Mammospheres derived from MUC1+ or CD10+ progenitors from reduction mammoplasties comprise hollow and solid structures consistent with luminal- and myoepithelial-like morphologies.

<p>(<b>A</b>) <b>Strategy for isolating luminal and myoepithelial progenitor-enriched subpopulations from reduction mammoplasty tissue.</b> Tissues were physically and enzymatically processed to epithelial-rich, single cell suspensions, then cultured for 7d in mammary epithelial growth medium to generate enough cells for fluorescence-activated cell sorting (FACS) and sphere culture. Primary cultures were stained with fluorescent antibody conjugates (MUC1-FITC, CD10-PE-Cy5), the Sytox® Blue viability stain and ‘Lineage’ cocktail (‘Lin’: CD140b-PE, CD45-PE, CD31-PE; markers of stromal fibroblasts, leukocytes and endothelia respectively). CD10 and MUC1-sorted cells were placed in sphere-promoting culture for 10d before analysis. (<b>B</b>) <b>Gating strategy for enrichment of luminal- and myoepithelial-like progenitor cells.</b> Acellular particles and dead cells were excluded based on low light scatter and Sytox® blue positivity. Non-epithelial, PE⁺ cells were also excluded, then MUC1⁺ and CD10⁺ cells were collected for sphere culture. (<b>C</b>) <b>Light microscope images (i,iii) and H&E histological sections (ii,iv–vii) of spheres generated from CD10⁺ or MUC1⁺ primary breast epithelial cells.</b> Open arrows, MUC1+-derived spheres often had limited lateral connections giving a petal-like appearance. Solid arrows, shows single cell with signet ring secretory morphology. (<b>D</b>) <b>Immunophenotypic analysis of MUC1⁺ and CD10⁺ progenitor-derived primary breast mammospheres.</b> Spheres were generated as described in A/B. Representative images from immunohistochemical analysis of the indicated antigens on FFPE preparations of spheres are shown. Images taken at 200× magnification. Scale bar 100 µm. Black arrows indicate apical membranous staining of E-cadherin and EGFR in luminal-like spheres. Red arrows indicate the mesenchymal-like spheres found rarely amongst the dominant structures formed in both CD10+ and MUC1+ sorted populations.