Fig. 2. Breast epithelial cell types form distinct structures in organoid culture.

a Bright field microscopy image of a representative organoid culture demonstrating multiple structure types, highlighted by red arrowheads. b Representative examples of organoid morphology for each of the indicated mammary cell lineages. c Cells from an organoid culture (representative of n = 9 cultures) were stained for EpCAM and CD49f, and sorted using FACS into mature luminal (ML, red), luminal progenitor (LP, orange), and basal/stem cell (BS, green) populations. Cells were recultured as organoids, and then assessed by bright field microscopy (n = 9), or confocal microscopy after staining for the indicated markers (column 1, columns 2–3, and columns 4–6 are from separate images, n = 3). Scale bars = 100 µm. d, e Cells of the indicated types were recultured after sorting for >6 weeks, then imaged by bright field microscopy in d, (representative of n = 9, scale bar = 100 µm), or analyzed for EpCAM and CD49f expression by flow cytometry in e. Cultures with luminal progenitor cells that differentiated to generate other mammary cell types were ORG7, ORG43, ORG46, ORG48, ORG49, and ORG51. ORG41, ORG42, and ORG50 had luminal progenitor cells that did not differentiate to generate other mammary cell types. f Luminal progenitor cells (EpCAM⁺ CD49f⁺) sorted from two different organoid cultures were recultured as organoids, stained for CK8 (green) or EpCAM (green), CK14 (red), and with DAPI (blue), and imaged by confocal microscopy (representative of n = 6, scale bar = 100 µm). g Cells were double-sorted for EpCAM and CD49f, then re-grown and assayed as in e.