Objective Human intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited. Design We performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology. Results We discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models. Conclusions Our study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.

中文翻译：

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DNA甲基化定义了人类肠道上皮类器官的区域特性，并在发育过程中发生动态变化

目的 人类肠道上皮类器官（IEO）越来越被认为是一种极具前景的转化研究工具。然而，我们对它们的表观遗传分子特征和文化行为的理解仍然有限。设计 我们对源自儿童/成人和胎儿小肠和大肠的人类 IEO 以及匹配的纯化人类肠道上皮细胞进行了全基因组 DNA 甲基化和转录组分析。此外，使用 CRISPR/Cas9 技术对类器官进行体外分化和基因组编辑。结果我们发现了稳定的表观遗传特征，这些特征定义了肠道上皮功能的区域差异，包括细胞分化过程中节段特异性基因的诱导。已建立的 DNA 甲基化谱与细胞环境无关，因为类器官在长时间培养期间保留了其区域 DNA 甲基化。与儿科和成人类器官相比，胎儿肠道来源的类器官在培养中表现出明显的 DNA 甲基化和基因表达动态变化，表明体外成熟。通过将 CRISPR/Cas9 基因组编辑应用于胎儿类器官，我们证明该过程部分受 TET1 调节，TET1 是一种参与 DNA 去甲基化过程的酶。最后，从被诊断患有胃异位症的儿童中生成 IEO，揭示了与疾病相关的持续且明显的 DNA 甲基化差异，突出了类器官作为疾病特异性研究模型的使用。结论 我们的研究表明粘膜来源的 IEO 与匹配的原代上皮细胞的表观遗传特征具有惊人的相似性。此外，这些结果表明肠道干细胞固有的 DNA 甲基化模式建立和维持区域肠道规范，并参与早期上皮发育和疾病。