In Vitro Modeling of Age-Related Methylation Changes with Intestinal Organoids [RRBS]

Intestinal organoids, three-dimensional cultures from intestinal stem cells, are a powerful model for studying aging, and DNA methylation is an accurate biological clock. Consequently, we hypothesized that organoid DNA methylation could serve as an aging metric and a valuable tool for in vitro aging research. Our initial study revealed significant DNA methylation changes, during organoid culture, with 27% of total CpG sites undergoing hypomethylation, and 11% gaining hypermethylation. Hypomethylation occurred predominantly in aging-associated genomic regions, including non-promoter, non-CpG island regions (e.g., transposable elements), while hypermethylation, in CpG islands, significantly (p < 0.001) correlated with aging. Comparison of aging-methylated sites with differentiation-specific CpG sites showed minimal overlap, indicating negligible association. Early-passage (P0 and P2) organoids, derived from 4- and 24-month-old mice, preserved aging-specific methylation patterns, with a correlation coefficient of 0.48 (p < 0.001) between methylation differences in old versus young primary cells. Conversely, long-term passaging revealed distinct methylation changes, specific to each organoid line. Some early passage organoids exhibited more hypomethylation, clustering with mid-passage (P10 - P13) organoids, while late-passage (P24 and P27) organoids entered a crisis stage with severe hypomethylation, growth arrest, and distant clustering. Transposable elements remained hypomethylated, compared to primary cells. Aging-related methylation sites continued to change with passage, and linear modeling predicted that organoids age at a rate of 0.46 months per week in culture. Treatment with decitabine reversed the methylation age of organoids, derived from 24-month-old mice. These findings suggest that organoids effectively model aging, with further research needed to assess culture-associated influences. Overall design: Intestinal crypts from 4-month-old and 24-month-old mice were isolated to generate organoids, which were then used for DNA and RNA extraction. These organoids underwent RRBS (Reduced Representation Bisulfite Sequencing) and RNA-seq analysis to investigate age- and passage-related changes in DNA methylation and gene expression. This approach allowed us to assess how aging and extended culturing influence the molecular landscape of the organoids

肠道类器官（intestinal organoids）是源自肠道干细胞的三维培养体系，是研究衰老的强效模型；而DNA甲基化（DNA methylation）则是精准的生物时钟。据此，我们提出假设：类器官的DNA甲基化可作为衰老量化指标，同时也是体外衰老研究的实用工具。
我们的初步研究显示，类器官培养过程中存在显著的DNA甲基化改变：全体CpG位点中27%发生低甲基化（hypomethylation），11%出现高甲基化（hypermethylation）。低甲基化主要发生在衰老相关基因组区域，包括非启动子、非CpG岛区域（例如转座元件（transposable elements））；而CpG岛内的高甲基化则与衰老呈显著相关性（p < 0.001）。
将衰老相关甲基化位点与分化特异性CpG位点进行比对，发现二者重叠度极低，提示二者关联可忽略不计。源自4月龄与24月龄小鼠的早期传代（P0及P2）类器官保留了衰老特异性甲基化模式，老年与幼年原代细胞（primary cells）的甲基化差异间相关系数为0.48（p < 0.001）。反之，长期传代则出现不同类器官系特有的特异性甲基化改变。
部分早期传代类器官呈现更显著的低甲基化，与中期传代（P10-P13）类器官聚为一类；而晚期传代（P24及P27）类器官则进入危机阶段，表现为严重低甲基化、生长停滞，并形成独立聚类。相较于原代细胞，转座元件仍维持低甲基化状态。衰老相关甲基化位点随传代持续改变，线性模型预测类器官在培养过程中的衰老速率为每周0.46个月龄。
用地西他滨（decitabine）处理可逆转源自24月龄小鼠的类器官的甲基化衰老时钟。上述结果表明，类器官可有效模拟衰老过程，后续仍需开展研究以评估培养过程带来的相关影响。

整体实验设计：分离4月龄与24月龄小鼠的肠道隐窝以构建类器官，随后用于DNA与RNA提取。对这些类器官进行RRBS（简化代表性亚硫酸氢盐测序，Reduced Representation Bisulfite Sequencing）及RNA-seq（RNA测序）分析，以探究DNA甲基化与基因表达的衰老及传代相关变化。该实验方案可用于评估衰老与长期培养如何影响类器官的分子图谱。