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      Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis

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          Abstract

          Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5 + stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

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          Most cited references 44

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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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              R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling.

              The Wnt/β-catenin signaling system plays essential roles in embryonic development and in the self-renewal and maintenance of adult stem cells. R-spondins (RSPOs) are a group of secreted proteins that enhance Wnt/β-catenin signaling and have pleiotropic functions in development and stem cell growth. LGR5, an orphan receptor of the G protein-coupled receptor (GPCR) superfamily, is specifically expressed in stem cells of the intestinal crypt and hair follicle. Knockout of LGR5 in the mouse results in neonatal lethality. LGR4, a receptor closely related to LGR5, also has essential roles in development, as its knockout leads to reduced viability and retarded growth. Overexpression of both receptors has been reported in several types of cancer. Here we demonstrate that LGR4 and LGR5 bind the R-spondins with high affinity and mediate the potentiation of Wnt/β-catenin signaling by enhancing Wnt-induced LRP6 phosphorylation. Interestingly, neither receptor is coupled to heterotrimeric G proteins or to β-arrestin when stimulated by the R-spondins, indicating a unique mechanism of action. The findings provide a basis for stem cell-specific effects of Wnt/β-catenin signaling and for the broad range of functions LGR4, LGR5, and the R-spondins have in normal and malignant growth.
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                Author and article information

                Journal
                EMBO J
                EMBO J
                The EMBO Journal
                Nature Publishing Group
                0261-4189
                1460-2075
                16 October 2013
                17 September 2013
                17 September 2013
                : 32
                : 20
                : 2708-2721
                Affiliations
                [1 ]Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht , Utrecht, The Netherlands
                [2 ]Diabetes Research Center, Vrije Universiteit Brussel , Brussels, Belgium
                [3 ]Department of Nephrology, Leiden University Medical Center, Albinusdreef 2 , Leiden, The Netherlands
                Author notes
                [a ]Hubrecht Institute for Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584CT Utrecht & University Medical Centre Utrecht , Utrecht, The Netherlands. Tel.:+31 30 212 1800; Fax:+31 30 251 6464; E-mail: h.clevers@ 123456hubrecht.eu
                [b ]Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103 , D2, B1090 Brussel, Belgium. Tel.:+3224774477; Fax:+3224774472; E-mail: Harry.Heimberg@ 123456vub.ac.be
                [*]

                These authors contributed equally to this work.

                [†]

                Present address: Department of Gastroenterology, School of Medicine, Keio University, 35 Shinanomachi, Shinnjukuku, Tokyo 160-8582, Japan

                [‡]

                Present address: Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

                Article
                emboj2013204
                10.1038/emboj.2013.204
                3801438
                24045232
                Copyright © 2013, European Molecular Biology Organization

                This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/.

                Categories
                Article

                Molecular biology

                beta cell, duct cell, pancreas, wnt, stem cell

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