Fig. 5: ALA delays ISC aging through a Paneth cell-dependent mechanism.

a–f Lgr5^hi cells from young Lgr5-EGFP mice and CD24⁺ Paneth cells from both young and old mice were sorted and co-cultured in matrigel. Schematic diagram illustrating the sorting procedure of ISCs and Paneth cells using flow cytometry (a). Representative FACS plots of Lgr5-EGFP⁺ cells and CD24⁺ Paneth cells from the intestinal organoids of young Lgr5-EGFP mice and old C57BL/6 mice (b). (n = 3 biologically independent mice per group). Representative images of Lgr5^hi ISCs co-cultured with Paneth cells from young and old mice at day 3 (c) and 8 (d). (scale bars: 250 μm). The number of colonies (e) and buds (f) at day 8. (e): n = 3 biologically independent co-culture replicates per group. (f): n = 9 organoids/group, obtained from 3 biologically independent mice. g–k The diagram (g) showing the workflow for preparing conditioned media from organoids of young and old mice. Lgr5^hi cells were cultured with conditioned media or conditioned media+ALA. Representative images (h, i, scale bars, 250 μm) of organoids derived from young Lgr5^hi ISCs. Quantification of organoids (j) and organoid buds (k) in each group. (j): n = 4 biologically independent mice replicates per group. (k): n = 12 organoids/group, obtained from 4 biologically independent mice. Unless otherwise indicated, data are presented as the mean ± SD. one-way ANOVA with Tukey’s multiple comparison test (e, f, j, k); for (k), p = 1e−7 for young CM vs old CM. Source data are provided as a Source Data file.