Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier

Sayed, Ibrahim M.; Tindle, Courtney; Fonseca, Ayden G.; Ghosh, Pradipta; Das, Soumita

doi:10.1016/j.xpro.2021.100680

Cited by 9 publications

(17 citation statements)

References 17 publications

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“…EDMs were infected apically at a multiplicity of infection of 1:10 in Dulbecco's modified Eagle's medium/F12 with HEPES, 10% fetal bovine serum and 1% GlutaMAX (Thermo Fisher Scientific) as reported previously (Sayed et al . 2021). After 1 h of bacterial invasion, cultures were washed and treated with gentamicin (50 μg/ml, Thermo Fisher Scientific) in wash buffer to kill remaining extracellular bacteria.…”

Section: Methodsmentioning

confidence: 99%

Diarrhoeal pathogenesis in Salmonella infection may result from an imbalance in intestinal epithelial differentiation through reduced Notch signalling

Quach

Jayaratne

Lee

et al. 2022

The Journal of Physiology

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Section: Methodsmentioning

confidence: 99%

Diarrhoeal pathogenesis in Salmonella infection may result from an imbalance in intestinal epithelial differentiation through reduced Notch signalling

Quach

Jayaratne

Lee

et al. 2022

The Journal of Physiology

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“…To prepare EDMs, single cells from enteroids were plated in the presence of 5% conditioned media and diluted Matrigel (1:40) as done before 47,48 . For all functional assays, experiments were performed multiple times with EDMs-derived from enteroids collected from at least 3 different mice, including both the genders.…”

Section: Methodsmentioning

confidence: 99%

“…Intestinal crypts were isolated from the colonic tissue specimen by digesting with Collagenase type I [2 mg/ml; Invitrogen] and cultured in stem-cell enriched conditioned media with WNT 3a, R-spondin and Noggin as described before [42][43][44][45][46][47] .…”

Section: Isolation Of Enteroids From the Small Intestine Of The Mousementioning

confidence: 99%

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The crosstalk between microbial sensors ELMO1 and NOD2 shape intestinal immune responses

Sharma

Achi

Ibeawuchi

et al. 2022

Preprint

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Microbial sensors play an essential role in maintaining cellular homeostasis. Our knowledge is limited on how microbial sensing helps in differential immune response and its link to inflammatory diseases. Recently, we have shown that cytosolic sensor ELMO1 (Engulfment and Cell Motility Protein-1) binds to effectors from pathogenic bacteria and controls intestinal inflammation. Here, we show that ELMO1 interacts with another sensor, NOD2 (Nucleotide-binding oligomerization domain-containing protein 2), that recognizes bacterial cell wall component muramyl dipeptide (MDP). The polymorphism of NOD2 is linked to Crohns disease (CD) pathogenesis. Interestingly, we found that overexpression of ELMO1 and mutant NOD2 (L1007fs) were not able to clear the CD-associated adherent invasive E. coli (AIEC-LF82). To understand the interplay of microbial sensing of ELMO1-NOD2 in epithelial cells and macrophages, we used enteroid-derived monolayers (EDMs) from ELMO1 and NOD2 KO mice and ELMO1 and NOD2-depleted murine macrophage cell lines. The infection of murine EDMs with AIEC-LF82 showed higher bacterial load in ELMO1-KO, NOD2 KO EDMs, and ELMO1 KO EDMs treated with NOD2 inhibitors. The murine macrophage cells showed that the downregulation of ELMO1 and NOD2 is associated with impaired bacterial clearance that is linked to reduced pro-inflammatory cytokines and reactive oxygen species. Our results indicated that the crosstalk between microbial sensors in enteric infection and inflammatory diseases impacts the fate of the bacterial load and disease pathogenesis.

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“…They can be differentiated towards intestinal, 39 pancreatic ductlike, 227 liver 33 228 and gastric 223 Available techniques ⇒ Apical-out (polarity inversion, allows access to the apical but not basal site). 114 241 ⇒ Planar cultures: monolayer, cells adherent to plate (easy to perform, allows access to the apical but not basal site), [241][242][243] air-liquid Interface (epithelial injury responses, host-microbe interaction, allows access to both, the apical and basal site). 121 241 243 ⇒ Microinjection (technique equipment is needed, hostmicrobe interaction).…”

Section: Key Information and Available Techniquesmentioning

confidence: 99%

Organoids in gastrointestinal diseases: from experimental models to clinical translation

Günther

Winner

Stappenbeck

2022

Gut

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We are entering an era of medicine where increasingly sophisticated data will be obtained from patients to determine proper diagnosis, predict outcomes and direct therapies. We predict that the most valuable data will be produced by systems that are highly dynamic in both time and space. Three-dimensional (3D) organoids are poised to be such a highly valuable system for a variety of gastrointestinal (GI) diseases. In the lab, organoids have emerged as powerful systems to model molecular and cellular processes orchestrating natural and pathophysiological human tissue formation in remarkable detail. Preclinical studies have impressively demonstrated that these organs-in-a-dish can be used to model immunological, neoplastic, metabolic or infectious GI disorders by taking advantage of patient-derived material. Technological breakthroughs now allow to study cellular communication and molecular mechanisms of interorgan cross-talk in health and disease including communication along for example, the gut–brain axis or gut–liver axis. Despite considerable success in culturing classical 3D organoids from various parts of the GI tract, some challenges remain to develop these systems to best help patients. Novel platforms such as organ-on-a-chip, engineered biomimetic systems including engineered organoids, micromanufacturing, bioprinting and enhanced rigour and reproducibility will open improved avenues for tissue engineering, as well as regenerative and personalised medicine. This review will highlight some of the established methods and also some exciting novel perspectives on organoids in the fields of gastroenterology. At present, this field is poised to move forward and impact many currently intractable GI diseases in the form of novel diagnostics and therapeutics.

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Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier

Cited by 9 publications

References 17 publications

Diarrhoeal pathogenesis in Salmonella infection may result from an imbalance in intestinal epithelial differentiation through reduced Notch signalling

Diarrhoeal pathogenesis in Salmonella infection may result from an imbalance in intestinal epithelial differentiation through reduced Notch signalling

The crosstalk between microbial sensors ELMO1 and NOD2 shape intestinal immune responses

Organoids in gastrointestinal diseases: from experimental models to clinical translation

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