In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells |
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Authors: | T.?León-Quinto,J.?Jones,A.?Skoudy,M.?Burcin,B.?Soria author-information" > author-information__contact u-icon-before" > mailto:bernat.soria@umh.es" title=" bernat.soria@umh.es" itemprop=" email" data-track=" click" data-track-action=" Email author" data-track-label=" " >Email author |
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Affiliation: | (1) Institute of Bioengineering, School of Medicine, Miguel Hernández University, San Juan Campus, Carretera Alicante-Valencia Km 87, 03550 Alicante, Spain;(2) Cellular and Molecular Biology Unit, Municipal Institute of Medical Research (IMIM), Pompeu Fabra University, Barcelona;(3) Cardion, Erkrath, Germany;(4) Department of Surgery, National University of Singapore, Singapore |
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Abstract: | Aims/hypothesis We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. Methods Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice.Results The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols.Conclusions/interpretation These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.Abbreviations Anti-Shh antibody against sonic hedgehog - D3 undifferentiated D3 stem cell line - EB embryoid bodies - ES embryonic stem - FBS fetal bovine serum - LIF leukaemia inhibitory factor - mES mouse embryonic stem - Ngn3 neurogenin 3 - P gelatine-coated plates - Pdx-1 pancreatic duodenum homeobox 1 |
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Keywords: | Diabetes Differentiation Embryonic stem cells Exogenous agents Insulin-producing cells Islet progenitors |
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