Hypoxia-tolerant apical-out intestinal organoids to model host-microbiome interactions

Abstract

Microbiome is an integral part of the gut and is essential for its proper function. Imbalances of the microbiota can be devastating and have been linked with several gastrointestinal conditions. Current gastrointestinal models do not fully reflect the in vivo situation. Thus, it is important to establish more advanced in vitro models to study host-microbiome/pathogen interactions. Here, we developed for the first time an apical-out human small intestinal organoid model in hypoxia, where the apical surface is directly accessible and exposed to a hypoxic environment. These organoids mimic the intestinal cell composition, structure and functions and provide easy access to the apical surface. Co-cultures with the anaerobic strains Lactobacillus casei and Bifidobacterium longum showed successful colonization and probiotic benefits on the organoids. These novel hypoxia-tolerant apical-out small intestinal organoids will pave the way for unraveling unknown mechanisms related to host-microbiome interactions and serve as a tool to develop microbiome-related probiotics and therapeutics.

Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death

“…Similarly, CHGA and MUC2 show no change. Interestingly, Co et al show increased expression of enterocyte marker gene sucrose isomaltase ( SI ), while Kakni et al demonstrate slightly decreasing levels of enterocyte marker gene villin‐1 ( VIL1 ) 12,18 . Using our canine organoids, we demonstrate that overall, key marker genes are relatively stably expressed in our basal‐out and apical‐out organoids.…”

“…show increased expression of enterocyte marker gene sucrose isomaltase (SI), while Kakni et al demonstrate slightly decreasing levels of enterocyte marker gene villin-1 (VIL1). 12,18 Using our canine organoids, we demonstrate that overall, key marker genes are relatively stably expressed in our basal-out and apical-out organoids. While LGR5 and MUC2 expression decreases slightly, but non-significantly, VIL1 expression is slightly increased 72 h after polarity reversal.…”

“…Apical‐out organoids were first published by Co et al, who have demonstrated that human intestinal organoids that usually grow embedded in a three‐dimensional extracellular matrix (ECM) can reverse their polarity when they are cultured in a floating manner in medium without ECM 12 . This method has now been adopted by many other labs and expanded to different animal species, for example, pigs, 13 chickens, 14 cows 15 and dogs, 16 and also organoids derived from human iPSCs and embryonic stem cells 17,18 . Remarkably, regardless of the species from which the organoids were derived, the majority of these studies utilised their apical‐out organoids within the initial 7 days following the induction of polarity reversal 13,16,17,19–21 .…”

“…According to previous reports, whether the induction of polarity reversal is associated with organoid differentiation is to some extent controversial. Two studies show that apical‐out organoids do not present immensely different gene expression profiles compared to their basal‐out counterparts as long as they are cultured in their standard growth medium 12,18 . However, according to another study using porcine organoids, the expression of the stem cell marker termed leucine‐rich repeat‐containing G‐protein coupled receptor 5 ( LGR5 ) is diminished after 3 days of apical‐out culture and differentiation markers chromogranin A ( CHGA ; enteroendocrine cells), mucin‐2 ( MUC2 ; goblet cells) and intestinal alkaline phosphatase ( ALPI ; enterocytes) show strikingly elevated expression.…”

Modelling esophageal adenocarcinoma and Barrett’s esophagus with patient-derived organoids

“…Unsurprisingly, microinjection also requires precise technical skills and limits the throughput capacity for co-culture experiments. Alternatives to microinjection involve manipulation of cells to reverse the apical-basolateral polarity of cells ( Co et al, 2021 ; Kakni et al, 2023 ) or re-seeding of organoids as monolayers to expose the apical surface ( VanDussen et al, 2015 ). The former of these is technically challenging whilst the latter results in loss of 3D structure and organization, reducing the physiological relevance of the model.…”