rectal-organoid-morphology-analysis-roma-diagnostic-assay-cystic

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two ...

KU Leuven

Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix-cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix-integrin interactions during microglial migration in the embryonic brain ex vivo. We focused on microglia-fibronectin interactions mediated through the fibronectin receptor α5β1 integrin because in vitro work indirectly suggested a role for this ligand-receptor pair. Using 2-photon time-lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age-specific function of the α5β1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5β1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5β1 integrin in microglial migration during colonization of the embryonic brain.

Age-specific function of α5β1 integrin in microglial migration during early colonization of the developing mouse cortex.

Determining the exact pathogenesis of chronic gastrointestinal diseases remains difficult due to the complex in vivo environment. In this review we give an overview of the available epithelial cell culture systems developed to investigate pathophysiology of gastrointestinal diseases. Traditionally used two-dimensional (2D) immortalised (tumour) cell lines survive long-term, but are not genetically stable nor represent any human in particular. In contrast, primary cultures are patient unique, but short-lived. Three-dimensional (3D) organoid cultures resemble the crypt-villus domain and contain all cell lineages, are long-lived and genetically stable. Unfortunately, manipulation of the 3D organoid system is more challenging. Combining the 3D and 2D technologies may overcome limitations and offer the formation of monolayers on permeable membranes or flow-chambers. Determining the right model to use will depend on the pathology of interest and the focus of the research, defining which cell types need to be included in the model.

Human intestinal epithelium in a dish: Current models for research into gastrointestinal pathophysiology.

In vitro studies using immortalised cancer cell lines showed that butyrate has an overall positive effect on epithelial barrier integrity, but the physiological relevance of cancer cell lines is limited. We developed epithelial monolayers from human tissue samples of patients with ulcerative colitis [UC] to assess the effect of butyrate on epithelial barrier function.

Butyrate Does Not Protect Against Inflammation-induced Loss of Epithelial Barrier Function and Cytokine Production in Primary Cell Monolayers From Patients With Ulcerative Colitis.

We aimed to identify biomarkers that might be used to predict responses of patients with inflammatory bowel diseases (IBD) to vedolizumab therapy.

Expression Levels of 4 Genes in Colon Tissue Might Be Used to Predict Which Patients Will Enter Endoscopic Remission After Vedolizumab Therapy for Inflammatory Bowel Diseases.

Ex Vivo Mimicking of Inflammation in Organoids Derived From Patients With Ulcerative Colitis.

Patients with inflammatory bowel disease [IBD] are considered immunosuppressed, but do not seem more vulnerable for COVID-19. Nevertheless, intestinal inflammation has shown to be an important risk factor for SARS-CoV-2 infection and prognosis. Therefore, we investigated the role of intestinal inflammation on the viral intestinal entry mechanisms, including ACE2, in IBD.

Intestinal Receptor of SARS-CoV-2 in Inflamed IBD Tissue Seems Downregulated by HNF4A in Ileum and Upregulated by Interferon Regulating Factors in Colon.

The gut microbiota appears to play a central role in health, and alterations in the gut microbiota are observed in both forms of inflammatory bowel disease [IBD], namely Crohn's disease and ulcerative colitis. Yet, the mechanisms behind host-microbiota interactions in IBD, especially at the intestinal epithelial cell level, are not yet fully understood. Dissecting the role of host-microbiota interactions in disease onset and progression is pivotal, and requires representative models mimicking the gastrointestinal ecosystem, including the intestinal epithelium, the gut microbiota, and immune cells. New advancements in organoid microfluidics technology are facilitating the study of IBD-related microbial-epithelial cross-talk, and the discovery of novel microbial therapies. Here, we review different organoid-based ex vivo models that are currently available, and benchmark their suitability and limitations for specific research questions. Organoid applications, such as patient-derived organoid biobanks for microbial screening and 'omics technologies, are discussed, highlighting their potential to gain better mechanistic insights into disease mechanisms and eventually allow personalised medicine.

Organoid-based Models to Study the Role of Host-microbiota Interactions in IBD.

Diagnosing cystic fibrosis (CF) when sweat chloride is not in the CF range and less than 2 disease-causing  mutations are found requires physiological CFTR assays, which are not always feasible or available. We developed a new physiological CFTR assay based on the morphological differences between rectal organoids from subjects with and without CF. In organoids from 167 subjects with and 22 without CF, two parameters derived from a semi-automated image analysis protocol (rectal organoid morphology analysis, ROMA) fully discriminated CF subjects with two disease-causing mutations from non-CF subjects (p<0.001). ROMA, feasible at all ages, can be centralised to improve standardisation.

Rectal organoid morphology analysis (ROMA) as a promising diagnostic tool in cystic fibrosis.

Inflammatory Bowel Disease (IBD) has a multifactorial origin and originates from a complex interplay of environmental factors with the innate immune system at the intestinal epithelial interface in a genetically susceptible individual. All these factors make its etiology intricate and largely unknown. Multi-omic datasets obtained from IBD patients are required to gain further insights into IBD biology. We here review the landscape of multi -omic data availability in IBD and identify barriers and gaps for future research. We also outline the various technical and non-technical factors that influence the utility and interpretability of multi -omic datasets and thereby the study design of any research project generating such datasets. Co-ordinated generation of multi-omic datasets and their systemic integration with clinical phenotypes and environmental exposures will not only enhance understanding of the fundamental mechanisms of IBD but also improve therapeutic strategies. Finally, we provide recommendations to enable and facilitate generation of multi -omic datasets.

Article	Year
Age-specific function of α5β1 integrin in microglial migration during early colonization of the developing mouse cortex. Glia\| PubMed ID: 28417486	2017
Human intestinal epithelium in a dish: Current models for research into gastrointestinal pathophysiology. United European gastroenterology journal\| PubMed ID: 29238585	2017
Butyrate Does Not Protect Against Inflammation-induced Loss of Epithelial Barrier Function and Cytokine Production in Primary Cell Monolayers From Patients With Ulcerative Colitis. Journal of Crohn's & colitis\| PubMed ID: 30919886	2019
Expression Levels of 4 Genes in Colon Tissue Might Be Used to Predict Which Patients Will Enter Endoscopic Remission After Vedolizumab Therapy for Inflammatory Bowel Diseases. Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association\| PubMed ID: 31446181	2020
Ex Vivo Mimicking of Inflammation in Organoids Derived From Patients With Ulcerative Colitis. Gastroenterology\| PubMed ID: 32474118	2020
Intestinal Receptor of SARS-CoV-2 in Inflamed IBD Tissue Seems Downregulated by HNF4A in Ileum and Upregulated by Interferon Regulating Factors in Colon. Journal of Crohn's & colitis\| PubMed ID: 32915959	2021
Organoid-based Models to Study the Role of Host-microbiota Interactions in IBD. Journal of Crohn's & colitis\| PubMed ID: 33341879	2021
Rectal organoid morphology analysis (ROMA) as a promising diagnostic tool in cystic fibrosis. Thorax\| PubMed ID: 33859053	2021
Tailoring multi-omics to inflammatory bowel diseases: all for one and one for all. Journal of Crohn's & colitis\| PubMed ID: 35150242	2022

Kaline Arnauts

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