Abstract
Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2+SOX17+ endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2+ mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1–3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.
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Acknowledgements
This work was supported by the Juvenile Diabetes Research Foundation (JDRF-2-2003-530 to J.M.W.) and the US National Institutes of Health (R01GM072915, R01DK080823A1 and S1 to J.M.W.). This work was also supported in part by PHS grants P30 DK078392 (to J.R.S.) and K01 DK091415 (to J.R.S.). J.C.H. is supported by an Endocrine Fellows Foundation Developmental Research Grant in Diabetes, Obesity and Fat Cell Biology. K.W.M. is partially supported by the Training Program in Organogenesis award 5T32HD046387-05 and by award no. T32-GM063483 from the National Institute of General Medical Sciences. We also acknowledge core support from the Pluripotent Stem Cell Facility of Cincinnati Children's Hospital (supported by grant no. U54 RR025216). We thank J. Whitsett (Cincinnati Children's Hospital Medical Center) for rabbit anti-FOXA2 antibodies and the Pluripotent Stem Cell Facility of Cincinnati Children's Hospital for iPSCs.
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J.M.W., J.R.S., J.C.H. and K.W.M. conceived the study and experimental design. J.M.W. and J.R.S. analyzed data and co-wrote the manuscript. J.C.H. and K.W.M. performed experiments.
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J.M.W. and J.R.S. are inventors on a patent involving the system described in this protocol.
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McCracken, K., Howell, J., Wells, J. et al. Generating human intestinal tissue from pluripotent stem cells in vitro. Nat Protoc 6, 1920–1928 (2011). https://doi.org/10.1038/nprot.2011.410
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DOI: https://doi.org/10.1038/nprot.2011.410
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