Introduction
Patient-derived organoid culture is a powerful and versatile system for investigating the molecular mechanisms underlying a wide range of gastrointestinal diseases, including inflammatory bowel disease (IBD) [1]. In the context of IBD, intestinal organoids derived from patients retain key genetic, epigenetic, and disease-specific features, offering a unique platform to model individual responses to inflammatory triggers [2, 3]. This approach also shows promise in predicting personalized responses to biologic and small-molecule therapies, advancing precision medicine strategies for IBD [4]. Although intestinal organoids represent a major advancement in personalized ex vivo cellular modeling, the detailed effects of differentiation and inflammation on the cellular landscape remain poorly understood.
Aims & Methods
In this study, we analyzed differentiated and undifferentiated intestinal epithelial organoids derived from both an IBD patient with active ulcerative colitis and a healthy donor (non-IBD) (n = 4). Inflammation was induced by treating organoids with the pro-inflammatory cytokines TNF-α (10 ng/mL) and IFN-γ (10 ng/mL). Organoids were dissociated using TrypLE Express Enzyme supplemented with Y27632, and viable cells were enriched by fluorescence-activated cell sorting (FACS) using 7-AAD staining. Cell viability and concentration were assessed using the Countess 3 automated cell counter and Trypan Blue staining. Single-cell suspensions were then processed using the 10x Genomics GEM-X Universal 3’ Gene Expression application, and libraries were sequenced on the NovaSeq X Plus platform. Primary data processing was performed using Cell Ranger (10X Genomics), followed by downstream visualization in Loupe Browser (10X Genomics) and an in-house pipeline.
Results
The mean number of recovered cells per sample was 1,957, with an average of 142,015 reads per cell and 27,127 genes detected across all samples. Single-cell transcriptome analysis revealed five distinct clusters in undifferentiated organoids (both treated and untreated), and six to seven clusters in differentiated organoids (treated and untreated). The majority of cells expressed the epithelial marker EPCAM, confirming their epithelial origin. Inflammatory stimulation led to a significant upregulation of TNF-α and IFN-γ gene expression (p < 0.05) in both differentiated and undifferentiated samples. Furthermore, differentiated organoids displayed a significant increase in specific epithelial subtypes, including Paneth cells, enteroendocrine cells, and proliferative cells (p < 0.05), compared to their undifferentiated counterparts.
Conclusion
To conclude, single-cell transcriptomic profiling of patient-derived intestinal organoids revealed a dynamic and heterogeneous cellular landscape shaped by both differentiation and inflammatory signals. The observed upregulation of inflammatory markers underscores the organoid model's utility in mimicking inflammatory responses. Additionally, the potential expansion of distinct epithelial subpopulations in differentiated conditions highlights the model's ability to faithfully recapitulate the complexity of the intestinal epithelium.
References
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2. Dotti, I., Mora-Buch, R., Ferrer-Picón, E., Planell, N., Jung, P., Masamunt, M. C., ... & Salas, A. (2017). Alterations in the epithelial stem cell compartment could contribute to permanent changes in the mucosa of patients with ulcerative colitis. Gut, 66(12), 2069-2079.
3. Sato, T., Stange, D. E., Ferrante, M., Vries, R. G., Van Es, J. H., Van Den Brink, S., ... & Clevers, H. (2011). Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology, 141(5), 1762-1772.
4. Arnauts, K., Verstockt, B., Santo Ramalho, A., Vermeire, S., Verfaillie, C., & Ferrante, M. (2020). Ex vivo mimicking of inflammation in organoids derived from patients with ulcerative colitis. Gastroenterology, 159(4), 1564-1567.
