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

Durczak, Paulina M; Fair, Kathryn L; Jinks, Nicholas; Cuevas Ocaña, Sara; Sainz Zuñiga, Carlos B; Hannan, Nicholas R F

Abstract

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-β, 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.

Citation

Durczak, P. M., Fair, K. L., Jinks, N., Cuevas Ocaña, S., Sainz Zuñiga, C. B., & Hannan, N. R. F. (2024). Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications. Journal of Visualized Experiments, 2024(205), Article e61199. https://doi.org/10.3791/61199

• Journal Article Type: Article
• Acceptance Date: May 25, 2020
• Online Publication Date: Mar 8, 2024
• Publication Date: Mar 8, 2024
• Deposit Date: Nov 5, 2021
• Publicly Available Date: Mar 14, 2024
• Journal: Journal of Visualized Experiments
• Electronic ISSN: 1940-087X
• Publisher: Journal of Visualized Experiments
• Peer Reviewed: Peer Reviewed
• Volume: 2024
• Issue: 205
• Article Number: e61199
• DOI: https://doi.org/10.3791/61199
• Keywords: Intestines, Cell Differentiation, Intestinal Mucosa, Induced Pluripotent Stem Cells, Organoids, Humans, Inflammatory Bowel Diseases
• Public URL: https://nottingham-repository.worktribe.com/output/6613527
• Publisher URL: https://www.jove.com/t/61199/generation-hipsc-derived-intestinal-organoids-for-developmental