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Updated: Mar 7, 2026
Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids
Published on: June 23, 2023
Modeling age-related methylation changes in vitro with intestinal organoids
Himani Vaidya1, Gennaro Calendo1, Kelsey Keith1,2
1Coriell Institute for Medical Research, Camden, NJ, 08013, USA.
Background:
Intestinal organoids, three-dimensional cultures derived from intestinal stem cells, serve as a promising model for studying aging. DNA methylation is recognized as a biological clock of aging, and we hypothesized the value of organoid DNA methylation for aging research. To test this, we compared DNA methylation patterns in organoids to those observed in primary aging intestinal epithelium.
Results:
Our genome-wide analysis revealed significant DNA methylation changes during organoid establishment and culture. Specifically, 27% of CpG sites exhibited hypomethylation, while 11% gained methylation (hypermethylation). The observed hypermethylation primarily affected CpG islands (14-25% of CpG sites) and corresponded with sites susceptible to age-related methylation in vivo (p < 0.001). Early-passage (P0 and P2) organoids derived from 4- and 24-month-old mice retained aging-specific methylation patterns, with a correlation coefficient of 0.48 (p < 0.001) between in vivo and ex vivo methylation differences. Furthermore, age-related hypermethylation continued to change with passage, with linear modeling using CpG sites that strongly associated with age from a previous dataset indicating that organoids age at an approximate rate of 2 weeks per week in culture. In contrast, hypomethylation primarily (30-36% of CpG sites) occurred in non-aging-associated genomic regions, such as non-promoter, non-CpG island regions (including transposable elements), and showed no correlation with differentiation or aging in vivo.
Conclusions:
Our findings suggest that DNA methylation changes in organoids reflect two distinct biological processes: aging, as marked by CpG island hypermethylation, and an unexplained stochastic hypomethylation defect. Additionally, treatment with the DNA methylation inhibitor decitabine reduced the methylation age of organoids from 22 to 10 months, demonstrating the potential of organoids as a model for studying extrinsic influences on age-related DNA hypermethylation.
