Introduction

The usage of intestinal stem cell-derived organoids for intestinal diseases studies is emerging. The main reason for this is that they can be generated from the intestinal tissue and cultured in vitro for prolonged period. It has been shown that long-term cultured intestinal organoids maintain stable genetic and transcriptome profiles, spatial and compositional cellular arrangement and reflect the in vivostate of the tissue of origin [1-3]. However, the data on DNA methylation levels and its stability in colon organoids derived and sub-cultured from adult intestinal stem cells remain very scarce, especially in the context of ulcerative colitis (UC) [4-5].

Aims & Methods

The aim of the study was to evaluate the epigenetic stability of human intestinal organoids obtained and cultured from colonic biopsies of UC patients and healthy controls during long-term culture by assessing quantitative methylation level of LINE-1.
Study included control subjects (CON, n=6), patients with active UC (aUC, n=6) and quiescent UC (qUC, n=7) recruited at the Department of Gastroenterology, Hospital of Lithuanian University of Health Sciences. Bisulfite conversion of DNA samples of colon biopsies, crypts primary and sub-cultured organoids was performed (in total, n=95 DNA samples), followed by PCR amplification using biotin-labeled LINE-1 primers. Quantitative determination of methylation of LINE-1 CpG islands was performed by pyrosequencing on PyroMark Q24 machine. Pyrograms were analyzed using the PyroMark Q24 software (v. 2.0.8, Qiagen), statistical analysis of quantitative methylation data (Wilcox-test) and data visualization were performed using R studio (R version 4.0.3).

Results

LINE-1 region was highly methylated in all studied biological sample groups of all conditions. The average LINE-1 methylation level when assessing all collected biological samples of corresponding group was identical in CON (66.4% ± 5.1) and qUC (66.6% ± 4.0) groups while it was slightly lower in the group of aUC (64.5% ± 4.7). Further, the tendency indicating that regardless the condition, methylation level of the initial colon biopsy samples differ significantly when comparing them to the respective epithelial colon organoid cultures was observed. In CON group, LINE-1 methylation level of late-passage organoids decreased significantly when compared to colon biopsy (by 8.1%), crypts (by 8.0%), and passage 0 (by 6.3%) and passage 1 (by 5.9%) organoids. Similarly, methylation levels of passage 1 and passage 5 organoids also differed from primary biological material in the groups of qUC (by 4.0 and 5.0%, respectively) and aUC (by 4.9 and 4.6%, respectively). Finally, LINE-1 methylation level comparisons of organoids-derived data revealed significant differences between health conditions. Methylation level of LINE-1 region was lower in the passage 1 organoids of aUC patients when compared to either CON or qUC group (by 4.7 and 3.7%, respectively). Interestingly, sizable decrease in LINE-1 methylation level was observed in the late-passage organoids of CON group, which, in turn, resulted in significant difference of 3.8% when compared to passage 5 organoids of qUC.

Conclusion

LINE-1 region of both healthy control and UC patients colon tissues and corresponding intestinal organoids is highly methylated, and long-term culturing of these organoids results in decrease of LINE-1 methylation level which proceeds at different pace depending on the inflammation status of primary tissue.

References

1. Dotti I, Salas A. Potential use of human stem cell-derived intestinal organoids to study inflammatory bowel diseases. Inflamm Bowel Dis. 2018;24(12):2501–9.
2. Middendorp S, Schneeberger K, Wiegerinck CL, Mokry M, Akkerman RDL, van Wijngaarden S, et al. Adult stem cells in the small intestine are intrinsically programmed with their location-specific function: Intestinal stem cells are intrinsically programmed. Stem Cells. 2014;32(5):1083–913.
3. van der Hee B, Madsen O, Vervoort J, Smidt H, Wells JM. Congruence of transcription programs in adult stem cell-derived jejunum organoids and original tissue during long-term culture. Front Cell Dev Biol. 2020;8:375.
4. Howell KJ, Kraiczy J, Nayak KM, Gasparetto M, Ross A, Lee C, et al. DNA methylation and transcription patterns in intestinal epithelial cells from pediatric patients with inflammatory bowel diseases differentiate disease subtypes and associate with outcome. Gastroenterology. 2018;154(3):585–98.
5. Edgar RD, Perrone F, Foster AR, Payne F, Lewis S, Nayak KM, et al. Culture-associated DNA methylation changes impact on cellular function of human intestinal organoids. Cell Mol Gastroenterol Hepatol. 2022;14(6):1295–310.

Introduction

Maintaining intestinal homeostasis relies on the interplay between individuals and their unique gut microbiota. Disruptions may result in chronic inflammation and Inflammatory Bowel Disease (IBD) [1]. This study delves into this interplay, emphasizing the characterization of diet-derived microbiome-associated metabolites and prioritizing them based on their effects on epithelial barrier integrity and inflammatory status.

Aims & Methods

Diet-derived microbiome-associated metabolites – epigallocatechin-gallate, gallic acid (GA), protocatechuic acid (PCA), ellagic acid (EA), and urolithins (Uro-A, B, C, D) – were selected through extensive literature research. In the commercial colon cell model (Caco-2) and 3D epithelial organoids from primary adult intestinal-epithelial stem cells, inflammation was induced with TNF-alpha and IFN-gamma (25ng/ml and each) to mimic IBD conditions. The expression of inflammatory cytokines genes (TNF-alpha, CXCL-9, MCP-1, IL-8) and epithelial barrier integrity markers (ZO-1 and OCLN) were evaluated by qRT-PCR. Epithelial barrier integrity was visualized using FITC-dextran assay.

Results

The induction of inflammation significantly increased in key inflammatory markers by an average 7.5-fold change (±1.8SD) in Caco-2 and 3D IBD organoid models. Metabolite analysis in Caco-2 cells showed that GA exhibited a 1.24-fold increase in TNF-alpha (p=0.036) and a -7.22-fold decrease in MCP-1 (p=0.049) gene expressions. EA decreased the expression of TNF-alpha and MCP-1 genes by -1.59-fold (p=0.045) and -3.59-fold (p=0.036) respectively, whereas IL-8 was reduced by -7.62-fold (p=0.01). Uro-A uniquely decreased MCP-1 gene expression by -7.16-fold (p=0.004) but increased IL-8 by 8-fold (p=0.01). PCA demonstrated a statistically significant decrease in all tested inflammatory markers (p<0.05). Analysis of 3D IBD organoids highlighted unique effects in restoring the epithelial barrier and expression of inflammatory marker genes.

Conclusion

Our findings demonstrate the anti-inflammatory properties of gallic acid, ellagic acid, protocatechuic acid, and urolithin-A in both Caco-2 and 3D organoid models. This research elucidates the complex interrelationships within the nutrition-microbiome-metabolome axis and its consequential impact on gut health.

References

1. Zheng, D., Liwinski, T. & Elinav, E. Interaction between microbiota and immunity in health and disease. Cell Res 30, 492–506 (2020). https://doi.org/10.1038/s41422-020-0332-7

Disclosure

No conflict of interest to declare.

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

1. Günther, C., Winner, B., Neurath, M. F., & Stappenbeck, T. S. (2022). Organoids in gastrointestinal diseases: from experimental models to clinical translation. Gut, 71(9), 1892-1908.\
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.

Introduction

A compromised intestinal epithelial barrier plays a central role in inflammatory bowel diseases (IBD) by increasing intestinal permeability and promoting inflammation. Therefore, the restoration of intestinal barrier has become critical in IBD management ¹. Methylxanthine caffeine and its metabolites are widely consumed bioactive compounds that are known to influence various physiological processes ². However, their impact on gut health, particularly in modulating inflammation and epithelial permeability, remains underexplored.

Aims & Methods

The aim of this study was to investigate potential effects of caffeine (CAF) and its primary metabolites paraxanthine (PRX), theobromine (TBR) and theophylline (TPHL) on inflammatory responses and barrier integrity in colonic epithelial organoids, providing insights into their potential role in IBD management. Primary screening of different concentrations of CAF (1/2.5/5 mM) and its metabolites PRX (10/50/100 µM), TBR (10/20/50 µM) and TPHL (25/100/500 µM) was performed in Caco-2 cells. Selected metabolites and concentrations were further tested on inflamed 3D intestinal epithelial organoids derived from ulcerative colitis patient (n = 1) and non-IBD individual (n = 1). Both Caco-2 cells and organoids were pre-treated with metabolites for 1 hour before inflammation induction and were further co-treated with TNF-α/IFN-γ cytokine mix (10 ng/ml each) and metabolites for 24 hours. The effects on inflammation status and epithelial barrier integrity were tested through targeted gene expression analysis using RT-PCR and FITC-Dextran (4kDa) assay, while the effects on generation of reactive oxygen species were investigated by Flow cytometry and DHE assay. The study was approved by the Kaunas Regional Biomedical Research Ethics Committee (protocol no. BE-2-31).

Results

Primary screening revealed that both CAF and its metabolite PRX affected the expression of genes encoding inflammatory cytokines (TNF, CXCL9; p ≤ 0.05) and tight junction proteins (OCLN, TJP1, CLDN1; p ≤ 0.05) and were selected for further analysis in 3D intestinal organoids. Neither CAF, nor PRX reduced the expression of inflammatory cytokine-encoding genes in inflamed intestinal organoids. However, both CAF and PRX increased expression of tight junction proteins in inflamed intestinal organoids derived from control non-IBD (TJP1 – 3.16-fold by CAF and 1.33-fold by PRX (p ≤ 0.05); OCLN – 1.57-fold by CAF and 1.38-fold by PRX (p ≤ 0.05)) and ulcerative colitis (TJP1 – 1.32-fold by PRX; OCLN - 1.14-fold by CAF and 1.27-fold by PRX (p ≤ 0.05)) patient-derived organoids. Mean percentages of FITC-Dextran positive organoids were 43 %, 51 %, and 34 % in untreated, inflamed, and inflamed + PRX groups, respectively, revealing a trend in barrier restoration, though it did not reach statistical significance (p = 0.16 in inflamed vs. inflamed + PRX). A tendency of reduction in reactive oxygen species production was also observed after exposure to caffeine or paraxanthine (by 17 % and 11 %, respectively (p > 0.05)).

Conclusion

Methylxanthine paraxanthine might have a positive effect on gut epithelial barrier function through restoration of the expression of tight junction protein-coding genes.
The study was funded by the European Union and the State Secretariat for Education, Research and Innovation (SERI) (project: miGut-Health, grant no. 101095470).

References

1. Neurath MF et al. (2025) The intestinal barrier: a pivotal role in health, inflammation, and cancer. The Lancet Gastroeneterology & Hepatology, S2468-1253(24)00390-X, https://doi.org/10.1016/S2468-1253(24)00390-X
2. Reddy VS et al. (2024) Pharmacology of caffeine and its effects on the human body. European Journal of Medicinal Chemistry Reports, 10 (100138), https://doi.org/10.1016/j.ejmcr.2024.100138.