Background: This study aimed to investigate how aging alters the homeostasis of the colonic intestinal epithelium and regeneration after tissue injury using organoid models and to identify its underlying molecular mechanism. Methods: To investigate aging-related changes in the colonic intestinal epithelium, we conducted organoid cultures from old (older than 80 weeks) and young (6–10 weeks) mice and compared the number and size of organoids at day 5 of passage 0 and the growth rate of organoids between the two groups. Results: The number and size of organoids from old mice was significantly lower than that from young mice (p < 0.0001) at day 5 of passage 0. The growth rate of old-mouse organoids from day 4 to 5 of passage 0 was significantly slower than that of young-mouse organoids (2.21 times vs. 1.16 times, p < 0.001). RNA sequencing showed that TGF-β- and cell cycle-associated genes were associated with the aging effect. With regard to mRNA and protein levels, Smad3 and p-Smad3 in the old-mouse organoids were markedly increased compared with those in the young-mouse organoids. Decreased expression of ID1, increased expression of p16INK4a, and increased cell cycle arrest were observed in the old mouse-organoids. Treatment with SB431542, a type I TGF-β receptor inhibitor, significantly increased the formation and growth of old-mouse organoids, and TGF-β1 treatment markedly suppressed the formation of young-mouse organoids. In the acute dextran sulfate sodium-colitis model and its organoid experiments, the colonic epithelial regeneration after tissue injury in old mice was significantly decreased compared with young mice. Conclusions: Aging reduced the formation ability and growth rate of colonic epithelial organoids by increasing cell cycle arrest through TGF-β-Smad3-p16INK4a signaling.
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