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Cell Journal، جلد ۱۷، شماره Suppl ۱، صفحات ۱۱-۱۱
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عنوان انگلیسی Is-5: Generation of Functional Islets of Langerhans from Human Exocrine Pancreas
چکیده انگلیسی مقاله Objective: Cell therapy in the form of human islet transplantation has been a successful form of treatment for patients with type 1 diabetes for over 15 years, but is significantly limited by lack of suitable donor material. During the islet isolation procedure, 98% of the pancreas, comprising acinar and ductal tissue (the exocrine tissue) is normally discarded. The aim of this study was to optimize a protocol whereby this exocrine material could be efficiently reprogrammed to provide an additional supply of islets for transplantation. Materials and Methods: On arrival in the lab the exocrine- enriched fraction was immediately plated on to tissue culture dishes. The cells attached to the dish and formed a monolayer, which if left unchecked would undergo the epithelial to mesenchymal transition (EMT). This mesenchymal monolayer could be repeatedly passaged. During preliminary experiments, we found that inhibiting the process of EMT (day0 – day1) using TGFBeta1 and Rho kinase inhibitors enhanced the efficiency of reprogramming. The cells were also treated with chromatin modifying agents at this stage. Reprogramming was then initiated by treatment with adenoviruses containing the pancreatic transcription factors Pdx1, MafA, Ngn3 and Pax4. Four days later (D4) the cells were treated with an siRNA to inhibit expression of the endogenous transcription factor ARX. The protocol was complete by D10. From D3-D10 the media was supplanted with Betacellulin, EGF, and nicotinamide. Results: Immunocytochemistry revealed that the reprogrammed cell population was monohormonal, comprising 40% C-peptide-positive, 4% glucagon-positive, and < 2% somatostatin-positive cells. The resultant Betalike cells exhibited glucose responsive insulin secretion, expressed insulin protein levels (33.5 ± 7.3 pg/ ug protein) at around 15% of that in adult human islets, packaged insulin into secretory granules, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. Conclusion: We describe here a highly reproducible and robust protocol that generates reprogrammed islet cells from the human exocrine pancreatic tissue that is normally discarded after the islet isolation procedure. The reprogrammed cells share many of the properties of adult endogenous Beta cells and compare well with surrogate Beta cells generated from human embryonic stem or iPS cells. We estimate that around 1.5 x 108 reprogrammed cells would have a therapeutic effect if transplanted in human diabetics; thus one recipient pancreas could provide numerous islet grafts.
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نویسندگان مقاله k docherty | k docherty


نشانی اینترنتی http://celljournal.org/journal/article/abstract/29
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