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Background <p>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.</p> Results <p>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 (<i>p</i> &lt; 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 (<i>p</i> &lt; 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&#xa0;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.</p> Conclusions <p>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&#xa0;months, demonstrating the potential of organoids as a model for studying extrinsic influences on age-related DNA hypermethylation.</p>

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Modeling age-related methylation changes in vitro with intestinal organoids

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月6日

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  • Himani Vaidya,
  • Gennaro Calendo,
  • Kelsey Keith,
  • Anthony Pompetti,
  • Curt Balch,
  • Jozef Madzo,
  • Jaroslav Jelinek,
  • Jean-Pierre J. Issa

摘要

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.