Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications.

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of the human intestinal epithelium, in which they exhibit crypt/villus-like structures. Here, we describe the generation of hiPSC-derived intestinal organoids by the stepwise differentiation of hiPSCs into definitive endoderm, which is then posteriorized to form hindgut epithelium before being transferred into 3D culture conditions. The 3D culture environment consists of extracellular matrix (ECM) (e.g., Matrigel or other compatible ECM) supplemented with SB202190, A83-01, Gastrin, Noggin, EGF, R-spondin-1 and CHIR99021. Organoids undergo passaging every 7 days, where they are mechanically disrupted before transfer to fresh extracellular matrix and allowed to expand. QPCR and immunocytochemistry confirm that hiPSC-derived intestinal organoids contain mature intestinal epithelial cell types including goblet cells, Paneth cells and enterocytes. Additionally, organoids show evidence of polarization by expression of villin localized on the apical surface of epithelial cells. The resulting organoids can be used to model human intestinal development as well as numerous human intestinal diseases including inflammatory bowel disease. To model intestinal inflammation, organoids can be exposed to inflammatory mediators such as TNF-α, TGF-ß, and bacterial LPS. Organoids exposed to proinflammatory cytokines display an inflammatory and fibrotic phenotype in response. Pairing of healthy versus hiPSCs derived from patients with IBD may be useful in understanding mechanisms driving IBD. This may reveal novel therapeutic targets and novel biomarkers to assist in early disease diagnosis.

In Vitro and in Vivo Assays for Testing Retinoids Effect on Intestinal Progenitors' Lineage Commitments.

The intestine consists of epithelial cells surrounded by a complex environment as mesenchymal cells and the gut microbiota. With its impressive stem cell regeneration capability, the intestine is able to constantly replenish cells lost through apoptosis or abrasion by food passing through. Over the past decade, researchers have identified signaling pathways involved in stem cell homeostasis such as retinoids pathway. Retinoids are also involved in cell differentiation of healthy and cancer cells. In this study, we describe several approaches in vitro and in vivo to further investigate the effect of retinoids on stem cells, progenitors, and differentiated intestinal cells.

Intestinal organoids for modelling intestinal development and disease.

Gastrointestinal diseases are becoming increasingly prevalent in developed countries. Immortalized cells and animal models have delivered important but limited insight into the mechanisms that initiate and propagate these diseases. Human-specific models of intestinal development and disease are desperately needed that can recapitulate structure and function of the gut in vitro Advances in pluripotent stem cells and primary tissue culture techniques have made it possible to culture intestinal epithelial cells in three dimensions that self-assemble to form 'intestinal organoids'. These organoids allow for new, human-specific models that can be used to gain insight into gastrointestinal disease and potentially deliver new therapies to treat them. Here we review current in vitro models of intestinal development and disease, considering where improvements could be made and potential future applications in the fields of developmental modelling, drug/toxicity testing and therapeutic uses.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.