Development of Physiologically Responsive Human iPSC-Derived Intestinal Epithelium to Study Barrier Dysfunction in IBD

In inflammatory bowel disease (IBD), the intestinal epithelium is characterized by increased permeability both in active disease and remission states. The genetic underpinnings of this increased intestinal permeability are largely unstudied, in part due to a lack of appropriate modelling systems. Our aim is to develop an in vitro model of intestinal permeability using induced pluripotent stem cell (iPSC)-derived human intestinal organoids (HIOs) and human colonic organoids (HCOs) to study barrier dysfunction. iPSCs were generated from healthy controls, adult onset IBD, and very early onset IBD (VEO-IBD) patients and differentiated into HIOs and HCOs. EpCAM+ selected cells were seeded onto Transwell inserts and barrier integrity studies were carried out in the presence or absence of pro-inflammatory cytokines TNFα and IFNγ. Quantitative real-time PCR (qRT-PCR), transmission electron microscopy (TEM), and immunofluorescence were used to determine altered tight and adherens junction protein expression or localization. Differentiation to HCO indicated an increased gene expression of CDX2, CD147, and CA2, and increased basal transepithelial electrical resistance compared to HIO. Permeability studies were carried out in HIO- and HCO-derived epithelium, and permeability of FD4 was significantly increased when exposed to TNFα and IFNγ. TEM and immunofluorescence imaging indicated a mislocalization of E-cadherin and ZO-1 in TNFα and IFNγ challenged organoids with a corresponding decrease in mRNA expression. Comparisons between HIO- and HCO-epithelium show a difference in gene expression, electrophysiology, and morphology: both are responsive to TNFα and IFNγ stimulation resulting in enhanced permeability, and changes in tight and adherens junction architecture. This data indicate that iPSC-derived HIOs and HCOs constitute an appropriate physiologically responsive model to study barrier dysfunction and the role of the epithelium in IBD and VEO-IBD.

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Dynamic effects of genetic variation on gene expression revealed following hypoxic stress in cardiomyocytes

eLife ◽

10.7554/elife.57345 ◽

2021 ◽

Vol 10 ◽

Cited By ~ 1

Author(s):

Michelle C Ward ◽

Nicholas E Banovich ◽

Abhishek Sarkar ◽

Matthew Stephens ◽

Yoav Gilad

Keyword(s):

Gene Expression ◽

Complex Traits ◽

Induced Pluripotent Stem Cell ◽

Chromatin Accessibility ◽

Regulatory Changes ◽

Life Threatening ◽

Vitro Model ◽

Culturing Conditions ◽

Induced Pluripotent

One life-threatening outcome of cardiovascular disease is myocardial infarction, where cardiomyocytes are deprived of oxygen. To study inter-individual differences in response to hypoxia, we established an in vitro model of induced pluripotent stem cell-derived cardiomyocytes from 15 individuals. We measured gene expression levels, chromatin accessibility, and methylation levels in four culturing conditions that correspond to normoxia, hypoxia, and short- or long-term re-oxygenation. We characterized thousands of gene regulatory changes as the cells transition between conditions. Using available genotypes, we identified 1,573 genes with a cis expression quantitative locus (eQTL) in at least one condition, as well as 367 dynamic eQTLs, which are classified as eQTLs in at least one, but not in all conditions. A subset of genes with dynamic eQTLs is associated with complex traits and disease. Our data demonstrate how dynamic genetic effects on gene expression, which are likely relevant for disease, can be uncovered under stress.

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Food Contaminants Effects on an In Vitro Model of Human Intestinal Epithelium

Toxics ◽

10.3390/toxics9060135 ◽

2021 ◽

Vol 9 (6) ◽

pp. 135

Author(s):

Marion Guibourdenche ◽

Johanna Haug ◽

Noëllie Chevalier ◽

Madeleine Spatz ◽

Nicolas Barbezier ◽

...

Keyword(s):

Gene Expression ◽

Intestinal Permeability ◽

Intestinal Epithelium ◽

In Vitro Model ◽

Food Contaminants ◽

Gut Barrier ◽

Gut Barrier Function ◽

Vitro Model ◽

Human Intestinal Epithelium

Pesticide residues represent an important category of food contaminants. Furthermore, during food processing, some advanced glycation end-products resulting from the Maillard reaction can be formed. They may have adverse health effects, in particular on the digestive tract function, alone and combined. We sought to validate an in vitro model of the human intestinal barrier to mimic the effects of these food contaminants on the epithelium. A co-culture of Caco-2/TC7 cells and HT29-MTX was stimulated for 6 h with chlorpyrifos (300 μM), acrylamide (5 mM), Nε-Carboxymethyllysine (300 μM) alone or in cocktail with a mix of pro-inflammatory cytokines. The effects of those contaminants on the integrity of the gut barrier and the inflammatory response were analyzed. Since the co-culture responded to inflammatory stimulation, we investigated whether this model could be used to evaluate the effects of food contaminants on the human intestinal epithelium. CPF alone affected tight junctions’ gene expression, without inducing any inflammation or alteration of intestinal permeability. CML and acrylamide decreased mucins gene expression in the intestinal mucosa, but did not affect paracellular intestinal permeability. CML exposure activated the gene expression of MAPK pathways. The co-culture response was stable over time. This cocktail of food contaminants may thus alter the gut barrier function.

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Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes as an in vitro model in toxicology: strengths and weaknesses for hazard identification and risk characterization

Expert Opinion on Drug Metabolism & Toxicology ◽

10.1080/17425255.2021.1894122 ◽

2021 ◽

Author(s):

Sarah D. Burnett ◽

Alexander D. Blanchette ◽

Weihsueh A. Chiu ◽

Ivan Rusyn

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

In Vitro Model ◽

Induced Pluripotent Stem Cell ◽

Hazard Identification ◽

Risk Characterization ◽

Vitro Model ◽

Induced Pluripotent

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Abstract 246: Human Induced Pluripotent Stem Cells Reveal Mitophagy as an Essential Process Against Diabetic Cardiomyopathy

Circulation Research ◽

10.1161/res.117.suppl_1.246 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Sang-Ging Ong ◽

Won Hee Lee ◽

Kazuki Kodo ◽

Haodi Wu ◽

Joseph C Wu

Keyword(s):

Oxidative Stress ◽

Diabetic Cardiomyopathy ◽

Mitochondrial Fission ◽

Induced Pluripotent Stem Cell ◽

Mitochondrial Fragmentation ◽

Normal Cells ◽

Mitochondrial Pathology ◽

Vitro Model ◽

Induced Pluripotent

Diabetic cardiomyopathy is a common consequence of diabetes and associated with mitochondrial pathology. Using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as an in vitro model of diabetes, we sought to understand the role of mitophagy, a process that selectively degrades mitochondria through the autophagy-lysosome pathway as a crucial quality control pathway against diabetic cardiomyopathy. We first showed that iPSC-CMs exposed to a diabetic milieu demonstrated increased hypertrophy, impaired calcium signaling, and higher oxidative stress. Flow cytometry analysis of iPSC-CMs subjected to diabetic conditions revealed two distinct population of cells (normal and hypertrophied), suggesting a heterogeneous response to hyperglycemia. In these cells, hypertrophied iPSC-CMs were found to have reduced mitophagy compared to normal cells when exposed to hyperglycemia. In addition, we showed that mitochondrial fragmentation was also decreased in the hypertrophied iPSC-CMs compared to normal cells upon exposure to hyperglycemia, demonstrating a link between mitochondrial fragmentation and mitophagy. Surprisingly, pretreatment of iPSC-CMs with a non-selective antioxidant, N-(2-mercaptopropionyl)-glycine, not only failed to limit the deleterious effects of hyperglycemia, but actually led to increased hypertrophy and cell death. We found that mitophagy was significantly reduced in iPSC-CMs following antioxidant treatment, suggesting the need of mild oxidative stress as a trigger for mitophagy. Future studies are warranted to further investigate the association between oxidative stress, mitochondrial fragmentation, and mitochondrial fission as targets against diabetic cardiomyopathy.

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Human Cardiac Organoids for Modeling Genetic Cardiomyopathy

Cells ◽

10.3390/cells9071733 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1733 ◽

Cited By ~ 1

Author(s):

Michele Filippo Buono ◽

Lisa von Boehmer ◽

Jaan Strang ◽

Simon P. Hoerstrup ◽

Maximilian Y. Emmert ◽

...

Keyword(s):

Cardiac Fibroblasts ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Promising Tool ◽

Vitro Model ◽

Human Cardiac Fibroblasts ◽

Induced Pluripotent ◽

First Time ◽

Design And Testing

Genetic cardiomyopathies are characterized by changes in the function and structure of the myocardium. The development of a novel in vitro model could help to better emulate healthy and diseased human heart conditions and may improve the understanding of disease mechanisms. In this study, for the first time, we demonstrated the generation of cardiac organoids using a triculture approach of human induced pluripotent stem-cell-derived cardiomyocytes (hiPS-CMs)—from healthy subjects and cardiomyopathy patients—human cardiac microvascular endothelial cells (HCMECs) and human cardiac fibroblasts (HCFs). We assessed the organoids’ suitability as a 3D cellular model for the representation of phenotypical features of healthy and cardiomyopathic hearts. We observed clear differences in structure and beating behavior between the organoid groups, depending on the type of hiPS-CMs (healthy versus cardiomyopathic) used. Organoids may thus prove a promising tool for the design and testing of patient-specific treatments as well as provide a platform for safer and more efficacious drug development.

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