Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro

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Abstract
Using a sequence of growth-factor manipulations to mimic embryonic intestinal development in culture, a new study has successfully generated human intestinal tissue in vitro from embryonic and induced pluripotent stem cells. The resulting epithelium was uniformly intestinal, with villus-like structures and progenitor domains, and contained all of the functional cell types of the gut, including brush borders that were indistinguishable from adult intestine. This approach may provide therapeutic benefit for disease studies. Using a temporal series of growth factor manipulations to mimic embryonic intestinal development in culture, this study has successfully directed the differentiation of human pluripotent stem cells (both embryonic stem cells and induced pluripotent stem cells) into intestinal tissue. This approach may provide therapeutic benefit for disease studies. 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.