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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.

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Authors

Paulina M. Durczak

Kathryn L. Fair

Sara Cuevas Ocaña

Carlos B. Sainz Zuñiga

NICK HANNAN NICK.HANNAN@NOTTINGHAM.AC.UK
Associate Professor



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. (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 General Immunology and Microbiology; General Biochemistry, Genetics and Molecular Biology; General Chemical Engineering; General Neuroscience
Public URL https://nottingham-repository.worktribe.com/output/6613527
Publisher URL https://www.jove.com/t/61199/generation-hipsc-derived-intestinal-organoids-for-developmental

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