Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro1,2. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells3,4,5,6 that have therapeutic efficacy in animal models of liver disease7,8 and diabetes9, respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development10. This involved activin-induced definitive endoderm formation11, FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis12,13,14, and a pro-intestinal culture system15,16 to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal ‘organoids’ consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers17. The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis18, is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.
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Gene Expression Omnibus
Data have been deposited at NCBI under accession number GSE25557.
We thank members of the laboratory, D. Wiginton and C. Wylie for input. We also thank M. Kofron, T. Stefader and R. Lang for assistance with imaging. Vectors and antibodies were from D. Melton (Addgene no. 19410, 19413), S. Yamanaka (17217–17220), C. Baum (OCT4, KLF4, SOX4, MYC lenti), and I. Manabe (KLF5 antibody). This work was supported by the Juvenile Diabetes Research Foundation JDRF-2-2003-530 (J.M.W.) and NIH, R01GM072915 (J.M.W.); R01DK080823A1 and S1 (A.M.Z. and J.M.W.); R03 DK084167 and R01 CA142826 (N.F.S.), F32 DK83202-01 and T32 HD07463 (J.R.S.). We also acknowledge core support for viral vectors, microarrays (supported by P30 DK078392), karyotyping and the Pluripotent Stem Cell Facility (supported by U54 RR025216).
This file contains Supplementary Figures 1-15 with legends, Supplementary Tables 1-3 and additional references.
About this article
Nature Reviews Genetics (2018)