Human breast mesenchymal and epithelial precursor cells maintain their identities in ex vivo cell culture conditions. (A) Schematic of the experimental procedure for the isolation of mesenchymal stem cells human adipose tissue-derived mesenchymal stem cells (hAMSCs) and primary human breast epithelial cells (PHBECs) from normal breast reduction mammoplasty tissues. Arrows: mechanical (black) and enzymatic (gray) digestion steps. Isolated epithelial cells can be grown either as monolayer cultures (two-dimensional) or as three-dimensional cultures. (B) Colony formation assay with single cells derived from mammospheres grown in M5 medium. Representative images of K19 (red) and K14 (green) immunofluorescent staining of PHBEC colonies: 4',6-diamidino-2-phenylindole (DAPI)- (blue) stained nuclei. Scale bar 100 μm. (C) Differentiation assays of mesenchymal precursor cells in M5 medium. Representative images of adipocytes and osteocytes stained with lipid specific Oil Red O or alkaline phosphatase (AP), respectively. Scale bars 10 μm. (D) Schematics of ex vivo monoculture conditions of PHBEC (upper) and hAMSC (lower) with representative phase-contrast images (left) and fluorescence images of PHBECs expressing enhanced green fluorescent protein (EGFP) and hAMSCs expressing red fluorescent protein (DsRed2) after 30 days growth on an extracellular matrix (ECM)-coated mesh. Scale bars 200 μm. (E) Representative flow cytometry dot plots showing separated DsRed2-expressing mesenchymal cells (left) and EGFP-expressing epithelial cells (right) prior to RNA extraction and transcription profiling. (F) Gene set enrichment analysis (GSEA) with hAMSC-specific genes and signatures of EpCAM- CD49f- CD10+ and EpCAM + breast cells isolated from normal breast tissue. hAMSC/EpCAM- CD49f- CD10+: enrichment score (ES) = 0.87, normalized enrichment score (NES) = 3.2, false discovery rate (FDR) <0.0001, P <0.0001; hAMSC/EpCAM+: ES = −0.87, NES = −3.18, FDR <0.0001, P <0.0001. (G) GSEA with PHBEC-specific genes and signatures of EpCAM + and EpCAM- CD49f- CD10+ breast cells. PHBEC/EpCAM+: ES = 0.85, NES = 3.29, FDR <0.0001, P <0.0001; PHBEC/ EpCAM- CD49f- CD10+: ES = −0.85, NES = −3.32, FDR <0.0001, P <0.0001.