Figure 2

Heterotypic three-dimensional cultures maintain tri-potent mammary epithelial precursor cells. (A) Schematic of primary human breast epithelial cells (PHBECs) and human adipose tissue-derived mesenchymal stem cells (hAMSCs) co-culture, phase-contrast (left) and fluorescence images of clusters of the co-cultures grown for 30 days on an extracellular matrix (ECM)-coated mesh. Scale bar 100 μm. (B) Three-dimensional reconstruction of 28 z-sections (0.6 μm) from confocal images of a 30-day-old co-culture of tagged-PHBECs (VenusNLS, green) and hAMSCs (DsRed2, red) on ECM-coated mesh. Autofluorescence of nylon mesh (blue). Scale bar 10 μm. (C) Confocal images of K14- and K19-stained 60-day-old co-cultures of untagged cells: 4',6-diamidino-2-phenylindole (DAPI)- (blue) stained nuclei. Arrows indicate double-positive putative bi-potent epithelial progenitor cells. Scale bar 50 μm. (D) Three-dimensional reconstruction of 48 z-sections (0.4 μm) from confocal images of K14-, K19- and DAPI-stained co-cultures (cells without fluorescent protein tags were stained). Scale bar 10 μm. Arrows indicate double-positive cells. (E) Percentages of K14, K19 and K14/K19 double-positive cells in co-cultures. K14 versus K19, P = 0.10714; K19 versus K14K19, P = 0.00019; K14 versus K14K19, P = 0.01444. (F) Colony formation assay. Fluorescence images of K14- and K19-stained colonies formed by single cells derived from co-cultures of untagged PHBECs and hAMSCs. DAPI- (blue) stained nuclei. Scale bar 50 μm. (G) Percentage of colonies formed by single cells derived from co-cultures. Total: all colonies (n >8 cells); large: large mixed colonies (n >32 cells, >4 doublings); small: small colonies (n = 8 to 32 cells, 2 to 4 doublings). Small versus large, P = 0.46563; total versus large P = 0.13752; total versus small, P = 0.29473. (H) Lactogenesis assay. Fluorescence images of K14-, K18- and β-casein-stained colonies derived from single cells from co-cultures grown in lactogenic differentiation conditions. DAPI- (blue) stained nuclei. Lower right corner: 2.5-fold magnification of merged image. Scale bars 50 μm.