Î²-Cell replacement therapy represents the most promising approach to restore Î²-cell mass and glucose homeostasis in patients with type 1 diabetes. Safety and ethical issues associated with pluripotent stem cells stimulated the search for adult progenitor cells with endocrine differentiation capacities. We have already described amodel for expansion and differentiation of human pancreatic duct-derived cells (HDDCs) into insulin-producing cells. Here we show an innovative and robust in vitro system for large-scale production of Î²-like cells from HDDCs using a nonintegrative RNA based reprogramming technique. Synthetic modified RNAs for pancreatic transcription factors (pancreatic duodenal homeobox 1, neurogenin3, and V-Mafmusculoaponeurotic fibrosarcoma oncogene homolog A [MAFA]) were manufactured and daily transfected in HDDCs without strongly affecting immune response and cell viability. MAFA overexpression was efficient and sufficient to induce Î²-cell differentiation of HDDCs, which acquired a broad repertoire of mature Î²-cell markers while downregulating characteristic epithelial-mesenchymal transition markers. Within 7 days, MAFA-reprogrammed HDDC populations contained 37% insulin-positive cells and a proportion of endocrine cells expressing somatostatin and pancreatic polypeptide. Ultrastructure analysis of differentiated HDDCs showed both immature and mature insulin granules with lightbackscattering properties. Furthermore, in vitro HDDC-derived Î² cells (called Î²-HDDCs) secreted human insulin and C-peptide in response to glucose, KCl, 3-isobutyl-1-methylxanthine, and tolbutamide stimulation. Transplantation of Î²-HDDCs into diabetic SCID-beige mice confirmed their functional glucose-responsive insulin secretion and their capacity to mitigate hyperglycemia. Our data describe a new, reliable, and fast procedure in adult human pancreatic cells to generate clinically relevant amounts of new Î² cells with potential to reverse diabetes.
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