (TDLUs). We here assess whether this heterogeneity impacts on oncogenic PIK3CA transformation. Single cell RNA sequencing (scRNA-seq) and multicolor imaging reveal that terminal ducts represent the most enriched source of cells with ductal MEP markers including α-smooth muscle actin (α-SMA), keratin K14, K17 and CD200. Furthermore, we find neighboring CD200^high and CD200^low progenitors within terminal ducts. When sorted and kept in ground state conditions, their CD200^low and CD200^high phenotypes are preserved. Upon differentiation, progenitors remain multipotent and bipotent, respectively. Immortalized progenitors are transduced with mutant PIK3CA on an shp53 background. Upon transplantation, CD200^low MEP progenitors distinguish from CD200^high by the formation of multilayered structures with a hyperplastic inner layer of luminal epithelial cells. We suggest a model with spatially distributed MEP progenitors as founder cells of biphasic breast lesions with implications for early detection and prevention strategies.