Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids

Gopalakrishnan, Shreya; Bakke, Ingrid; Hansen, Marianne Doré; Skovdahl, Helene Kolstad; Granlund, Atle van Beelen; Sandvik, Arne K.; Bruland, Torunn

doi:10.3389/fimmu.2023.1097383

Cited by 3 publications

(1 citation statement)

References 38 publications

(26 reference statements)

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“…While ISCs serve as the main cell source for these organoids, Pluripotent Stem Cells (PSCs), Embryonic Stem Cells (ESCs) and induced Pluripotent Stem Cells (iPSCs) have also found applications in constructing 3D models [1]. Several protocols currently exist that outline how various organoid types, such as those derived from normal tissue, colorectal cancers, pluripotent stem cells and cancer-associated mutations, can be generated and transplanted to answer clinical questions [18,19]. Application of organoids allow for detailed investigations into the structure, growth, development, function and interactions of the intestinal epithelium with surrounding cells [1,9].…”

Section: Discussionmentioning

confidence: 99%

The Application of Intestinal Stem Cell Organoids in the Treatment of Inflammatory Bowel Disease

Gallicchio

2024

JRMBR

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Inflammatory Bowel Disease (IBD) comprises chronic inflammatory disorders affecting the gastrointestinal tract, such as Crohn’s Disease (CD) and Ulcerative Colitis (UC). The escalating global incidence of IBD is multifactorial, involving genetic, microbial, environmental and immunological factors. Despite current therapies emphasizing immune suppression, sustained efficacy remains elusive. Recently, Stem Cell Therapy (SCT) has emerged as a potential avenue for inducing remission in IBD patients. Intestinal Stem Cells (ISCs) have gained attention for their recent emergence in research and potential cultivation into organoids, offering a promising source for IBD treatment. This review focuses on the potential of ISC SCT, emphasizing its organoid culturing capabilities. It highlights organoids’ applications in IBD research, monitors advancements in animal and human trials and examines current limitations and future directions in organoid research for IBD treatment.

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Section: Discussionmentioning

confidence: 99%

The Application of Intestinal Stem Cell Organoids in the Treatment of Inflammatory Bowel Disease

Gallicchio

2024

JRMBR

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The effect of rs2910686 on ERAP2 expression in IBD and epithelial inflammatory response

Sæterstad,

Østvik,

Hansen

et al. 2024

J Transl Med

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Background ERAP2 is an aminopeptidase involved in antigen processing and presentation, and harbor genetic variants linked to several inflammatory diseases such as Inflammatory Bowel Disease (IBD). The lack of an ERAP2 gene homologue in mice has hampered functional studies, and most human studies have focused on cells of hematopoietic origin. Using an IBD biobank as vantage point, this study explores how genetic variation in ERAP2 affects gene expression in human-derived epithelial organoids upon proinflammatory stimulation. Methods An IBD patient cohort was genotyped with regards to two single nucleotide polymorphisms (SNP) (rs2910686/rs2248374) associated with ERAP2 expression levels, and we examined the correlation between colon gene expression and genotype, specifically aiming to establish a relationship with ERAP2 expression proficiency. Human-derived colon organoids (colonoids) with known ERAP2 genotype were established and used to explore differences in whole genome gene expression between ERAP2-deficient (n = 4) and -proficient (n = 4) donors upon pro-inflammatory encounter. Results When taking rs2910686 genotype into account, ERAP2 gene expression is upregulated in the inflamed colon of IBD patients. Colonoids upregulate ERAP2 upon IFNɣ stimulation, and ERAP2 expression proficiency is dependent on rs2910686 genotype. Colonoid genotyping confirms that mechanisms independent of the frequently studied SNP rs2248374 can cause ERAP2-deficiency. A total of 586 genes involved in various molecular mechanisms are differentially expressed between ERAP2 proficient- and deficient colonoids upon proinflammatory stimulation, including genes encoding proteins with the following molecular function: catalytic activity (AOC1, CPE, ANPEP and MEP1A), regulator activity (TNFSF9, MDK, GDF15, ILR6A, LGALS3 and FLNA), transmembrane transporter activity (SLC40A1 and SLC5A1), and extracellular matrix structural constituents (FGL2, HMCN2, and MUC17). Conclusions ERAP2 is upregulated in the inflamed IBD colon mucosa, and expression proficiency is highly correlated with genotype of rs2910686. While the SNP rs2248374 is commonly used to determine ERAP2 expressional proficiency, our data confirms that mechanisms independent of this SNP can lead to ERAP2 deficiency. Our data demonstrates that epithelial ERAP2 presence affects the inflammatory response in colonoids, suggesting a pleiotropic role of ERAP2 beyond MHC class I antigen processing.

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Review of New Approach Methodologies for Application in Risk Assessment of Nanoparticles in the Food and Feed Sector: Status and Challenges

Usmani,

Bremer‐Hoffmann,

Cheyns

et al. 2024

EFS3

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New Approach Methodologies (NAMs), broadly understood to include in silico, in chemico, in vitro and ex vivo methods, show great potential in advancing risk assessment albeit their regulatory implementation is lagging. The EFSA Guidance on risk assessment of nanomaterials (EFSA Guidance on Nano‐RA) suggests nano‐specific risk assessment is best achieved through Integrated Approaches to Testing and Assessment (IATAs) with NAMs as the first choice to generate new information. Integrating NAMs in risk assessment promises several advantages such as a better human focus, more detailed insights into molecular mechanisms and a higher efficacy. However, applying NAMs to NMs also poses considerable challenges such as issues related to dispersion stability, dosimetry, agglomeration, dissolution, transformations or assay interferences. Significant efforts are being undertaken by standardisation organisations and research projects to establish various NAMs for NMs. Here a thorough review is provided covering NAMs that will be potentially useful for risk assessment of NMs in the food and feed sector. It follows the structure of the EFSA Guidance on Nano‐RA and expands it, where needed, to support decision‐making in selection of NAMs for NM risk assessment. The review begins with an overview on nano‐specific NAM‐frameworks, followed by a description of individual NAMs including those relevant to NM physicochemical characterisation, exposure and hazard assessment covering toxicodynamics and toxicokinetics. The focus is on NAMs concerning NM degradation/dissolution, genotoxicity, cytotoxicity, oxidative stress, (pro‐)inflammation, and barrier integrity as those are important endpoints for initial screening according to the EFSA framework. As a result, in total 267 individual nano‐relevant NAMs, mostly “not validated” (with a few notable exceptions), were included in this review. Validation notwithstanding, NAMs could already prove relevant and reliable for risk assessment of NMs, especially in integrated approaches.

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Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids

Cited by 3 publications

References 38 publications

The Application of Intestinal Stem Cell Organoids in the Treatment of Inflammatory Bowel Disease

The Application of Intestinal Stem Cell Organoids in the Treatment of Inflammatory Bowel Disease

The effect of rs2910686 on ERAP2 expression in IBD and epithelial inflammatory response

Review of New Approach Methodologies for Application in Risk Assessment of Nanoparticles in the Food and Feed Sector: Status and Challenges

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