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. 2017 Nov 15;68(1):49–61. doi: 10.1136/gutjnl-2017-314817

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DNA methylation profiling of human intestinal epithelial organoids (IEOs) and primary intestinal epithelial cells (IECs). (A) Summary of experimental setup. Mucosal biopsies were obtained from distal small bowel (terminal ileum=TI) and distal large bowel (sigmoid colon=SC) and used for IEC purification or generation of IEOs. Brightfield images showing established IEOs in culture. (B) Multidimensional scaling plot of genome-wide DNA methylation profiles of purified IEC (labelled as ‘x’), mucosa-derived IEOs (labelled as ‘o’), whole biopsies from SC (labelled as ‘b’) and non-epithelial cell fraction of mucosal biopsies (labelled as ‘n’). (C) Venn diagram indicating the number of significant differentially methylated positions (DMPs) as well as overlap between TI and SC purified primary cells and respective organoids (adjusted p<0.01, n=16 IEC, n=10 IEO for each gut location.) Organoids were passages 1–11. (D) Scatterplots of average DNA methylation beta values of overlapping gut-segment specific DMPs (see figure 1C) in matched IECs and IEOs of the respective gut segment. Pearson correlation r=0.948 (TI) and r=0.955 (SC), p<2.2e-16. (E) Examples of differentially methylated regions displaying gut segment-specific methylation levels in both organoids and primary epithelium. Left panel region upstream of interleukin-6 receptor (IL6R), right panel special AT-rich sequence-binding protein 2-antisense 1 (SATB2-AS1). Chromosomal location, CpGs and DNA methylation (expressed as average beta value (ranging from 0=unmethylated to 1=fully methylated) of sample groups) are displayed. (F) Validation of DNA methylation profiles by pyrosequencing of bisulfite-converted DNA, showing per cent of CpG methylation in genomic regions of IL6R and SATB2-AS1. Data are presented as mean+SD of n=4 per group and representative for two independent experiments. (G) Heatmap of top 1000 DMPs, identified by comparing purified TI versus SC epithelium, in the respective organoids profiled at various passages (P). See also online supplementary figures S1 and S2. SCo, SC organoids; SCp, SC purified; TIo, TI organoids; TIp, TI purified.