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

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          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 1, 2. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells 36 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 1214; 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 markers 17. 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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          Most cited references 24

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          Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.

          Development of a cell therapy for diabetes would be greatly aided by a renewable supply of human beta-cells. Here we show that pancreatic endoderm derived from human embryonic stem (hES) cells efficiently generates glucose-responsive endocrine cells after implantation into mice. Upon glucose stimulation of the implanted mice, human insulin and C-peptide are detected in sera at levels similar to those of mice transplanted with approximately 3,000 human islets. Moreover, the insulin-expressing cells generated after engraftment exhibit many properties of functional beta-cells, including expression of critical beta-cell transcription factors, appropriate processing of proinsulin and the presence of mature endocrine secretory granules. Finally, in a test of therapeutic potential, we demonstrate that implantation of hES cell-derived pancreatic endoderm protects against streptozotocin-induced hyperglycemia. Together, these data provide definitive evidence that hES cells are competent to generate glucose-responsive, insulin-secreting cells.
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            Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

            Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.
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              Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.

              The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                0028-0836
                1476-4687
                24 November 2010
                12 December 2010
                3 February 2011
                1 August 2011
                : 470
                : 7332
                : 105-109
                Affiliations
                [1 ] Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039
                [2 ] Division of Hematology and Oncology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039
                [3 ] Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039
                [4 ] Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039
                Author notes
                [* ]Corresponding Author: Correspondence and requests for material should be addressed to: james.wells@ 123456cchmc.org
                Article
                nihpa254446
                10.1038/nature09691
                3033971
                21151107

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                Funding
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Funded by: National Institute of General Medical Sciences : NIGMS
                Funded by: National Cancer Institute : NCI
                Award ID: R03 DK084167-02 ||DK
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Funded by: National Institute of General Medical Sciences : NIGMS
                Funded by: National Cancer Institute : NCI
                Award ID: R01 GM072915-01A2 ||GM
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Funded by: National Institute of General Medical Sciences : NIGMS
                Funded by: National Cancer Institute : NCI
                Award ID: R01 DK080823-01A1S1 ||DK
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Funded by: National Institute of General Medical Sciences : NIGMS
                Funded by: National Cancer Institute : NCI
                Award ID: R01 DK080823-01A1 ||DK
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Funded by: National Institute of General Medical Sciences : NIGMS
                Funded by: National Cancer Institute : NCI
                Award ID: R01 CA142826-02 ||CA
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Funded by: National Institute of General Medical Sciences : NIGMS
                Funded by: National Cancer Institute : NCI
                Award ID: F32 DK083202-01 ||DK
                Categories
                Article

                Uncategorized

                drug transport, progenitor cell, fgf, wnt, transplantation, colon, intestine, posterior endoderm

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