Extended Data Fig. 7: Organoids employ revSCs for functional regeneration following damage.
a) Representative images of Clu-tdTom;Lgr5-GFP organoids treated with TGFβ1 (200 pM) and 4-OHT (schematic), stained for Paneth (Lyz1), Enteroendocrine (ChgA) and Goblet cells (Muc2). Red arrows indicate cells of interest. Scale bars, 10 µm (Crypt) and 50 µm (whole organoid). N = 3 replicates. b) Schematic (top) and representative IF images (bottom) of control and 5-FU-treated (20 µM; 24 h) Lgr5-GFP-expressing organoids. Scale bar, 10 µm (Crypts), 50 µm (whole organoids). N = 3 replicates. c) Schematic (top) and brightfield images (bottom) showing organoid morphology following a 24-hour pulse of 5-FU (20 µM) and after 2 successive passages. Scale bar, 10 µm. N = 3 replicates. d) Expression of indicated ISC signature genes in organoids treated with 5-FU (20 µM; 48 h) was quantified by RT-qPCR and normalized to Ywhaz expression. Data are plotted as mean ± SEM. P= Lgr5, 0.011; Olfm4, 0.0038; Ascl2 = 0.055; Lrig1, 0.088. N = 3 replicates. e) Expression of indicated revSC signature genes in organoids treated with 5-FU (20 µM; 48 h), quantified by RT-qPCR, and normalized to Ywhaz expression. Data are plotted as mean ± SEM. P= Clu, 0.05; Anxa1, 0.003; F3, 9.6E-3. N = 3 experiments performed in triplicate (d-e). f) Clu-CreERT2;lsl-tdTom;Lgr5-GFP organoids treated with 5-FU (20 µM) and 4-OHT (top) as indicated (Schematic), and lineage-traced offspring visualized after damage, as indicated. Scale bars, 10 µm. Unpaired, one-tailed t-test; *p ≤ 0.05; ** p < 0.01; ***p < 0.001 (d and e). P, passage; d, day; h, hour; 4-Hydroxytamoxifen, 4-OHT; Clu-CreERT2;lsl-tdTom;Lgr5-GFP-DTR, ClutdTom;Lgr5-GFP. N = 3 replicates. Source numerical data available in source data.
