title: Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.
abstract: 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 vitro. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes, 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 development. This involved activin-induced definitive endoderm formation, FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis, and a pro-intestinal culture system 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 markers. 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 anendocrinosis, 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.
authors.affiliation: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Spence
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Mayhew
authors.forename: Scott A
authors.initials: SA
authors.lastname: Rankin
authors.forename: Matthew F
authors.initials: MF
authors.lastname: Kuhar
authors.forename: Jefferson E
authors.initials: JE
authors.lastname: Vallance
authors.forename: Kathryn
authors.initials: K
authors.lastname: Tolle
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Hoskins
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Kalinichenko
authors.forename: Susanne I
authors.initials: SI
authors.lastname: Wells
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Zorn
authors.forename: Noah F
authors.initials: NF
authors.lastname: Shroyer
authors.forename: James M
authors.initials: JM
authors.lastname: Wells
mesh_terms: D028341:Activins / Q000494:pharmacology; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D019521:Body Patterning / Q000187:drug effects; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D004707:Endoderm / Q000166:cytology / Q000187:drug effects / Q000196:embryology; D051519:Fibroblast Growth Factor 4 / Q000494:pharmacology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007422:Intestines / Q000033:anatomy & histology / Q000166:cytology* / Q000187:drug effects / Q000196:embryology; D008871:Microvilli / Q000187:drug effects; D009024:Morphogenesis / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D038081:Organogenesis / Q000187:drug effects; D013997:Time Factors; D051153:Wnt Proteins / Q000494:pharmacology; D060508:Wnt3 Protein; D060509:Wnt3A Protein
chemical_list: D028341:Activins; D051792:Basic Helix-Loop-Helix Proteins; D003470:Culture Media; D051519:Fibroblast Growth Factor 4; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C496445:FGF4 protein, human; C498695:NEUROG3 protein, human; C555510:WNT3A protein, human; C422405:activin A