Intestinal Organoids Research Articles

MAGIC matrices: freeform bioprinting materials to support complex and reproducible organoid morphogenesis.

Organoids are powerful models of tissue physiology, yet their applications remain limited due to their relatively simple morphology and high organoid-to-organoid structural variability. To address these limitations we developed a soft, composite yield-stress extracellular matrix that supports optimal organoid morphogenesis following freeform 3D bioprinting of cell slurries at tissue-like densities. The material is designed with two temperature regimes: at 4 °C it exhibits reversible yield-stress behavior to support long printing times without compromising cell viability. When transferred to cell culture at 37 °C, the material cross-links and exhibits similar viscoelasticity and plasticity to basement membrane extracts such as Matrigel. We first characterize the rheological properties of MAGIC matrices that optimize organoid morphogenesis, including low stiffness and high stress relaxation. Next, we combine this material with a custom piezoelectric printhead that allows more reproducible and robust self-organization from uniform and spatially organized tissue "seeds." We apply MAGIC matrix bioprinting for high-throughput generation of intestinal, mammary, vascular, salivary gland, and brain organoid arrays that are structurally similar to those grown in pure Matrigel, but exhibit dramatically improved homogeneity in organoid size, shape, maturation time, and efficiency of morphogenesis. The flexibility of this method and material enabled fabrication of fully 3D microphysiological systems, including perfusable organoid tubes that experience cyclic 3D strain in response to pressurization. Furthermore, the reproducibility of organoid structure increased the statistical power of a drug response assay by up to 8 orders-of-magnitude for a given number of comparisons. Combined, these advances lay the foundation for the efficient fabrication of complex tissue morphologies by canalizing their self-organization in both space and time.

Xenotransplanted human organoids identify transepithelial zinc transport as a key mediator of intestinal adaptation

Short bowel syndrome (SBS) leads to severe morbidity and mortality. Intestinal adaptation is crucial in improving outcomes. To understand the human gene pathways associated with adaptation, we perform single-cell transcriptomic analysis of human small intestinal organoids explanted from mice with experimental SBS. We show that transmembrane ion pathways, specifically the transepithelial zinc transport pathway genes SLC39A4 and SLC39A5, are upregulated in SBS. This discovery is corroborated by an external dataset, bulk RT-qPCR, and Western blots. Oral zinc supplementation is shown to improve survival and weight gain of SBS mice and increase the proliferation of intestinal crypt cells in vitro. Finally, we identify the upregulation of SLC39A5 and associated transcription factor KLF5 in biopsied intestinal tissue specimens from patients with SBS. Thus, we identify zinc supplementation as a potential therapy for SBS and describe a xenotransplantation model that provides a platform for discovery in other intestinal diseases.

AP-1B regulates interactions of epithelial cells and intraepithelial lymphocytes in the intestine

Intraepithelial lymphocytes (IELs) reside in the epithelial layer and protect against foreign pathogens, maintaining the epithelial barrier function in the intestine. Interactions between IEL and epithelial cells are required for IELs to function effectively; however, the underlying molecular machinery remains to be elucidated. In this study, we found that intestinal epithelium-specific deficiency of the clathrin adaptor protein (AP)-1B, which regulates basolateral protein sorting, led to a massive reduction in IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins (E-cadherin, butyrophilin-like 2, and plexin B2), were decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among these proteins, CD166 interacted with CD6 on the surface of induced IEL. CD166 knockdown, using shRNA in intestinal organoid cultures, significantly inhibited IEL recruitment to the epithelial layer. These findings highlight the essential role of AP-1B-mediated basolateral sorting in IEL maintenance and survival within the epithelial layer. This study reveals a novel function of AP-1B in the intestinal immune system.

6686 Age-Associated Local GH Adversely Impacts the Microenvironmental Epithelial Landscape

Abstract Disclosure: V.M. Chesnokova: None. S. Zonis: None. T. Apaydin: None. C. Wang Valencia: None. R. Ainsworth: None. R. Barrett: None. A. Kettenbach: None. S. Melmed: None. The tissue microenvironmental homeostasis is disrupted with age-related changes. Knowledge of mechanisms enabling the age-associated tissue landscape is limited. Senescent cells increase with age, and the secretome released from senescence cells (SASP) affects neighboring cells, often creating a milieu favoring neoplasia. We previously showed that local non-pituitary epithelial growth hormone (GH) is induced and secreted from senescent cells, accumulates with age, and suppresses DNA repair, leading to abundant DNA damage. To examine SASP-derived autocrine GH actions, we analyzed normal human colon cells (hNCC) infected with lentivirus expressing hGH (lentiGH) or vector (lentiV). Mass spectrometry revealed significant changes in the Rho signaling pathway reflecting cell cytoskeletal rearrangements. To examine paracrine GH action, we co-cultured intact human 3D intestinal organoids with organoids infected with lentiGH for 5 weeks. Using whole-exome sequencing we found that local paracrine GH emanating from lentiGH organoids increases neighboring intact organoid cell chromosomal instability, inducing somatic mutations, including deletions, breakends, duplications, and insertions. Mechanisms for observed chromosomal instability include sequelae of GH-mediated suppressed DNA damage repair proteins including pATM, pATR, and pBRCA2, leading to DNA damage accumulation and favoring cell transformation over the long term. Furthermore, enriched gene ontology and KEGG pathway analysis of intact organoids exposed to paracrine GH identified distorted extracellular matrix (ECM) gene expression and focal adhesion pathways. We confirmed these genomic changes with Western blotting showing altered proteins associated with ECM and those responsible for cell structural rearrangements. Moreover, phospho-omics of GH-exposed cells revealed significant phosphorylation changes in proteins associated with cell skeletal rearrangement and cell migration pathways. We found that GH triggers these changes by EMT activation as shown by suppressing E-cadherin and inducing Twist2 and Snai1 transcription factors. Together, all these changes are consistent with observed increased migration, invasion, and anchorage-independent growth of hNCC infected with lentiGH or co-cultured with GH-secreting hNCC or with GH-secreting normal colon fibroblasts. As paracrine GH also facilitates metastases after intra-splenic injection of senescent cells in nude mice carrying xenografts secreting GH, our results indicate that accumulated and locally secreted GH in aging tissue enables a microenvironmental landscape favoring epithelial cell transformation. Presentation: 6/1/2024

GZMA suppressed GPX4-mediated ferroptosis to improve intestinal mucosal barrier function in inflammatory bowel disease

BackgroundOur previous study has demonstrated a decreased colonic CD8+CD39+ T cells, enrichment of granzyme A (GZMA), was found in pediatric-onset colitis and inflammatory bowel disease (IBD) characterized by impaired intestinal barrier function. However, the influence of GZMA on intestinal barrier function remains unknown.MethodsWestern blotting(WB), real-time PCR (qPCR), immunofluorescence (IF) and in vitro permeability assay combined with intestinal organoid culture were used to detect the effect of GZMA on intestinal epithelial barrier function in vivo and in vitro. Luciferase, immunoprecipitation (IP) and subcellular fractionation isolation were performed to identify the mechanism through which GZMA modulated intestinal epithelial barrier function.ResultsHerein, we, for the first time, demonstrated that CD8+CD39+ T cells promoted intestinal epithelial barrier function through GZMA, leading to induce Occludin(OCLN) and Zonula Occludens-1(ZO-1) expression, which was attributed to enhanced CDX2-mediated cell differentiation caused by increased glutathione peroxidase 4(GPX4)-induced ferroptosis inhibition in vivo and in vitro. Mechanically, GZMA inhibited intestinal epithelial cellular PDE4B activation to trigger cAMP/PKA/CREB cascade signaling to increase CREB nuclear translocation, initiating GPX4 transactivity. In addition, endogenous PKA interacted with CREB, and this interaction was enhanced in response to GZMA. Most importantly, administration of GZMA could alleviate DSS-induced colitis in vivo.ConclusionThese findings extended the novel insight of GZMA contributed to intestinal epithelial cell differentiation to improve barrier function, and enhacement of GZMA could be a promising strategy to patients with IBD.

Tuft cells act as regenerative stem cells in the human intestine

In mice, intestinal tuft cells have been described as a long-lived, postmitotic cell type. Two distinct subsets have been identified: tuft-1 and tuft-2 (ref. 1). By combining analysis of primary human intestinal resection material and intestinal organoids, we identify four distinct human tuft cell states, two of which overlap with their murine counterparts. We show that tuft cell development depends on the presence of Wnt ligands, and that tuft cell numbers rapidly increase on interleukin-4 (IL-4) and IL-13 exposure, as reported previously in mice2–4. This occurs through proliferation of pre-existing tuft cells, rather than through increased de novo generation from stem cells. Indeed, proliferative tuft cells occur in vivo both in fetal and in adult human intestine. Single mature proliferating tuft cells can form organoids that contain all intestinal epithelial cell types. Unlike stem and progenitor cells, human tuft cells survive irradiation damage and retain the ability to generate all other epithelial cell types. Accordingly, organoids engineered to lack tuft cells fail to recover from radiation-induced damage. Thus, tuft cells represent a damage-induced reserve intestinal stem cell pool in humans.

Cultivating complexity: Advancements in establishing in vitro models for the mucus-adhering gut microbiota.

A healthy mucus is essential for maintaining intestinal homeostasis and overall well-being. In recent years, extensive research focused on understanding the intricate interactions between mucus and the gut microbiota. Mucus-adhering bacteria play crucial roles in preserving barrier integrity, epithelial permeability and mucus architecture, as well as in the colonization resistance against pathogens. Unravelling the significance of these microorganisms in human health and disease is challenging, primarily because most of the studies on the human gut microbiota rely on faecal samples, which do not fully represent the microecological complexity found in the intestinal mucosa. This review discusses novel strategies to specifically target and evaluate the mucosal microbiota, such as culturomics applied to mucosal biopsies or brushings, intestinal organoids and artificial in vitro models incorporating mucus.

Long-term three-dimensional high-resolution imaging of live unlabeled small intestinal organoids via low-coherence holotomography

Organoids, which are miniature in vitro versions of organs, possess significant potential for studying human diseases and elucidating their underlying mechanisms. Live imaging techniques play a crucial role in organoid research and contribute to elucidating the complex structure and dynamic biological phenomena of organoids. However, live, unlabeled high-resolution imaging of native organoids is challenging, primarily owing to the complexities of sample handling and optical scattering inherent in three-dimensional (3D) structures. Additionally, conventional imaging methods fail to capture the real-time dynamic processes of growing organoids. In this study, we introduce low-coherence holotomography as an advanced, label-free, quantitative imaging modality designed to overcome several technical obstacles for long-term live imaging of 3D organoids. We demonstrate the efficacy of low-coherence holotomography by capturing high-resolution morphological details and dynamic activities within mouse small intestinal organoids at subcellular resolution. Moreover, our approach facilitates the distinction between viable and nonviable organoids, significantly enhancing its utility in organoid-based research. This advancement underscores the critical role of live imaging in organoid studies, offering a more comprehensive understanding of these complex systems.

CSF1 is expressed by the intestinal epithelial cells to regulate Mφ macrophages and maintain epithelial homeostasis and is downregulated in neonates with necrotizing enterocolitis

BackgroundColony stimulating factor 1 (CSF1) is generally expressed by immune cells in response to pro-inflammatory stimuli. The CSF1 receptor (CSFR) is activated by CSF1, and plays a key role in macrophage homeostasis. Furthermore, the CSF1R+ macrophages maintain homeostasis in the intestinal epithelium. The aim of this study was to explore the functions of CSF1-expressing and CSF1R+ macrophages in necrotizing enterocolitis (NEC), which commonly affects the ileum of neonates.MethodsIn-situ CSF1 expression in the intestines of neonates with NEC or intestinal atresia (n = 4 each) was detected by immunofluorescence staining. The CSF1 levels in the intestinal crypt-derived organoid cultures were measured by ELISA. Peripheral blood monocyte-derived Mφ macrophages were co-cultured with the organoids and stimulated with lipopolysaccharide (LPS) to mimic the inflamed state of the ileum in NEC patients.ResultsCSF1 was expressed in the intestinal epithelial cells of the fetal and neonatal samples, but suppressed in the NEC samples. Furthermore, CSF1 expression was downregulated in the intestinal crypt-derived organoids by LPS. CSF1R+ macrophages were detected near the intestinal crypts in the non-inflamed intestines but were absent in tissues obtained from pediatric NEC patients. Peripheral blood monocyte-derived macrophages promoted intestinal organoid proliferation in vitro following CSF1 stimulation. Finally, low concentrations of LPS slightly enhanced the proliferation of organoids co-cultured with the macrophages, whereas higher doses had a significant inhibitory effect.ConclusionsIntestinal epithelial cells express CSF1 to regulate the resident macrophages, maintain epithelial homeostasis, and resist infection. The abundant CSF1R+ macrophages in the fetal intestine may overexpress TNF-α upon activation of the TLR4/NF-κB pathway, resulting in epithelial damage and NEC induction.

Estimation of Chloride Channel Residual Function and Assessment of Targeted Drugs Efficiency in the Presence of a Complex Allele [L467F;F508del] in the CFTR Gene.

Complex alleles of the CFTR gene complicate the diagnosis of cystic fibrosis (CF), the classification of its pathogenic variants, affect the clinical picture of the disease and can affect the efficiency of targeted drugs. The total frequency of complex allele [L467F;F508del] in the Russian population of patients with CF is 0.74%, and in patients with the F508del/F508del genotype, its frequency reaches 8%. This article presents multi-faceted study of the complex allele [L467F;F508del] in a cohort of patients with genotypes [L467F;F508del]/class I (c.3532_3535dup, c.1766+2T>C, W1310X, 712-1G>T), and data for a unique patient with the genotype [L467F;F508del]/[L467F;F508del]. Using the intestinal current measurement method, it was demonstrated the absence of CFTR function for [L467F;F508del]/class I and [L467F;F508del]/[L467F;F508del] genotypes. In intestinal organoids, it was shown that [L467F;F508del] in combination with class I variants and in the homozygotes abolishes the efficacy of both two-component (ivacaftor+lumacaftor; ivacaftor+tezacaftor) and three-component (ivacaftor+tezacaftor+elexacaftor) targeted drugs. When prescribing ivacaftor+tezacaftor+elexacaftor to three patients, they did not have a clinical effect after 6-12 months.

Cataloguing the postnatal small intestinal transcriptome during the period of susceptibility to necrotizing enterocolitis.

In the first postnatal month, the developing mouse intestine shifts from an immature to a mature intestine that will sustain the organism throughout the lifespan. Here, we surveyed the mouse intestine in C57Bl/6 mice by RNA-Seq to evaluate the changes in gene expression over time from the day of birth through 1 month of age in both the duodenum and ileum. We analyzed gene expression for changes in gene families that correlated with the periods of NEC susceptibility or resistance. We highlight that increased expression of DNA processing genes and vacuolar structure genes, tissue development and morphogenesis genes, and cell migration genes all correlated with NEC susceptibility, while increases in immunity gene sets, intracellular transport genes, ATP production, and intracellular metabolism genes correlated with NEC resistance. Using trends identified in the RNA analyses, we further evaluated expression of cellular markers and epithelial regulators, immune cell markers, and adenosine metabolism components. We confirmed key changes with qRT-PCR and immunofluorescence. In addition, we compared some findings to humans using human intestinal biopsies and organoids. This dataset can serve as a reference for other groups considering the role of single molecules or molecular families in early intestinal and postnatal development.

MiR-27a-3p regulates intestinal cell proliferation and differentiation through Wnt/β-catenin signalling.

Intestinal stem cells differentiate into absorptive enterocytes, characterised by increased brush border enzymes such as intestinal alkaline phosphatase (IAP), making up the majority (95%) of the terminally differentiated cells in the villus. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here, we show that the intestinal microRNA (miR)-27a-3p is an important regulator of intestinal epithelial cell proliferation and enterocyte differentiation. Repression of endogenous miR-27a-3p leads to increased enterocyte differentiation and decreased intestinal epithelial cell proliferation in mouse and human small intestinal organoids. Mechanistically, miR-27a-3p regulates intestinal cell differentiation and proliferation at least in part through the regulation of retinoic acid receptor α (RXRα), a modulator of Wnt/β-catenin signalling. Repression of miR-27a-3p increases the expression of RXRα and concomitantly, decreases the expression of active β-catenin and cyclin D1. In contrast, overexpression of miR-27a-3p mimic decreases the expression of RXRα and increases the expression of active β-catenin and cyclin D1. Moreover, overexpression of the miR-27a-3p mimic results in impaired enterocyte differentiation and increases intestinal epithelial cell proliferation. These alterations were attenuated or blocked by Wnt inhibition. Our study demonstrates an miR-27a-3p/RXRα/Wnt/β-catenin pathway that is important for the maintenance of enterocyte homeostasis in the small intestine.

ReBiA-Robotic Enabled Biological Automation: 3D Epithelial Tissue Production.

The Food and Drug Administration's recent decision to eliminate mandatory animal testing for drug approval marks a significant shift to alternative methods. Similarly, the European Parliament is advocating for a faster transition, reflecting public preference for animal-free research practices. In vitro tissue models are increasingly recognized as valuable tools for regulatory assessments before clinical trials, in line with the 3R principles (Replace, Reduce, Refine). Despite their potential, barriers such as the need for standardization, availability, and cost hinder their widespread adoption. To address these challenges, the Robotic Enabled Biological Automation (ReBiA) system is developed. This system uses a dual-arm robot capable of standardizing laboratory processes within a closed automated environment, translating manual processes into automated ones. This reduces the need for process-specific developments, making in vitro tissue models more consistent and cost-effective. ReBiA's performance is demonstrated through producing human reconstructed epidermis, human airway epithelial models, and human intestinal organoids. Analyses confirm that these models match the morphology and protein expression of manually prepared and native tissues, with similar cell viability. These successes highlight ReBiA's potential to lower barriers to broader adoption of in vitro tissue models, supporting a shift toward more ethical and advanced research methods.

Wuzhishan miniature pig-derived intestinal 2D monolayer organoids to investigate the enteric coronavirus infection.

Intestinal organoids are valuable tools for investigating intestinal physiology and pathology ex vivo. In previous studies, intestinal organoids of commercial pigs have been developed. Here, we established intestinal organoids derived from Wuzhishan miniature pigs (WZS pigs), a unique kind of pig in the Hainan province of China. Three-dimensional (3D) intestinal organoids and organoid monolayers were developed and assessed. Furthermore, the susceptibility of organoid monolayers of WZS pigs to transmissible gastroenteritis virus (TGEV) was demonstrated. An RNA-seq analysis revealed that the TGEV infection stimulated antiviral and inflammatory immune responses in organoid monolayer models. The study implied the transmission risk of swine enteric coronavirus on WZS pigs and provided useful tools for further research on WZS pigs as laboratory miniature pig models.

Dual color carbon dots for simultaneous dynamic fluorescence tracking of mitochondria and lysosomes

The development of fluorescent probes to monitor mitochondria and lysosomes will be helpful not only to realize the status monitoring of the organelles, but also to gain insights into their functions in both healthy and diseased states. Carbon Dots (CDs), as a nanomaterial with excellent properties, have provided new tools and methods for research in organelle imaging in recent years. In this study, we synthesized green fluorescence-emitting carbon dots (G-CDs) with mitochondria-targeting ability and red fluorescence-emitting carbon dots (R-CDs) with lysosome-targeting ability by using the same precursors. G-CDs are brightly with a quantum yield of up to 51.8 % and R-CDs have a long-wavelength emission of 611 nm. Based on the unique properties of G-CDs and R-CDs, we observed the changes of mitochondria during apoptosis and mitochondrial autophagy in HeLa cells by using G-CDs. And the dynamics of lysosomes in the inhibition of cellular autophagy and cellular necrosis were observed by utilizing the R-CDs. Furthermore, we observed human intestinal organoids samples stained by G-CDs and R-CDs, extending the relevant applications of carbon dots imaging to the organoids field, which brings forth new possibilities for research and drug development of related diseases.

IFNγ induces epithelial reprogramming driving CXCL11-mediated T cell migration.

The cytokine interferon-gamma (IFNγ) plays a multifaceted role in intestinal immune responses ranging from anti- to pro-inflammatory depending on the setting. Here, using a 3D co-culture system based on human intestinal epithelial organoids, we explore the capacity of IFNγ-exposure to reprogram intestinal epithelia and thereby directly modulate lymphocyte responses. IFNγ treatment of organoids led to transcriptional reprogramming, marked by a switch to a pro-inflammatory gene expression profile, including transcriptional upregulation of the chemokines CXCL9, CXCL10, and CXCL11. Proteomic analysis of organoid-conditioned medium post-treatment confirmed chemokine secretion. IFNγ-treatment of organoids led to enhanced T cell migration in a CXCL11-dependent manner without affecting T cell activation status. Taken together, our results suggest a specific role for CXCL11 in T cell recruitment that could be targeted to prevent T cell trafficking to the inflamed intestine.

Anisakis extracellular vesicles elicit immunomodulatory and potentially tumorigenic outcomes on human intestinal organoids

BackgroundAnisakis spp. are zoonotic nematodes causing mild to severe acute and chronic gastrointestinal infections. Chronic anisakiasis can lead to erosive mucosal ulcers, granulomas and inflammation, potential tumorigenic triggers. How Anisakis exerts its pathogenic potential through extracellular vesicles (EVs) and whether third-stage infective larvae may favor a tumorigenic microenvironment remain unclear.MethodsHere, we investigated the parasite's tumorigenic and immunomodulatory capabilities using comparative transcriptomics, qRT-PCR and protein analysis with multiplex ELISA on human intestinal organoids exposed to Anisakis EVs. Moreover, EVs were characterized in terms of shape, size and concentration using classic TEM, SEM and NTA analyses and advanced interferometric NTA.ResultsAnisakis EVs showed classic shape features and a median average diameter of around 100 nm, according to NTA and iNTA. Moreover, a refractive index of 5–20% of non-water content suggested their effective biological cargo. After treatment of human intestinal organoids with Anisakis EVs, an overall parasitic strategy based on mitigation of the immune and inflammatory response was observed. Anisakis EVs impacted gene expression of main cytokines, cell cycle regulation and protein products. Seven key genes related to cell cycle regulation and apoptosis were differentially expressed in organoids exposed to EVs. In particular, the downregulation of EPHB2 and LEFTY1 and upregulation of NUPR1 genes known to be associated with colorectal cancer were observed, suggesting their involvement in tumorigenic microenvironment. A statistically significant reduction in specific mediators of inflammation and cell-cycle regulation from the polarized epithelium as IL-33R, CD40 and CEACAM1 from the apical chambers and IL-1B, GM-CSF, IL-15 and IL-23 from both chambers were observed.ConclusionsThe results here obtained unravel intestinal epithelium response to Anisakis EVs, impacting host’s anthelminthic strategies and revealing for the first time to our knowledge the host-parasite interactions in the niche environment of an emerging accidental zoonosis. Use of an innovative EV characterization approach may also be useful for study of other helminth EVs, since the knowledge in this field is very limited.Graphical

Using integrated transcriptomics and metabolomics to explore the effects of infant formula on the growth and development of small intestinal organoids.

Infant formulas are designed to provide sufficient energy and the necessary nutrients to support the growth and development of newborns. Currently, research on the functions of formula milk powder focuses on clinical research and cell experiments, and there were many cell experiments that investigated the effect of infant formulas on cellular growth. However, most of the cells used are tumor cell lines, which are unable to simulate the real digestion process of an infant. In this study, we innovatively proposed a method that integrates human small intestinal organoids (SIOs) with transcriptomics and metabolomics analysis. We induced directed differentiation of human embryonic stem cells into SIOs and simulated the intestinal environment of newborns with them. Then, three kinds of 1-stage infant formulas from the same brand were introduced to simulate the digestion, absorption, and metabolism of the infant intestine. The nutritional value of each formula milk powder was examined by multi-omics sequencing methods, including transcriptomics and metabolomics analysis. Results showed that there were significant alterations in gene expression and metabolites in the three groups of SIOs after absorbing different infant formulas. By analyzing transcriptome and metabolome data, combined with GO, KEGG, and GSEA analysis, we demonstrated the ability of SIOs to model the different aspects of the developing process of the intestine and discovered the correlation between formula components and their effects, including Lactobacillus lactis and lactoferrin. The study reveals the effect and mechanisms of formula milk powder on the growth and development of infant intestines and the formation of immune function. Furthermore, our method can help to construct a multi-level assessment model, detect the effects of nutrients, and evaluate the interactions between nutrients, which is helpful for future research and development of infant powders.

324 Use of organoids as in vitro model to study intestinal function

Abstract Organoids are tridimensional structures cultured in the lab from stem cells. Organoids have been developed for many different organs, particularly those with Lgr5+ stem cells. The organs of the digestive system belong to this group of organs and to date, organoids have been established from all the digestive tract tissues and organs including liver, gall bladder and pancreas. Although most protocols to generate organoids have focused on human and mouse organs, organoids from domestic and production animals like dogs, cats, rabbits, pigs, chickens, turkeys, and horses are actively used in research. In this presentation, the principles of developing intestinal organoids will be covered, along with a summary of how they have been used to address intestinal function and disease in our lab and by others. In our lab, we use swine enteroids (intestinal organoids) to study the intestinal epithelial responses to nutrients and pathogens. We have explored differential responses of the intestinal epithelium to organic and inorganic sources of dietary zinc, and we are currently investigating the mechanism of pathogenesis of Lawsonia intracellularis, the causative agent of porcine intestinal adenopathy. We will also discuss methods for modeling intestinal physiology using enteroids, and the advantages and challenges of working with swine enteroids.

CFTR modulators response of S737F and T465N CFTR variants on patient-derived rectal organoids

BackgroundPredictions based on patient-derived materials of CFTR modulators efficacy have been performed lately in patient-derived cells, extending FDA-approved drugs for CF patients harboring rare variants. Here we developed intestinal organoids from subjects carrying S737F- and T465N-CFTR in trans with null alleles to evaluate their functional impact on CFTR protein function and their restoration upon CFTR modulator treatment. The characterization of S737F-CFTR was performed in two subjects recently assessed in nasal epithelial cells but not in colonoids.ResultsOur functional analysis (Ussing chamber) confirmed that S737F-CFTR is a mild variant with residual function as investigated in colonoids of patients with S737F/Dele22-24 and S737F/W1282X genotypes. An increase of current upon Elexacaftor/Tezacaftor/Ivacaftor (ETI) treatment was recorded for the former genotype. T465N is a poorly characterized missense variant that strongly impacts CFTR function, as almost no CFTR-mediated anion secretion was registered for T465N/Q39X colonoids. ETI treatment substantially improved CFTR-mediated anion secretion and increased the rescue of mature CFTR expression compared to either untreated colonoids or to dual CFTR modulator therapies.ConclusionsOur study confirms the presence of a residual function of the S737F variant and its limited response to CFTR modulators while predicting for the first time the potential clinical benefit of Trikafta® for patients carrying the rare T465N variant.