Monolayer culture of intestinal epithelium sustains Lgr5+ intestinal stem cells

a Representative bright-field and Lgr5-EGFP fluorescence images of small intestinal epithelial cells cultured in 2D system with blebbistatin, EGF, Noggin, and R-spondin1 (BENR) for 2 days, followed by culturing in BLRC for 4 days. b E-cadherin staining of epithelium and GFP of Lgr5+ stem cells in 2D-cultured monolayers. c Confocal images of monolayers stained for EpCam (green) and DAPI (blue). Right and bottom panels suggest the horizontal (H) and vertical (V) projections. d Confocal images staining with EphB2 (red) showed the location of crypt, stem cells and Paneth cells. Asterisks mark the Paneth cells. e Confocal images stained for proliferation (Ki67+), Enterocytes (Vil+), Paneth cells (Lyz+), Goblet cells (Muc2+), enteroendocrine cells (Chga+), and apoptosis (TUNEL+). f, g Representative FACS analysis (f) and gene expression (g) in 2D or 3D system. The data were analyzed by Student’s t-test and shown as mean？±？SD. *P？<？0.05, **P？<？0.01. h Muc2 staining of Goblet cells from cells initially cultured in 2D system (7 d) and then transferred to ENR or ENR plus ID (5 d). I: IWP-2. D: DAPT. Scale bars, 50 μ