Fig. 2

Human breast tissue undergoes morphogenesis and differentiation in hydrogels. a Bright-field images (top) and schematic representation (bottom) of the dramatic expansion and maturation of organoids over the course of 12 days. Scale bar represents 200 μm. b Quantification of the number of ducts per organoid, lobules per organoid, and cross-sectional area of organoids during a 12-day time course (N = 9 organoids). c Immunofluorescence of luminal CK8/18 (red) and myoepithelial CK14 (green) marker expression at seeding (left), 7 days (middle), or 11 days (right) after seeding. Upon seeding cells are disorganized, but they self-organize into bilayered organoids. By 11 days after seeding, outgrowths had matured and the CK8/18⁺ cells fully lined the luminal layer, while the CK14⁺ cells were basally localized. Nuclei were stained with DAPI (blue). Inset scale bar represents 50 μm. All other scale bars represent 200 μm. Also see Additional file 2: Figure S7 and Additional file 3: Movie S1. d Immunohistochemical staining of MUC1 and GATA3 expression in organoids after 21 days of culture. Representative image from one patient is shown. e Bright-field and fluorescence overlaid image of an organoid outgrowth following infection of cells with multicolored lentivirus. Clonal tracking shows heterogeneous localization of clones during organoid morphogenesis and ductal elongation. Also see Additional file 4: Movie S2, Additional file 5: Movie S3, Additional file 6: Movie S4, and Additional file 7: Movie S5. Error bars represent standard error of the mean. A.U. arbitrary units, DAPI 4′,6-diamidino-2-phenylindole, CK cytokeratin