Development of a Personalized Model of Intestinal Fibrosis Using Human Intestinal Organoids Derived From Induced Pluripotent Stem Cells

Abstract

Background Intestinal fibrosis is a serious complication of inflammatory bowel disease (IBD) with > 20% of Crohn’s disease patients developing this complication within 10 years of diagnosis. Despite improvements in anti-inflammatory medication, its incidence remains stubbornly high and thus far surgical intervention remains the only treatment option. Numerous cell types including intestinal epithelial and mesenchymal cells are implicated in this process, yet studies are hampered by the lack of personalized in vitro models. One potential avenue that would permit a personalized approach is to utilize human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs). iPSCs can be generated from any individual, faithfully recapitulate the genetics of the host and can be directed to form HIOs that contain both epithelial and mesenchymal cells. Our goal was to determine the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. Methods iPSCs from two control individuals and two very early onset-IBD (VEOIBD) patients with stricturing complications were obtained and directed to form HIOs. Given HIOs are heterogeneous in terms of size, shape and ratio of mesenchymal to epithelial cells, they were firstly dissociated to a single cell suspension and EpCAM was used to positively select for epithelial cells using magnetic activated cellular sorting. These EpCAM+ cells were then seeded onto transwells and EpCAM- cells were seeded as monolayers in 10% serum containing media. Both cell types were treated with the profibrotic cytokine TGFβ, and changes in the expression of selected genes were analyzed. Results iPSCs from all 4 individuals could be directed to form HIOs containing both epithelial (E-cadherin+) and mesenchymal (vimentin+) cells (see Fig. 1). In the TGFβ-treated mesenchymal cell population, expression of N-cadherin and Col1a1 was significantly increased in all four lines after 8 and 48hrs respectively, with the highest increase occurring in cells derived from VEOIBD patient 2 (see Table 1). In the TGFβ-treated epithelial cell population, Col1a1 and fibronectin expression was increased in all lines after 96hrs with the highest fold change occurring in cells derived from VEOIBD patient 1 (fibronectin) and 2 (Col1a1). Conclusion We demonstrate the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. We show that iPSCs generated from all selected individuals could be directed to form HIOs and that responses to the profibrotic cytokine TGFβ can be examined in both intestinal epithelial and mesenchymal cells. This now permits the generation of near unlimited quantities of patient specific cells that could be used to reveal cell and environmental specific mechanisms underpinning intestinal fibrosis which may ultimately lead to personalized treatments. Fluorescent images of human intestinal organoids generated from A) Control 1, B) Control 2, C) VEOIBD patient 1 and D) VEOIBD patient 2 that were immunostained with vimentin (green), E-cadherin (red) and counterstainied with DAPI (blue). All images X20

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“…The pathogenesis of CD fibrosis is not completely understood, which is largely due to the lack of human-specific intestinal fibrosis models. Excitingly, in 2021, Estrada et al 10 generated human intestinal organoids (HIOs) using induced pluripotent stem cells from CD patients with strictures, and epithelial and mesenchymal cells derived from HIOs under the stimulation of transforming growth factor beta (TGF-β) were able to replicate intestinal fibrotic responses. This model shed new insight on the exploration of mechanisms underlying intestinal fibrosis.…”

Intersection of stem cell biology and engineering towards next generation in vitro models of human fibrosis

“…Abnormal fibrotic deposition of ECM proteins and myofibroblast activation were observed when Duchenne muscular dystrophy (DMD) fibroblasts were embedded in the 3D vascularized muscle environment ( Bersini et al, 2018 ) ( Table 1 ). Estrada et al (2021) reported the generation of human intestinal organoids derived from patient derived-induced pluripotent stem cells. These 3D organoids provided a personalized approach for modeling intestinal fibrotic responses in vitro ( Table 1 ).…”

The role of Cdc42 Rho GTPase in the cystic fibrosis intestinal epithelial barrier

“…Human iPSCs are self-renewing pluripotent cells that can produce cells from any of the three germ layers (endoderm, ectoderm, and mesoderm) 186,188 . Human iPSCs can be differentiated down the intestinal epithelial lineage and used to grow human intestinal organoids (HIOs) 189 , which have been used extensively to study IBD 190,191 , host-microbe interactions 189,192 , and, more recently, as a method to develop CFTR modulator therapies 193 .…”