PT - JOURNAL ARTICLE AU - Zachary K. Criss II AU - Nobel Bhasin AU - Sara C. Di Rienzi AU - Anubama Rajan AU - Kali Deans-Fielder AU - Ganesh Swaminathan AU - Nabiollah Kamyabi AU - Xi-Lei Zeng AU - Deepavali Chakravarti AU - Clarissa Estrella AU - Xiaomin Yu AU - Ketki Patil AU - James C. Fleet AU - Michael P. Verzi AU - Sylvia Christakos AU - Michael A. Helmrath AU - Sumimasa Arimura AU - Ronald A. DePinho AU - Robert Britton AU - Anthony Maresso AU - Jane Grande-Allen AU - Sarah E. Blutt AU - Sue E. Crawford AU - Mary K. Estes AU - Sasirekha Ramani AU - Noah F. Shroyer TI - Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids AID - 10.1101/2021.06.02.446644 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.06.02.446644 4099 - http://biorxiv.org/content/early/2021/06/03/2021.06.02.446644.short 4100 - http://biorxiv.org/content/early/2021/06/03/2021.06.02.446644.full AB - 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.Competing Interest StatementThe authors have declared no competing interest.