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

John P Gleeson,Michifumi Yamashita,Clive N Svendsen,Robert J Barrett,Hannah Q Estrada,Stephan R Targan

doi:10.3390/ijms21041438

John P Gleeson, Michifumi Yamashita + Show 4 more

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https://doi.org/10.3390/ijms21041438

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Abstract

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.

Highlights

Inflammatory bowel disease (IBD) refers to a spectrum of complex polygenic disorders that are thought to result from dysregulated immune responses to commensal microbes in genetically susceptible hosts
To confirm the feasibility of our approach and its applicability to the IBD field, we generated induced pluripotent stem cell (iPSC) from control individuals, adult onset IBD patients, and very early onset IBD (VEO-IBD) patients
Given that iPSC-derived human intestinal organoids (HIOs) contain both an epithelial and mesenchymal cell population and we wished to seed only epithelial cells onto Transwells, HIOs were subsequently disassociated to a single cell suspension and epithelial cell adhesion molecule (EpCAM/CD326) was used to positively select for HIO-derived epithelial cells using magnetic-activated cell sorted (MACS). 2 × 105 EpCAM+ cells were incorporated into 0.33 cm2 Transwell inserts and after 18 days, monolayers had a Transepithelial electrical resistance (TEER)

Summary

Introduction

Inflammatory bowel disease (IBD) refers to a spectrum of complex polygenic disorders that are thought to result from dysregulated immune responses to commensal microbes in genetically susceptible hosts. In IBD, the intestinal epithelium is characterized by increased permeability both in active disease and remission states, and this increased permeability has been associated with elevated risk of relapse [1,2,3]. While it is known that various inflammatory cytokines and microbes can increase permeability, the finding of increased permeability among a proportion of unaffected first-degree relatives of IBD patients suggests a genetic association [4,5,6]. Given there is increased permeability in both the small [1] and large intestine [3], coupled with enormous genetic heterogeneity among IBD patients, there is an urgent need to develop personalized in vitro cellular models to allow each of these components to be studied in isolation

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