A Human 2D Primary Organoid-Derived Epithelial Monolayer Model to Study Host-Pathogen Interaction in the Small Intestine

Gut organoids are stem cell derived 3D models of the intestinal epithelium that are useful for studying interactions between enteric pathogens and their host. While the organoid model has been used for both bacterial and viral infections, this is a closed system with the luminal side being inaccessible without microinjection or disruption of the organoid polarization. In order to overcome this and simplify their applicability for transepithelial studies, permeable membrane based monolayer approaches are needed. In this paper, we demonstrate a method for generating a monolayer model of the human fetal intestinal polarized epithelium that is fully characterized and validated. Proximal and distal small intestinal organoids were used to generate 2D monolayer cultures, which were characterized with respect to epithelial cell types, polarization, barrier function, and gene expression. In addition, viral replication and bacterial translocation after apical infection with enteric pathogens Enterovirus A71 and Listeria monocytogenes were evaluated, with subsequent monitoring of the pro-inflammatory host response. This human 2D fetal intestinal monolayer model will be a valuable tool to study host-pathogen interactions and potentially reduce the use of animals in research.


Thomas Roodsant*, Marit Navis*, Ikrame Aknouch, Ingrid B. Renes, Ruurd M. van Elburg, Dasja Pajkrt, Katja C. Wolthers, Constance Schultsz, Kees C. H. van der Ark, Adithya Sridhar and Vanesa Muncan

* equal contributors



A Novel Organoid Model of Damage and Repair Identifies HNF4α as a Critical Regulator of Intestinal Epithelial Regeneration

Background & aims: Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking.

Methods: We established and characterized an in vitro model of intestinal damage and repair by applying γ-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration.

Results: We identified hepatocyte nuclear factor 4α (HNF4α) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies.

Conclusions: In conclusion, we established and validated an in vitro damage-repair model and identified HNF4α as a crucial regulator of intestinal regeneration. Transcript profiling: GSE141515 and GSE141518.

Keywords: Irradiation; Organoids; Regeneration.


Paula S Montenegro-Miranda, Jonathan H M van der Meer, Christine Jones, Sander Meisner, Jacqueline L M Vermeulen, Jan Koster, Manon E Wildenberg, Jarom Heijmans, Francois Boudreau, Agnes Ribeiro, Gijs R van den Brink, Vanesa Muncan



ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair

Background & Aims

Activation factor-1 transcription factor family members activating transcription factors 2 and 7 (ATF2 and ATF7) have highly redundant functions owing to highly homologous DNA binding sites. Their role in intestinal epithelial homeostasis and repair is unknown. Here, we assessed the role of these proteins in these conditions in an intestine-specific mouse model.


We performed in vivo and ex vivo experiments using Villin-CreERT2Atf2fl/flAtf7ko/ko mice. We investigated the effects of intestinal epithelium-specific deletion of the Atf2 DNA binding region in Atf7-/- mice on cellular proliferation, differentiation, apoptosis, and epithelial barrier function under homeostatic conditions. Subsequently, we exposed mice to 2% dextran sulfate sodium (DSS) for 7 days and 12 Gy whole-body irradiation and assessed the response to epithelial damage.


Activating phosphorylation of ATF2 and ATF7 was detected mainly in the crypts of the small intestine and the lower crypt region of the colonic epithelium. Under homeostatic conditions, no major phenotypic changes were detectable in the intestine of ATF mutant mice. However, on DSS exposure or whole-body irradiation, the intestinal epithelium showed a clearly impaired regenerative response. Mutant mice developed severe ulceration and inflammation associated with increased epithelial apoptosis on DSS exposure and were less able to regenerate colonic crypts on irradiation. In vitro, organoids derived from double-mutant epithelium had a growth disadvantage compared with wild-type organoids, impaired wound healing capacity in scratch assay, and increased sensitivity to tumor necrosis factor-α–induced damage.


ATF2 and ATF7 are dispensable for epithelial homeostasis, but are required to maintain epithelial regenerative capacity and protect against cell death during intestinal epithelial damage and repair.


Bartolomeus J. Meijer, Francesca P. Giugliano, Bart Baan, Jonathan H.M. van der Meer, Sander Meisner, Manon van Roest, Pim K. Koelink, Ruben J. de Boer, Nic Jones, Wolfgang Breitwieser, Nicole N. van der Wel, Manon E. Wildenberg, Gijs R. van den Brink, Jarom Heijmans, and Vanesa Muncan



Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning

During the suckling-to-weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium-derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivo In vitro postnatal development of the fetal-derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo Together, our data show that organoids derived from fetal epithelium undergo suckling-to-weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.


Marit Navis, Tania Martins Garcia, Ingrid B Renes, Jacqueline Lm Vermeulen, Sander Meisner, Manon E Wildenberg, GIjs R van den Brink, Ruurd M van Elburg, Vanesa Muncan



Blimp1 regulates the transition of neonatal to adult intestinal epithelium

In many mammalian species, the intestinal epithelium undergoes major changes that allow a dietary transition from mother’s milk to the adult diet at the end of the suckling period. These complex developmental changes are the result of a genetic programme intrinsic to the gut tube, but its regulators have not been identified. Here we show that transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1) is highly expressed in the developing and postnatal intestinal epithelium until the suckling to weaning transition. Intestine-specific deletion of Blimp1 results in growth retardation and excessive neonatal mortality. Mutant mice lack all of the typical epithelial features of the suckling period and are born with features of an adult-like intestine. We conclude that the suckling to weaning transition is regulated by a single transcriptional repressor that delays epithelial maturation.


Vanesa Muncan, Jarom Heijmans, Stephen D Krasinski, Nike V Buller, Mano E Wildenberg, Sander Meisner, Marjana Radonjic, Kelly A Stapleton, Wout H Lamers, Izak Biemond, Marius A van den Bergh Weerman, Donal O’Carroll, James C Hardwick, Daniel W Hommes, Gijs R van den Brink