Single-cell analysis of intestinal epithelial organoids reveals aging-associated differentiation delay conserved between in vivo and ex vivo

Intestinal epithelial stem cells (ISCs) are essential for maintaining the structural and functional integrity of the gut by continuously replenishing the intestinal epithelium. However, the effects of aging on the identity and function of ISCs and their progeny remain poorly understood. In this study, we performed single-cell RNA sequencing (scRNA-seq) to analyze the cellular composition and transcriptional landscape of intestinal epithelial organoids derived from young and aged mice. To complement and validate our organoid findings, we also examined publicly available scRNA-seq datasets from intestinal epithelial tissues of mice across different age groups. Our analysis revealed that ISCs from aged mice exhibited impaired differentiation trajectories, with a pronounced delay in the transition to transit-amplifying (TA) cells and later stages. Remarkably, the same differentiation delay and transcriptional changes were observed in native intestinal tissues, indicating that organoids can serve as faithful models for studying aging of stem cell-derived epithelial tissue. Additionally, a delay in differentiation was also observed in organoids derived from aged mice, manifested as a delayed onset of budding structures. Our results presented fundamental insights into the aging of ISCs conserved between in vivo and ex vivo and established an ex vivo platform for evaluating therapeutic interventions targeting intestinal regeneration in aging.