Human cancer cells identified in the tumor-free mixed mammary outgrowths are hormonal responsive, secrete human milk proteins in the mammary ducts and do not show increased ploidy. A1) Human-specific immunocytochemical staining for CD133 (red) shows CD133-positive NTERA2 cells present within the confines of the fat pad containing regenerated mammary ducts. A2) Human-specific fluorescent in situ hybridization (green, nuclear; identified with green arrows) and human-specific immunocytochemical staining for CD133 (red) shows NTERA2 cells present within the mammary outgrowths. A3) CD133-positive NTERA2 cells (red) differentiate into ERα (green) luminal epithelial cells. A4) PCR shows human specific Y chromosome present in NTERA2 cells prior to transplantation (+) as well as in the CD133enriched fraction obtained by magnetic sorting but not in normal mouse mammary epithelial cells (-) and the CD133depleted fraction. B1-B2) Basal cells express human K14 (red) and mouse K14 (green) in consecutive sections of the same duct. C) Immunocytochemical staining of mixed mammary outgrowth for human α-lactalbumin (green) and mouse caseins (red). D) Flow cytometry of propidium iodide stained cells demonstrate that mixed mammary outgrowths (green) do not contain a greater proportion of cells with abnormal ploidy as compared to cultures of mouse mammary epithelial cells (blue) or NTERA2 (red). All sections are counterstained with DAPI (blue). Scale bars A1) 20 μm, A2) and A3) 15 μm, B1) and B2) 10 μm, and C) 25 μm. Figure from Rosenfield and Smith, 2013.