Fig. 1: Crypt-like regions are spatially controlled by Wnt3a and EphrinB1 micropatterns.

a Schematic of small intestine crypt, showing a decreasing Wnt3a gradient toward the crypt top and increasing EphrinB1 expression toward the villus. b Simplified model with on/off Wnt3a and EphrinB1 patterns in an intestinal monolayer culture, top view and cross-section. Yellow cells indicate the area covered by Wnt3a patterns, green cells indicate the area covered with EphrinB1, and stem cells are marked in light green. c Representative fluorescence images of micropatterns of EphB2 receptors and EphrinB1 ligands printed on treated rBM-coated substrates. Images were immunostained for EphB2, EphrinB1 and laminin (Ln). Scale bars: 50 µm. d Representative fluorescence images of micropatterns of Wnt3a and EphrinB1 printed on treated rBM-coated substrates. Images were immunostained for Wnt3a, EphrinB1, and laminin. Scale bars: 2 mm (left-panels) and 50 µm (right-panels). e Left-panel and middle-panel (zoom): Representative images of organoid-derived single cells seeded on treated rBM at 24 h of culture. Cells were immunostained for Ki67, GFP (Lgr5⁺ stem cells), F-Actin and cell nuclei (DNA). Scale bars: 50 μm. Right-Panel: Graph plotting the percentage of positive cells (from the total cell number counted) for GFP at 24, 72 and 120 h. Mean ± SEM. N = 3 experiments. f Representative images of monolayers of organoid-derived single cells seeded on treated rBM. Cells were immunostained for Ki67, EphB2 receptor and F-Actin. Scale bars: 50 μm. g Left-panel and middle-panel (zoom): Representative images of organoid-derived single cells seeded on treated rBM at 48 h. Cells were immunostained for Ki67, Lysozyme (Lyz), F-Actin and DNA. Scale bars: 50  μm. Right-panel: Graph plotting the number of Lysozyme⁺ cells per crypt at 48 h of culture. Mean ± SEM. N = 3 experiments. h Representative images of organoid-derived single cells seeded on treated rBM at 48 h. Cells were immunostained for Ki67, Wnt3a and EphrinB1. Scale bars: 20 μm. i Graph plotting the probability of hosting a crypt for Wnt3a and EphrinB1 patterns. Mean ± SEM. N_Wnt3a = 3 and N_EphrinB1 = 4 experiments. Source data are provided as Source Data file.