Drivers of transcriptional variance in human intestinal epithelial organoids.

TitleDrivers of transcriptional variance in human intestinal epithelial organoids.
Publication TypeJournal Article
Year of Publication2021
AuthorsCriss, ZK, Bhasin, N, Di Rienzi, SC, Rajan, A, Deans-Fielder, K, Swaminathan, G, Kamyabi, N, Zeng, X-L, Doddapaneni, H, Menon, VK, Chakravarti, D, Estrella, C, Yu, X, Patil, K, Petrosino, JF, Fleet, JC, Verzi, MP, Christakos, S, Helmrath, MA, Arimura, S, DePinho, RA, Britton, RA, Maresso, AW, K Grande-Allen, J, Blutt, SE, Crawford, SE, Estes, MK, Ramani, S, Shroyer, NF
JournalPhysiol Genomics
Date Published2021 Nov 01
KeywordsCalcitriol, Cell Culture Techniques, Collagen, Colon, Crohn Disease, Culture Media, Drug Combinations, Escherichia coli, Escherichia coli Infections, Extracellular Matrix, Gene Expression Regulation, Humans, Intestinal Mucosa, Intestine, Small, Laminin, Organoids, Proteoglycans, RNA-Seq, Transcriptome, Virus Diseases, Viruses

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human 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. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; 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 versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, 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 epithelial organoids.

Alternate JournalPhysiol Genomics
PubMed ID34612061
PubMed Central IDPMC8616596
Grant ListU01 DK103117 / DK / NIDDK NIH HHS / United States
U19 AI116497 / AI / NIAID NIH HHS / United States
P30 DK056338 / DK / NIDDK NIH HHS / United States
U19 AI144297 / AI / NIAID NIH HHS / United States
R01 DK112365 / DK / NIDDK NIH HHS / United States
R01 DK118904 / DK / NIDDK NIH HHS / United States
/ HHMI / Howard Hughes Medical Institute / United States