TY - JOUR
T1 - Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids
AU - Martinez-Silgado, Adriana
AU - Yousef Yengej, Fjodor A.
AU - Puschhof, Jens
AU - Geurts, Veerle
AU - Boot, Charelle
AU - Geurts, Maarten H.
AU - Rookmaaker, Maarten B.
AU - Verhaar, Marianne C.
AU - Beumer, Joep
AU - Clevers, Hans
N1 - 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)
PY - 2022/9/16
Y1 - 2022/9/16
N2 - 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 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).
AB - 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 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).
KW - Cell differentiation
KW - CRISPR
KW - Organoids
KW - CRISPR-Cas Systems/genetics
KW - Intestines
KW - Genetic Engineering
KW - Humans
KW - Cell Differentiation/genetics
UR - http://www.scopus.com/inward/record.url?scp=85136198434&partnerID=8YFLogxK
U2 - 10.1016/j.xpro.2022.101639
DO - 10.1016/j.xpro.2022.101639
M3 - Article
C2 - 36042877
AN - SCOPUS:85136198434
SN - 2666-1667
VL - 3
JO - STAR protocols
JF - STAR protocols
IS - 3
M1 - 101639
ER -