Luminal Epithelial Cell Co-culture Assay

CD271^low/MUC1^high primary luminal epithelial cells were seeded at 6000 cells/cm² on confluent fibroblast feeders of CD105^high/CD26^low and CD105^low/CD26^high cells, respectively, in modified breastoid base medium without HEPES [9 (link)] (DMEM/F-12, 1:1), 1 μg/ml hydrocortisone (Sigma-Aldrich), 9 μg/ml insulin (Sigma-Aldrich), 5 μg/ml transferrin (Sigma-Aldrich), 5.2 ng/ml Na-Selenite (BD Industries), 100 μM ethanolamine (Sigma-Aldrich), 20 ng/ml basic fibroblast growth factor (PeproTech), 5 nM amphiregulin (R&D Systems), with the addition of 10 μM Y-27632 (Axon Medchem), 1.8 × 10⁻⁴ M adenine (Sigma Aldrich) and the serum replacement B27 (20 μl/ml, Life Technologies) [6 (link)]. hMSC feeders cultured under similar conditions were used for comparison.
To determine whether luminal progenitors and differentiated cells responded differently to co-culture, FACS-sorted CD166 (ALCAM)^high/laminin receptor 67LR^high (EpCAM^high/CD166^high/LNR67^high) or CD166^high (EpCAM^high/CD90^low/CD166^high) differentiated luminal cells versus 67LR^low (EpCAM^high/CD166^low/LNR67^low) or CD166^low (EpCAM^high/CD90^low/CD166^low) progenitors [6 (link)] were confronted with either CD105^high/CD26^low or CD105^low/CD26^high cells under similar conditions. Epithelial structure formation was observed for up to three weeks by phase contrast microscopy and photographed (Leica DM IL).