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DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development
  1. Judith Kraiczy1,
  2. Komal M Nayak1,
  3. Kate J Howell1,2,
  4. Alexander Ross1,3,
  5. Jessica Forbester4,
  6. Camilla Salvestrini5,
  7. Roxana Mustata6,7,
  8. Sally Perkins5,
  9. Amanda Andersson-Rolf6,7,
  10. Esther Leenen1,
  11. Anke Liebert1,
  12. Ludovic Vallier3,4,
  13. Philip C Rosenstiel8,
  14. Oliver Stegle2,
  15. Gordon Dougan4,
  16. Robert Heuschkel5,
  17. Bon-Kyoung Koo6,7,
  18. Matthias Zilbauer1,5,6
  1. 1 Department of Paediatrics, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK
  2. 2 European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, UK
  3. 3 Anne McLaren Laboratory & Department of Surgery, Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK
  4. 4 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK
  5. 5 Department of Paediatric Gastroenterology, Hepatology and Nutrition, Cambridge University Hospitals, Addenbrooke’s, Cambridge, UK
  6. 6 Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK
  7. 7 Department of Genetics, University of Cambridge, Cambridge, UK
  8. 8 Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Kiel, Germany
  1. Correspondence to Dr Bon-Kyoung Koo, Wellcome Trust MRC Stem Cell InstituteUniversity of Cambridge,Tennis Court Road, Cambridge,CB2 1QR, UK; bkk25{at} and Dr Matthias Zilbauer, Department of Paediatrics, University of Cambridge, Addenbrooke’s Hospital, Box 116, Cambridge,CB2 0QQ, UK; mz304{at}


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.

  • intestinal epithelium
  • intestinal stem cell
  • intestinal gene regulation
  • intestinal development
  • intestinal cell lines

This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See:

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  • B-KK and MZ contributed equally.

  • Contributors MZ, BK and JK designed the study; JK, KN, AAR, AR, RM, EL, AL and PR performed experiments; JK, KH, OS PR and MZ were involved in data analysis; CS, RH and SP were involved in sample acquisition; MZ, PR, GD, LV and RH were involved in funding acquisition; MZ, JK and BK wrote the manuscript. All authors contributed to the critical revision of the manuscript and approved the final version.

  • Funding This work was supported by funding from the following charitable organisations: Crohn’s in Childhood Research Association (CICRA), the Evelyn Trust, Crohn’s and Colitis in Childhood ('3Cs'), Addenbrooke’s Charitable Trust (ACT), and the Newlife Foundation for Disabled Children. JK was funded by a CICRA PhD studentship, KH was funded by an EBPOD EMBL-EBI/Cambridge Computational Biomedical Postdoctoral Fellowship. B-KK was supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [101241/Z/13/Z] and received core support from the Wellcome Trust and MRC to the WT-MRC Cambridge Stem Cell Institute. GD and JF received support from the Wellcome Trust. JF was supported by a studentship from the MRC. PR was supported by the Deutsche Forschungsgemeinschaft EXC306 Cluster ’Inflammation at Interfaces' and BMBF IHEC DEEP TP5.2.

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval NRES Committee East of England, Hertfordshire (REC-12/EE/0482) and (REC-96/085).

  • Provenance and peer review Not commissioned; externally peer reviewed.