Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids

Abstract

Background & Aims: Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. Methods: RNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways. Results: PERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures. Conclusions: Surprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.

Inter-gastruloid heterogeneity revealed by single cell transcriptomics time course: implications for organoid based perturbation studies

“…showed that brain organoids had more than randomly expected variability (Quadrato et al, 2017; Velasco et al, 2019), though this variability was comparable to the one observed in vivo (Velasco et al, 2019). Other studies in epithelial intestinal organoids similarly showed a high degree of inter-organoid variability (Criss et al, 2021; Hof et al, 2021; Mohammadi et al, 2021). These findings suggest that there is significant, uncharacterised interorganoid heterogeneity across organoid systems, which is important to understand and subsequently model ahead of perturbation.…”

The Emerging Role of Bile Acids in the Pathogenesis of Inflammatory Bowel Disease

“…Other recent important developments include the generation of apical-out (or reverse polarity) organoids which allows easier access to the apical membrane of the intestinal epithelium for exposure studies (120), and the ability to "inflame" organoids through treatment with a cocktail of inflammatory mediators (121). In addition, recently it has been demonstrated that as expected human ileal and colonic epithelial organoids express higher levels of the FXR and bile salt transporter genes in comparison to duodenal epithelial organoids (122). One of the major gaps in knowledge is how BA receptor activation can influence the aforementioned IBD-relevant downstream pathways such as autophagy and the inflammasome pathway in the human intestinal mucosa.…”

Making yourself at home in the intestinal tract - Dynamics of the microbiome, resistome and host-pathogen interactions Inwendig vertrouwd voelen - De dynamiek van de darmmicrobiota, het resistoom en van gastheer-pathogene interacties

“…In contrast to C. difficile and S. enterica serovar Typhimurium, important multi-resistant pathogens such as extended-spectrum beta-lactamase-producing ExPEC (ESBL-ExPEC) and VRE reside in the gut microbiome and do not seem to inflict damage to the host, up until the moment of invasion and infection in other body sites (75)(76)(77). ESBL-ExPEC and VRE are important examples of opportunistic multidrug resistant pathogens where gut colonization precedes infections (76,78).…”

“…In contrast to C. difficile and S. enterica serovar Typhimurium, important multi-resistant pathogens such as extended-spectrum beta-lactamase-producing ExPEC (ESBL-ExPEC) and VRE reside in the gut microbiome and do not seem to inflict damage to the host, up until the moment of invasion and infection in other body sites (75)(76)(77). ESBL-ExPEC and VRE are important examples of opportunistic multidrug resistant pathogens where gut colonization precedes infections (76,78). Especially in hospitalized patients, that frequently receive antibiotics, initial colonization by these opportunistic pathogens may lead to intestinal overgrowth increasing the risk of causing infections and outbreaks (75,76,(78)(79)(80)(81)(82)(83)(84).…”

“…ESBL-ExPEC and VRE are important examples of opportunistic multidrug resistant pathogens where gut colonization precedes infections (76,78). Especially in hospitalized patients, that frequently receive antibiotics, initial colonization by these opportunistic pathogens may lead to intestinal overgrowth increasing the risk of causing infections and outbreaks (75,76,(78)(79)(80)(81)(82)(83)(84). A factor contributing to their importance, is the shared trait to rapidly acquire genes, located on mobile genetic elements, such as plasmids, allowing rapid transmission of resistance to clinical important antibiotics (85)(86)(87)(88)(89).…”