Extended Data Fig. 1: Characterization of aged intestine.

a, Organoid-forming capacity of crypts from young and old mice (n = 4 mice per group). Student’s paired t-test. b, Frequency of organoids unable to form new crypts (fission deficiency) in young and old mice (n = 6 mice per group) analysed 5–9 days after isolation. Student’s paired t-test. c, Distribution of regenerative growth capacity of primary organoids from young and old mice (n = 6 mice per group). d, Regenerative growth of subcultured secondary mouse organoids (n = 6 mice per group). Student’s paired t-test. e, Distribution of regenerative growth capacity of subcultured secondary organoids from old and young mice (n = 6 mice per group). f, Representative H&E staining of mouse jejunal sections from young and old mice (four mice analysed per group). g, Quantification of EdU⁺ cells in jejunal crypts 2 h after administration. Only cells next to lysozyme⁺ Paneth cells were quantified as CBC stem cells. Crypt cells that were not touching lysozyme⁺ cells were quantified as transit-amplifying (TA) cells (n = 5 mice per group). Representative image of crypt stained for EdU (cyan), DAPI (nuclei, blue), lysozyme (white) and E-cadherin (red). Scale bar, 20 μm. h, Quantification of Ki67⁺ cells in human ileal biopsies. Cells at the crypt bottom with elongated nuclei next to postmitotic Paneth cells were counted as CBCs. Cells not at the crypt base were considered transit-amplifying cells. (n = 6 for 20–25-year-old donors, n = 10 for ≥ 75-year-old donors). i, Representative gating of Lgr5^hi, Lgr5^med, Lgr5^lo, Paneth and enteroendocrine cells (in relation to Fig. 1c). Quantification of enteroendocrine cells (n = 30 young, n = 26 old). For FACS gating strategy, see Supplementary Fig. 1. j, Analysis of human ileal biopsy material for lysozyme⁺ Paneth cells (n values for analysed samples shown). k, Immunostaining and quantification of Olfm4⁺ stem and progenitor cells (green background, n = 75 crypts from young and old. Five individuals per age group) and lysozyme⁺ Paneth cells in jejunal crypts (red background, n = 115 crypts from young and n = 117 crypts from old mice, five individuals per age group). Whiskers plotted according to Tukey’s method. Scale bars, 10 μm. l, Ratio of Lgr5^hi stem cells and Lgr5^med progenitor cells and ratio of Lgr5^hi stem cells and Paneth cells analysed by flow cytometry from isolated crypts (n = 30 young, n = 26 old). Whiskers plotted according to Tukey’s method. m, Regenerative growth of young Lgr5^hi stem cells co-cultured with young or old Paneth cells. Quantification at day 8–11 (n = 6). Representative images are from day 8. Scale bar, 100 μm. Student’s paired t-test. n, Long-term clonogenicity of young and old Lgr5^hi stem cells co-cultured with young and old Paneth cells. Serially passaged organoids were quantified 21 days after initial plating (n = 14 mice per age group). Combinations compared to average of young Lgr5^hi cells co-cultured with young and old Paneth cells. o, Fourteen-day co-culture of Paneth cells from tdTomato-expressing mouse (R26-mTmG) with Lgr5^hi stem cells from Lgr5-eGFP-IRES-creERT2 mouse show long-term niche interactions in organoid culture. Scale bar, 100 μm. Similar results were seen in three replicate wells from co-cultures of the same mice. Y, mice between 3 and 9 months of age; O, mice over 24 months of age in all experiments. In box plots, unless otherwise indicated, the line represents median, the box shows interquartile range and whiskers represent the range. All other data are mean ±  s.d.; two-tailed unpaired Student’s t-test; exact P values shown in corresponding panels.

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