Extended Data Fig. 4: Mini-gut tubes undergo rapid cell turnover and comprise key functional intestinal cell types.

a, Epithelial tissue turnover assessed through EdU pulse-chase experiments. Ten-day-old mini-guts were treated with EdU for 12 h basally and apically, followed by a chase period of four days. At 0 h after EdU removal, the majority of EdU⁺ cells resided within the crypts and adjacent regions. A 24-h EdU pulse-chase revealed distinct regions of cell proliferation that were to a large extent restricted to the crypts. Two days after the EdU pulse, numerous EdU⁺ cells were found in the lumen, suggesting the occurrence of intestinal epithelial cell migration from the crypts to the villus-like domains. Labelled cells were virtually absent four days after the EdU pulse, suggesting that tubular mini-guts underwent full turnover of the epithelium. Data are representative of one EdU labelling experiment with n = 2 replicates per condition. Scale bars, 50 μm. b, Micrograph of a mini-gut tube removed from the microchip for downstream histological sectioning and analysis. Scale bar, 200 μm. c, Histological cross-sections of 7-day-old mini-gut tubes stained with Alcian Blue showing acidic polysaccharides of the mucus layer (blue) counterstained with Nuclear Fast Red. The entire perpendicular section (left) and a higher magnification view of the goblet cells (right) are shown. Similar results were obtained for 10 sections from two biologically independent samples. Scale bars, 20 μm. d, Transmission electron microscopy cross-sectional views of 7-day-old mini-gut tube. Goblet cell (left; scale bar, 2 μm) and enterocyte brush border (right; scale bar, 0.3 μm). Data are representative of two samples. e, Gradual increase in aminopeptidase activity after induction of differentiation in mini-gut tubes. Mean ± s.d. from n = 3 biologically independent experiments.

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