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INTESTINAL Publication-derived

DNA Methylation Defines Regional Identity of Human Intestinal Epithelial Organoids and Undergoes Dynamic Changes During Development

Source Kraiczy et al., 2019 · University of Cambridge, Department of Paediatrics, Addenbrooke's Hospital · 10.1136/gutjnl-2017-314817

👤 Judith Kraiczy, Komal M Nayak, Kate J Howell, Alexander Ross, Jessica Forbester, Camilla Salvestrini, Roxana Mustata, Sally Perkins, Amanda Andersson-Rolf, Esther Leenen, Anke Liebert, Ludovic Vallier, Philip C Rosenstiel, Oliver Stegle, Gordon Dougan, Robert Heuschkel, Bon-Kyoung Koo, Matthias Zilbauer ⏱ 30 days 📋 10 phases 🧫 Human iPSC-derived intestinal epithelial stem cells; Human fetal and pediatric intestinal epithelium

Abstract

This protocol generates human intestinal epithelial organoids (IEOs) from pediatric, adult, and fetal intestinal biopsies to investigate DNA methylation patterns and their role in regional intestinal specification. IEOs retain stable, gut segment-specific epigenetic signatures that closely reflect primary epithelium; fetal IEOs additionally demonstrate in vitro maturation with dynamic DNA methylation changes, enabling investigation of epithelial development and disease-specific epigenetic alterations.

Cell source
Human iPSC-derived intestinal epithelial stem cells; Human fetal and pediatric intestinal epithelium
Application
Disease modeling; Developmental study; Epigenetic analysis of intestinal epithelial organoids

Protocol overview

28 steps across 10 phases

Human Intestinal Sample Collection and Epithelial Cell Purification Day 0–2
  1. 1 Obtain intestinal biopsies
  2. 2 Purify intestinal epithelial cells (IECs) by enzyme digestion
Human Intestinal Epithelial Organoid (IEO) Culture Establishment and Maintenance Day 0–28+ (passages 1–11+)
  1. 1 Culture pediatric and adult IEOs
  2. 2 Culture fetal gut-derived IEOs
  3. 3 Culture gastric organoids (optional, for disease models)
RNA and DNA Extraction Day 2–5
  1. 1 Extract DNA and RNA simultaneously from organoid samples
Genome-Wide DNA Methylation Analysis Day 5–10
  1. 1 Perform bisulfite conversion of DNA
  2. 2 Perform genome-wide DNA methylation array analysis
  3. 3 Validate DNA methylation by pyrosequencing
RNA Sequencing and Transcriptome Analysis Day 5–20
  1. 1 Perform RNA sequencing
  2. 2 Validate gene expression by quantitative PCR (qPCR)
In Vitro Differentiation of IEOs Day 1–5
  1. 1 Differentiate organoids in vitro
  2. 2 Collect differentiated organoids for analysis
DNA Methyltransferase (DNMT) Inhibitor Treatment Day 0–5
  1. 1 Short-term Aza-deoxycytidine (AdC) treatment
  2. 2 Long-term AdC treatment of fetal organoids
  3. 3 Assess methylation and gene expression after AdC treatment
Immunofluorescence and Imaging Day 0–2
  1. 1 Stain organoids with fluorescent antibodies
  2. 2 Acquire fluorescent and brightfield images
CRISPR-Cas9 Genome Editing of Fetal IEOs Day 0–30
  1. 1 Construct targeting vectors for CRISPR-Cas9
  2. 2 Electroporate organoids with CRISPR-Cas9 components
  3. 3 Confirm successful genome editing
  4. 4 Culture edited IEOs and monitor phenotype
  5. 5 Analyze DNA methylation changes in edited organoids
Bioinformatic and Statistical Analysis Day 10–30
  1. 1 Analyze DNA methylation data
  2. 2 Identify differentially methylated positions (DMPs) and regions (DMRs)
  3. 3 Analyze RNA-seq transcriptome data
  4. 4 Perform statistical analysis of qPCR and pyrosequencing data
  5. 5 Perform correlation and enrichment analyses

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kraiczy et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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