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      Pdx1 and Ngn3 Overexpression Enhances Pancreatic Differentiation of Mouse ES Cell-Derived Endoderm Population

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          Abstract

          In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin ( Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit + endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.

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          Most cited references 39

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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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              Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas.

              Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in today's diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                13 September 2011
                : 6
                : 9
                Affiliations
                [1 ]First Department of Internal Medicine, Nara Medical University, Kashihara, Nara, Japan
                [2 ]VistaGen Therapeutics, Inc., South San Francisco, California, United States of America
                [3 ]The Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, New York, United States of America
                [4 ]First Department of Surgery, Nara Medical University, Kashihara, Nara, Japan
                [5 ]McEwen Centre for Regenerative Medicine, Toronto, Canada
                University of Southern California, United States of America
                Author notes

                Conceived and designed the experiments: AK GK RS. Performed the experiments: AK RS MT KB MS . Analyzed the data: AK RS MT KB RS. Contributed reagents/materials/analysis tools: VG-E MI YS . Wrote the paper: AK GK RS.

                Article
                PONE-D-11-03745
                10.1371/journal.pone.0024058
                3172220
                21931641
                Kubo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 12
                Categories
                Research Article
                Biology
                Developmental Biology
                Stem Cells
                Embryonic Stem Cells
                Molecular Cell Biology
                Cellular Types
                Stem Cells
                Embryonic Stem Cells
                Medicine
                Anatomy and Physiology
                Endocrine System
                Endocrine Physiology
                Insulin
                Endocrinology
                Diabetic Endocrinology
                Gastroenterology and Hepatology
                Pancreas

                Uncategorized

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