Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids
Martinez-Silgado, Adriana; Yousef Yengej, Fjodor A.; Puschhof, Jens; Geurts, Veerle; Boot, Charelle; Geurts, Maarten H.; Rookmaaker, Maarten B.; Verhaar, Marianne C.; Beumer, Joep; Clevers, Hans
(2022) STAR protocols, volume 3, issue 3
(Article)
Abstract
Intestinal organoids are three-dimensional cultures that resemble key aspects of the epithelium of origin. Here, we describe how to differentiate human small intestinal organoids by combining growth media variations and genetic engineering. We detail the differentiation of human intestinal organoids in the presence and absence of BMP agonists to recapitulate
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a broader scope of functional cell states found in vivo. Using transient overexpression of the transcription factor Neurogenin-3, we describe the enhancement of differentiation toward rare enteroendocrine cells. For complete details on the use and execution of this protocol, please refer to Beumer et al. (2022).
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Keywords: Cell differentiation, CRISPR, Organoids, CRISPR-Cas Systems/genetics, Intestines, Genetic Engineering, Humans, Cell Differentiation/genetics, General Biochemistry,Genetics and Molecular Biology, General Immunology and Microbiology, General Neuroscience, Research Support, Non-U.S. Gov't, Journal Article
ISSN: 2666-1667
Publisher: Cell Press
Note: Funding Information: The development of the methods was supported by Netherlands Organ-on-Chip Initiative ( 024.003.001 ) from the Netherlands Organisation for Scientific Research (NWO) funded by the Ministry of Education, Culture and Science of the government of the Netherlands (A.M.S. and H.C.); the Oncode Institute (partly financed by the Dutch Cancer Society ); the European Research Council under ERC Advanced Grant (Guthormones; nr 101020405 ) (J.B. and H.C.); and NETRF / Petersen Accelerator (J.B. and H.C.). F.A.Y.Y., M.B.R., and M.C.V. acknowledge the Gravitation Program “Materials Driven Regeneration,” funded by the Netherlands Organization for Scientific Research (024.003.013) and the support of the partners of “Regenerative Medicine Crossing Borders” (RegMed XB), powered by Health∼Holland, Top Sector Life Sciences&Health. Publisher Copyright: © 2022 The Author(s)
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