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      Modeling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary

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          Organogenesis is a complex and inter-connected process, orchestrated by multiple boundary tissue interactions 17 . However, it is currently unclear how individual, neighboring components coordinate to establish an integral multi-organ structure. Here, we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cell (PSC). The boundary interactions between anterior and posterior gut spheroids differentiated from human PSCs enables autonomous emergence of hepato-biliary-pancreatic (HBP) organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factor supply. Whereas transplant-derived tissues were dominated by midgut derivatives, long-term culture of micro dissected HBP organoids develop into a segregated hepato-pancreato-biliary anlage, followed by the recapitulation of early morphogenetic events including the invagination and branching of three different and inter-connected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Missegregation of multi-organ domains incurred by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo 8, 9 . Together, we demonstrate that the experimental multi-organ integrated model can be established by the juxta-positioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily-accessible model for the study of complicated endoderm organogenesis in human.

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          Most cited references 34

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          An in vivo model of human small intestine using pluripotent stem cells.

          Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes, for pharmacologic studies and as a potential resource for therapeutic transplant. To date, limited in vivo models exist for human intestine, all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here, we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme, both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme, as demonstrated by differentiated intestinal cell lineages (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells), presence of functional brush-border enzymes (lactase, sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore, transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection, suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology, disease and translational studies.
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            Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro

            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 1,2. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells3–6 that have therapeutic efficacy in animal models of liver disease 7,8 and diabetes 9 respectively. However the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. We have established 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 10 (Summarized in supplementary Fig. 1). This involved activin-induced definitive endoderm (DE) formation 11, FGF/Wnt induced posterior endoderm pattering, hindgut specification and morphogenesis 12–14; and a pro-intestinal culture system 15,16 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 suggests that human intestinal stem cells form de novo during development. Lastly we determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis 18, is both necessary and sufficient for human enteroendocrine cell development in vitro. In conclusion, PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.
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              Vascularized and functional human liver from an iPSC-derived organ bud transplant.

              A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver-specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine.

                Author and article information

                30 August 2019
                25 September 2019
                October 2019
                05 November 2020
                : 574
                : 7776
                : 112-116
                [1. ]Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
                [2. ]Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
                [3. ]Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
                [4. ]Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
                [5. ]Department of Pediatrics, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
                [6. ]Institute of Research, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
                Author notes

                Author Contributions

                H.K., K.I. and R.O. carried out the experiment and analyzed data. M.M. and A.F. performed the organoid experiment. K.G. and N.S. performed computational analysis. H.K. and T.T. wrote the manuscript with support from K.I, R.O. and W.T.. M.K., J.M.W. and A.M.Z helped supervise the project. H.K and T.T. conceived the original idea. T.T. supervised the project.

                Corresponding Author: Takanori Takebe, takanori.takebe@ 123456cchmc.org

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