Fig. 1.

ISCs from aged mice exhibit significant deficiency in differentiation in primary culture. Freshly isolated crypts from 2 months old (young) and 24 months old (old) mice were plated at a density of 200 crypts per well (results show data from one representative experiment out of 2 independent experiments; n = 3 mice per group). (a, b) Absolute number (a) and percentage (b) of grown out structures on day 7 after plating. c Representative pictures of indicated groups on day 7 after plating. d,e Absolute number (d) and percentage (e) of grown out structures on day 10 after plating. f Representative pictures of indicated groups on day 10 after plating. Small cysts: diameters ≤70 μm; big cysts: diameters >70 μm; small organoids: with crypt-villus architectures, budding number ≤ 3 (for a and b) and budding number ≤ 5 (for d and e); big organoids: with crypt-villus architectures, budding number > 3 (for a and b) and budding number > 5 (for d and e). Arrow heads indicate typical organoids; arrows indicate typical big cysts. g mRNA expression of indicated genes in crypts cultured for 7 days (n = 3 independent experiments). mRNA expression of genes was normalized to beta-actin with the expression level of each gene in young crypts set to 1. Data are displayed as mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. ns, not significant. Unpaired two tailed Student’s t test was used. Scale bar: 200 μm