Intestinal Organoid Model Research Articles

Ex Vivo Intestinal Organoid Models: Current State-of-the-Art and Challenges in Disease Modelling and Therapeutic Testing for Colorectal Cancer

Despite improvements in participation in population-based screening programme, colorectal cancer remains a major cause of cancer-related mortality worldwide. Targeted interventions are desirable to reduce the health and economic burden of this disease. Two-dimensional monolayers of colorectal cancer cell lines represent the traditional in vitro models for disease and are often used for diverse purposes, including the delineation of molecular pathways associated with disease aetiology or the gauging of drug efficacy. The lack of complexity in such models, chiefly the limited epithelial cell diversity and differentiation, attenuated mucus production, lack of microbial interactions and mechanical stresses, has driven interest in the development of more holistic and physiologically relevant in vitro model systems. In particular, established ex vivo patient-derived explant and patient-derived tumour xenograft models have been supplemented by progress in organoid and microfluidic organ-on-a-chip cultures. Here, we discuss the applicability of advanced culturing technologies, such as organoid systems, as models for colorectal cancer and for testing chemotherapeutic drug sensitivity and efficacy. We highlight current challenges associated with organoid technologies and discuss their future for more accurate disease modelling and personalized medicine.

Dietary titanium dioxide nanoparticles impair intestinal epithelial regeneration by perturbating the function of intestinal stem cells

Intestinal health is closely linked to intestinal stem cells (ISCs), which are highly sensitive to the harmful substances in the lumen. However, there is limited knowledge regarding the effects of food additives on ISCs. This study aims to investigate the impact of dietary titanium dioxide nanoparticles (TiO2 NPs) compared with titanium dioxide microparticles (TiO2 MPs) on intestinal health associated with ISCs in response to dextran sodium sulfate (DSS)-induced enteritis in mice, as well as the related mechanism. We found that exposure to 1% (w/w) TiO2 NPs aggravated DSS-induced enteritis in mice, while this effect could not be observed under exposure to TiO2 MPs. Additionally, 1% (w/w) TiO2 NPs exposure under DSS-induced enteritis worsened the ISC-mediated regeneration of intestinal epithelium by decreasing the epithelial cell proliferation and epithelial turnover rate while increasing epithelial cell death. Meanwhile, using a 3D intestinal organoid model, we discovered that 20 μg/mL TiO2 NPs impaired ISC function and disrupted ISC fate specification both ex vivo and in vitro. Furthermore, TiO2 NPs hindered the nuclear translocation of β-catenin, reducing the overall output of Wnt signaling. Together, TiO2 NPs deteriorated the intestinal epithelial regeneration of mice with DSS-induced enteritis by perturbating ISC function and fate specification through a mechanism involving Wnt signaling. These findings highlight the adverse effect of dietary TiO2 NPs on ISCs and shed light on the particle size optimization of TiO2 food additive.

Establishment of an Apical-Out Organoid Model for Directly Assessing the Function of Postbiotics.

In vitro organoids that mimic the physiological properties of in vivo organs based on threedimensional cell cultures overcome the limitations of two-dimensional culture systems. However, because the lumen of a typical intestinal organoid is internal, we used an apical-out intestinal organoid model in which the lumen that absorbs nutrients is outside to directly assess the function of postbiotics. A composite culture supernatant of Lactiplantibacillus plantarum KM2 and Bacillus velezensis KMU01 was used as a postbiotic treatment. Expression of COX-2 decreased in apical-out organoids co-treated with a lipopolysaccharide (LPS) and postbiotics. Expression of tight-junction markers such as ZO-1, claudin, and Occludin increased, and expression of mitochondrial homeostasis factors such as PINK1, parkin, and PGC1a also increased. As a result, small and large intestine organoids treated with postbiotics protected tight junctions from LPS-induced damage and maintained mitochondrial homeostasis through mitophagy and mitochondrial biogenesis. This suggests that an apical-out intestinal organoid model can confirm the function of food ingredients.

Characterizing the effects of triclosan and triclocarban on the intestinal epithelial homeostasis using small intestinal organoids

Intestinal epithelium has the largest surface of human body, contributes dramatically to defense of toxicant-associated intestinal injury. Triclosan (TCS) and triclocarban (TCC), extensively employed as antibacterial agents in personal care products (PCPs) and healthcare facilities, caused serious damage to human intestine. However, the role of the intestinal epithelium in TCS/TCC-induced intestinal toxicity and its underlying toxic mechanisms remain incompletely understood. In this study, a novel 3D intestinal organoid model was utilized to investigate that exposure to TCS/TCC led to a compromised self-renewal and differentiation of intestinal stem cells (ISCs). Consequently, this disrupted intestinal epithelial homeostasis ultimately caused a reduction in nutrient absorption and deficient of epithelial defense to exogenous and endogenous pathogens stimulation. The inhibition of the Wnt signaling pathway in intestinal stem cell was contributed to the intestinal toxicity of TCS/TCC. These results were further confirmed in vivo with mice exposed to TCS/TCC. The findings of this study provide evidence that TCS/TCC possess the capacity to disturb the homeostasis of the intestinal epithelium, and emphasize the credibility of organoids as a valuable model for toxicological studies, as they could faithfully recapitulate in vivo phenomena.

Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn's Disease.

We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.

In vitro and in vivo evaluation of thapsigargin as an antiviral agent against transmissible gastroenteritis virus

Swine enteric coronaviruses (SeCoVs) pose a significant threat to the global pig industry, but no effective drugs are available for treatment. Previous research has demonstrated that thapsigargin (TG), an ER stress inducer, has broad-spectrum antiviral effects on human coronaviruses. In this study, we investigated the impact of TG on transmissible gastroenteritis virus (TGEV) infection using cell lines, porcine intestinal organoid models, and piglets. The results showed that TG effectively inhibited TGEV replication both in vitro and ex vivo. Furthermore, animal experiments demonstrated that oral administration of TG inhibited TGEV infection in neonatal piglets and relieved TGEV-associated tissue injury. Transcriptome analyses revealed that TG improved the expression of the ER-associated protein degradation (ERAD) component and influenced the biological processes related to secretion, nutrient responses, and epithelial cell differentiation in the intestinal epithelium. Collectively, these results suggest that TG is a potential novel oral antiviral drug for the clinical treatment of TGEV infection, even for infections caused by other SeCoVs.

Optimization of Vascularized Intestinal Organoid Model.

Vasculature is crucial for maintaining organ homeostasis and metabolism. Although 3D organoids can mimic organ structures and patterns, they still lack vascular systems, limiting the recapitulation ofphysiological complexities. Although vascularization of organoids has been demonstrated by mixing Matrigel in fibrin, how the mixed gel niche affects endothelial cells (ECs) and organoids remains unclear. Existing protocols rely on fibroblasts to promote vascular network formation. This study explores how varying the ratio of Matrigel in fibrin-Matrigel co-gel affects vascular network formation and intestinal organoid growth.A fine-tuned hydrogel is developed by adding aprotinin and 15% Matrigel in fibrin. Medium for co-culturing ECs and organoids is modified with basic fibroblast growth factor (bFGF) and heparin. In combination with fine-tuned hydrogel and modified medium, vascular network formation and organoid vascularization are successfully generated in the absence of fibroblast. Furthermore, structural cues and pore architectures are critical for angiogenesis and vascularization. By incorporating engineered thick collagen fiber bundles into the system, vascular network formation is guided by bundle architectures, enhancing interactions between vascular networks and organoids. The results demonstrate an optimized system that advances tissue and organoid vascularization by combining fiber bundles with fine-tuned hydrogel and modified medium.

Revitalizing gut health: Liangxue guyuan yishen decoction promotes akkermansia muciniphila -induced intestinal stem cell recovery post-radiation in mice

BackgroundThe efficacy of Liangxue Guyuan Yishen Decoction (LGYD), a traditional Chinese medicine, has been scientifically proven in the treatment of radiation-induced intestinal injury (RIII) and preservation of intestinal integrity and function following high-dose radiation exposure. However, further investigation is required to comprehensively elucidate the precise mechanisms underlying the therapeutic effects of LGYD in order to provide potential pharmaceutical options for radiation protection. PurposeThis study aims to elucidate the potential mechanism through which LGYD exerts its therapeutic effects on RIII by modulating the gut microbiota (GM). Methods16 s rRNA analysis was employed to assess the impact of varying doses of whole body irradiation (WBI) on GM in order to establish an appropriate model for this study. The effects of LGYD on GM and SCFA were evaluated using 16 s rRNA and Quantification of SCFA. UHPLC-QE-MS was utilized to identify the active components in LGYD as well as LGYD drug containing serum (LGYD-DS). Subsequently, immunofluorescence and immunohistochemical staining were conducted to validate the influence of LGYD and/or characteristic microbiota on RIII recovery in vivo. The effects of LGYD-DS, characteristic flora, and SCFA on intestinal stem cell (ISC) were assessed by measuring organoid surface area in intestinal organoid model. ResultsThe species composition and abundance of GM were significantly influenced by whole-body irradiation with a dose of 8.5 Gy, which was used as in vivo model. LGYD significantly improves the survival rate and promotes recovery from RIII. Additionally, LGYD exhibited a notable increase in the abundance of Akkermansia muciniphila (AKK) and levels of SCFA, particularly isobutyric acid. LGYD-DS consisted of seven main components derived from herbs of LGYD. In vivo experiments indicated that both LGYD and AKK substantially enhanced the survival rate after radiation and facilitated the recovery process for intestinal structure and function. In the organoid model, treatment with LGYD-DS, AKK supernatant or isobutyric acid significantly increased organoid surface area. ConclusionsLGYD has the potential to enhance RIII by promoting the restoration of intestinal stem cell, which is closely associated with the upregulation of AKK abundance and production of SCFA, particularly isobutyric acid.

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

A novel function of benzoic acid to enhance intestinal barrier defense against PEDV infection in Piglets

The intestinal barrier of newborn piglets is vulnerable and underdeveloped, making them susceptible to enteric virus infections. Benzoic acid (BA), employed as a growth promoter, exhibits the potential to enhance the gut health of piglets by modulating intestinal morphometry and tight junction dynamics. However, the extent to which BA regulates the intestinal mucus barrier through its impact on stem cells remains inadequately elucidated. Therefore, this study was conducted to investigate the effects of BA on the intestinal barrier and the differentiation of intestinal stem cells, employing in vivo piglet and in vitro intestinal organoid models. Our investigation revealed a significant increase in the number of goblet cells within the small intestine, as well as the strengthening of the mucus barrier in vivo following oral treatment with BA, providing partial protection against PEDV infection in piglets. Additionally, in vitro cultivation of enteroids with BA led to a notable increase in the number of MUC2+ GCs, indicating the promotion of GC differentiation by BA. Furthermore, transcriptome analysis revealed an upregulation of the number of GCs and the expression of cell vesicle transport-related genes during BA stimulation, accompanied by the downregulation of the Wnt and Notch signaling pathways. Mechanistically, MCT1 facilitated the transport of BA, subsequently activating the MAPK pathway to mediate GC differentiation. Overall, this study highlights a novel function for BA as a feed additive in enhancing the intestinal mucus barrier by promoting intestinal GC differentiation, and further prevents viral infection in piglets.

Divergent responses of human intestinal organoid monolayers using commercial in vitro cytotoxicity assays.

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.

Azoxymethane-induced carcinogenesis-like model of mouse intestine and mouse embryonic stem cell-derived intestinal organoids.

Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels. In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids. We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.

WS10.05 Subject selection plan for CHOICES clinical trial aiming to assess efficacy of newCFTR modulators and to validate the intestinal organoid model

WS10.05 Subject selection plan for CHOICES clinical trial aiming to assess efficacy of newCFTR modulators and to validate the intestinal organoid model

Oncostatin M Induces a Pro-inflammatory Phenotype in Intestinal Subepithelial Myofibroblasts.

Oncostatin-M (OSM) is associated with antitumor necrosis factor (anti-TNF)-α resistance in inflammatory bowel disease (IBD) and fibrosis in inflammatory diseases. We studied the expression of OSM and its receptors (OSMR, gp130) on intestinal subepithelial myofibroblasts (SEMFs) and the effect of OSM stimulation on SEMFs. The mRNA and protein expression of OSM, OSMR, gp130, and several fibrotic and chemotactic factors were studied in mucosal biopsies and isolated human intestinal SEMFs of patients with IBD and healthy controls (HCs) and in a model of human intestinal organoids (HIOs). Subepithelial myofibroblasts and HIOs were stimulated with OSM and interleukin (IL)-1α/TNF-α. RNAseq data of mucosal biopsies were also analyzed. Oncostatin-M receptors and gp130 were overexpressed in mucosal biopsies of patients with IBD (P < .05), especially in inflamed segments (P < .05). The expression of OSM, OSMR, and gp130 in SEMFs from HCs was increased after stimulation with IL-1α/TNF-α (P < .001; P < .01; P < .01). The expression of CCL2, CXCL9, CXCL10, and CXCL11 was increased in SEMFs from patients with IBD and HCs after stimulation with OSM in a dose-dependent manner (P < .001; P < .05; P < .001; P < .001) and was further increased after prestimulation with IL-1α/TNF-α (P < .01 vs OSM-alone). Similar results were yielded after stimulation of HIOs (P < .01). Oncostatin-M did not induce the expression of collagen I, III, and fibronectin. Oncostatin-M receptor expression was positively correlated with CCL2, CXCL9, CXCL10, and CXCL11 expression in mucosal biopsies (P < .001; P < .001; P = .045; P = .033). Human SEMFs overexpress OSMR in an inflammatory microenvironment. Oncostatin-M may promote inflammation in IBD via its stimulatory effects on SEMFs, which primarily involve chemoattraction of immune cells to the intestinal mucosa.

High-fat diet enhances cell proliferation and compromises intestinal permeability in a translational canine intestinal organoid model

BackgroundEmerging evidence underscores the responsiveness of the mammalian intestine to dietary cues, notably through the involvement of LGR5 + intestinal stem cells in orchestrating responses to diet-driven signals. However, the effects of high-fat diet (HFD) on these cellular dynamics and their impact on gut integrity remain insufficiently understood. Our study aims to assess the multifaceted interactions between palmitic acid (PA), cell proliferation, and the intestinal epithelial barrier using a canine colonoid model. Canine models, due to their relevance in simulating human intestinal diseases, offer a unique platform to explore the molecular mechanisms underlying HFD derived intestinal dysfunction.ResultsCanine colonoids were subjected to PA exposure, a surrogate for the effects of HFD. This intervention revealed a remarkable augmentation of cell proliferative activity. Furthermore, we observed a parallel reduction in transepithelial electrical resistance (TEER), indicating altered epithelium barrier integrity. While E-cadherin exhibited consistency, ZO-1 displayed a noteworthy reduction in fluorescence intensity within the PA-exposed group.ConclusionsBy employing canine intestinal organoid systems, we provide compelling insights into the impact of PA on intestinal physiology. These findings underscore the importance of considering both cell proliferative activity and epithelial integrity in comprehending the repercussions of HFDs on intestinal health. Our study contributes to a deeper understanding of the consequences of HFD on intestinal homeostasis, utilizing valuable translational in vitro models derived from dogs.

Time-dependent effects of tumor necrosis factor α on Ca2+-dependent secretion in murine small intestinal organoids.

Intestinal organoids are stem cell-derived, 3D "mini-guts" with similar functions as the native intestinal epithelium such as electrolyte transport or establishment of an epithelial barrier. During intestinal inflammation, epithelial functions are dysregulated by proinflammatory cytokines like tumor necrosis factor α (TNFα) and other messengers from the immune system resulting in a loss of electrolytes and water due to an impaired epithelial barrier and higher net secretion. A murine small intestinal organoid model was established to study (long-term) effects of TNFα on the intestinal epithelium in vitro using live imaging, immunohistochemical staining and qPCR. TNFα induced apoptosis in intestinal organoids as indicated by an increased number of cells with immunoreactivity for cleaved caspase 3. Furthermore, TNFα exposure led to swelling of the organoids which was inhibited by bumetanide and was concomitant with an upregulation of the bumetanide-sensitive Na+-K+-2Cl- symporter 1 (NKCC1) as shown by qPCR. Fura-2 imaging experiments revealed time-dependent changes in Ca2+ signaling consisting of a rise in the basal cytosolic Ca2+ concentration at day 1 and an increase of the carbachol-induced Ca2+ response after 3days TNFα exposure. This was prevented by preincubation with La3+, an inhibitor of non-selective cation channels, or by using a Ca2+-free buffer indicating an enhancement of the Ca2+ influx from the extracellular side by the cytokine. No significant changes in cDNA levels of epithelial barrier proteins could be observed in the presence of TNFα. Intestinal organoids are a useful tool to study the mechanism underlying the TNFα-induced secretion on enterocytes such as the regulation of NKCC1 expression or the modulation of cellular Ca2+ signaling.

Functional activity of the CFTR channel in a patient with the [L467F;F508del]/W1310X genotype

An important factor influencing the severity of cystic fibrosis (CF) and the effectiveness of treatment is the presence of complex alleles in the CFTR gene ≥ 2 variants in one allele. The influence of such alleles on the manifestations of CF has not been sufficiently studied.The aim of this study was to investigate the effect of the complex allele [L467F;F508del] on the phenotypic manifestations of CF and the efficacy of targeted therapy in an intestinal organoid (IO) model in a patient with the [L467F;F508del]/W1310X genotype.Methods. Methods for determining the difference in intestinal potentials (IDP), the KO method, and the forskolin test are presented using the medical history of a patient with the [L467F;F508del]/ W1310X genotype as an example.Results. The patient was diagnosed with progressive disease with obvious deterioration of pulmonary function. The ORKP method showed the absence of chlorine channel function. An assay with a KO culture from intestinal tissue showed a complete loss of functional activity of the chloride channel. In addition, the complex allele [L467F;F508del] is not sensitive to the effect of all tested CFTR modulators.Conclusion. The complex allele [L467F;F508del] causes a complete loss of functional CFTR protein and is not sensitive to the effect of any of the registered targeted drugs.

Notoginsenoside R1 promotes Lgr5+ stem cell and epithelium renovation in colitis mice via activating Wnt/β-Catenin signaling

Inflammatory bowel disease (IBD) is characterized by persistent damage to the intestinal barrier and excessive inflammation, leading to increased intestinal permeability. Current treatments of IBD primarily address inflammation, neglecting epithelial repair. Our previous study has reported the therapeutic potential of notoginsenoside R1 (NGR1), a characteristic saponin from the root of Panax notoginseng, in alleviating acute colitis by reducing mucosal inflammation. In this study we investigated the reparative effects of NGR1 on mucosal barrier damage after the acute injury stage of DSS exposure. DSS-induced colitis mice were orally treated with NGR1 (25, 50, 125 mg·kg−1·d−1) for 10 days. Body weight and rectal bleeding were daily monitored throughout the experiment, then mice were euthanized, and the colon was collected for analysis. We showed that NGR1 administration dose-dependently ameliorated mucosal inflammation and enhanced epithelial repair evidenced by increased tight junction proteins, mucus production and reduced permeability in colitis mice. We then performed transcriptomic analysis on rectal tissue using RNA-sequencing, and found NGR1 administration stimulated the proliferation of intestinal crypt cells and facilitated the repair of epithelial injury; NGR1 upregulated ISC marker Lgr5, the genes for differentiation of intestinal stem cells (ISCs), as well as BrdU incorporation in crypts of colitis mice. In NCM460 human intestinal epithelial cells in vitro, treatment with NGR1 (100 μM) promoted wound healing and reduced cell apoptosis. NGR1 (100 μM) also increased Lgr5+ cells and budding rates in a 3D intestinal organoid model. We demonstrated that NGR1 promoted ISC proliferation and differentiation through activation of the Wnt signaling pathway. Co-treatment with Wnt inhibitor ICG-001 partially counteracted the effects of NGR1 on crypt Lgr5+ ISCs, organoid budding rates, and overall mice colitis improvement. These results suggest that NGR1 alleviates DSS-induced colitis in mice by promoting the regeneration of Lgr5+ stem cells and intestinal reconstruction, at least partially via activation of the Wnt/β-Catenin signaling pathway.Schematic diagram of the mechanism of NGR1 in alleviating colitis. DSS caused widespread mucosal inflammation epithelial injury. This was manifested by the decreased expression of tight junction proteins, reduced mucus production in goblet cells, and increased intestinal permeability in colitis mice. Additionally, Lgr5+ ISCs were in obviously deficiency in colitis mice, with aberrant down-regulation of the Wnt/β-Catenin signaling. However, NGR1 amplified the expression of the ISC marker Lgr5, elevated the expression of genes associated with ISC differentiation, enhanced the incorporation of BrdU in the crypt and promoted epithelial restoration to alleviate DSS-induced colitis in mice, at least partially, by activating the Wnt/β-Catenin signaling pathway.

Assessment of cytochrome P450 induction in canine intestinal organoid models

Understanding cytochrome P450 (CYP) enzymes in the canine intestine is vital for predicting drug metabolism and developing safer oral medications. This study evaluates canine colonoids as a model to assess the expression and induction of essential intestinal CYP enzymes. Canine colonoids were cultured in expansion medium (EM) with Wnt-3A and in differentiation medium (DM) without Wnt-3A. We assessed the mRNA expression of CYP2B11, CYP2C21, CYP3A12, and CYP3A98 using qPCR and examined the effects of rifampicin and phenobarbital as inducers. Our findings show that DM significantly increased the mRNA expression of CYP3A98 and CYP2B11, but not CYP3A12, compared to EM. CYP2C21, not typically expressed in the intestine, remained unexpressed in colonoids. Rifampicin induced CYP3A98, aligning with pregnane x receptor (PXR) regulation, while phenobarbital did not, suggesting no constitutive androstane receptor (CAR) involvement. CYP2B11 did not respond to either inducer, suggesting alternative regulatory pathways in canine colonoids. This study is a pioneering effort to establish conditions for studying P450 expression in canine colonoids, confirming significant CYP3A98 expression in the canine intestine. It demonstrated colonoids can induce CYP activity post drug treatments. Further research is needed to enhance species-specific drug metabolism understanding and validate this model for broader applications.

P087 IFN-γ-Induced Intestinal Epithelial Cell Type-Specific Cytotoxic Responses of Human Enteroids: PANoptosis and The Protective Role of Selective JAK1 Inhibitors

Abstract Background Interferon-gamma (IFN-γ) plays an important role in the development of intestinal injury and inflammatory bowel disease (IBD). Using human intestinal organoid (enteorid) models, this study was investigated 1) what is the major response of intestinal epithelial cells (IECs) induced by IFN-γ to elucidate; 2) which programmed cell death (PCD) pathways cause IFN-γ-induced cell death on IECs in the absence of intestinal microbiota and immune cells; 3) how the expression of cell-cell adhesion molecules in epithelial cells is altered by IFN-γ; 4) how epithelial cell differentiation is altered by IFN-γ; 5) whether there is a difference in IFN-γ-induced PCD between different epithelial cell types; and 6) which molecules can block IFN-γ-induced PCD in IECs. Methods Using human enteroids, the major response of IECs induced by IFN-γ was evaluated, focusing on the IFN-γ-induced PCD pathway. The intestinal epithelial cell type-specific response to IFN-γ was assessed by bulk and single-cell RNA sequencing (RNA-seq). Furthermore, the molecules to block IFN-γ-induced PCD in IECs were evaluated. Results As the concentration of IFN-γ in the culture media increased, disruption of organoid structure, growth arrest, and cell death were observed in IFN-γ-treated enteroids on organoid reconstitution, MTT, and EdU assay. Bulk RNA-seq and western blot were identified the activation of pyroptosis, apoptosis, and necroptosis to form the collective inflammatory cell death pathway of PANoptosis. Single-cell RNA-seq indicated that IFN-γ altered epithelial cell differentiation in human enteroids, including expansion of the intstinal stem cell (ISC) population and depletion of the enterocyte population. PCD-associated gene expression was upregulated in enterocytes and goblet cells, but not in ISCs and paneth cells. Individual PCD inhibitors may be insufficient to prevent IFN-γ-induced cytotoxicity, whereas upadacitinib interferes with the downstream signaling of IFN-γ by inhibiting the activity of JAK1 kinase, resulting in effective blocking of PANoptosis. Conclusion PANoptosis is the major mechanism of IFN-γ-induced IEC damage, which was blocked by a selective JAK1 inhibitor. ECs were particularly susceptible to IFN-γ-induced PANoptosis. * This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2022R1A2B5B02002092 and 2021R1C1C2095192) and Future Medicine 2030 Project of the Samsung Medical Center (SMO1230011).