Corritore Elisa, Lee Yong-Syu, Pasquale Valentina, Liberati Daniela, Hsu Mei-Ju, Lombard Catherine Anne, Van Der Smissen Patrick, Vetere Amedeo, Bonner-Weir Susan, Piemonti Lorenzo, Sokal Etienne, Lysy Philippe A
Pediatric Research Laboratory, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium.
Diabetes Research Institute, Istituti di Ricovero e Cura a Carattere Scientifico, San Raffaele Scientific Institute, Milan, Italy.
Stem Cells Transl Med. 2016 Nov;5(11):1525-1537. doi: 10.5966/sctm.2015-0318. Epub 2016 Jul 12.
: β-Cell replacement therapy represents the most promising approach to restore β-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 a model 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 β-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-Maf musculoaponeurotic 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 β-cell differentiation of HDDCs, which acquired a broad repertoire of mature β-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 light-backscattering properties. Furthermore, in vitro HDDC-derived β cells (called β-HDDCs) secreted human insulin and C-peptide in response to glucose, KCl, 3-isobutyl-1-methylxanthine, and tolbutamide stimulation. Transplantation of β-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 β cells with potential to reverse diabetes.
β-Cell replacement therapy represents the most promising approach to restore glucose homeostasis in patients with type 1 diabetes. This study shows an innovative and robust in vitro system for large-scale production of β-like cells from human pancreatic duct-derived cells (HDDCs) using a nonintegrative RNA-based reprogramming technique. V-Maf musculoaponeurotic fibrosarcoma oncogene homolog A overexpression was efficient and sufficient to induce β-cell differentiation and insulin secretion from HDDCs in response to glucose stimulation, allowing the cells to mitigate hyperglycemia in diabetic SCID-beige mice. The data describe a new, reliable, and fast procedure in adult human pancreatic cells to generate clinically relevant amounts of new β cells with the potential to reverse diabetes.
β细胞替代疗法是恢复1型糖尿病患者β细胞数量和葡萄糖稳态最有前景的方法。与多能干细胞相关的安全性和伦理问题促使人们寻找具有内分泌分化能力的成体祖细胞。我们已经描述了一种将人胰腺导管来源细胞（HDDCs）扩增并分化为胰岛素分泌细胞的模型。在此，我们展示了一种创新且强大的体外系统，该系统使用基于非整合RNA的重编程技术从HDDCs大规模生产β样细胞。制备了用于胰腺转录因子（胰腺十二指肠同源盒1、神经生成素3和V-Maf肌腱膜纤维肉瘤癌基因同源物A [MAFA]）的合成修饰RNA，并每天转染到HDDCs中，且不会强烈影响免疫反应和细胞活力。MAFA的过表达有效且足以诱导HDDCs的β细胞分化，使其获得一系列成熟的β细胞标志物，同时下调特征性上皮-间质转化标志物。在7天内，经MAFA重编程的HDDC群体中含有37%的胰岛素阳性细胞以及一部分表达生长抑素和胰多肽的内分泌细胞。对分化后的HDDCs进行超微结构分析显示，存在具有光散射特性的未成熟和成熟胰岛素颗粒。此外，体外由HDDCs衍生的β细胞（称为β-HDDCs）在葡萄糖、氯化钾、3-异丁基-1-甲基黄嘌呤和甲苯磺丁脲刺激下分泌人胰岛素和C肽。将β-HDDCs移植到糖尿病SCID-米色小鼠体内证实了它们具有功能性葡萄糖反应性胰岛素分泌能力以及减轻高血糖的能力。我们的数据描述了一种在成人胰腺细胞中生成具有逆转糖尿病潜力的临床相关数量新β细胞的新的、可靠且快速的方法。
β细胞替代疗法是恢复1型糖尿病患者葡萄糖稳态最有前景的方法。本研究展示了一种创新且强大的体外系统，该系统使用基于非整合RNA的重编程技术从人胰腺导管来源细胞（HDDCs）大规模生产β样细胞。V-Maf肌腱膜纤维肉瘤癌基因同源物A的过表达有效且足以诱导HDDCs的β细胞分化以及使其在葡萄糖刺激下分泌胰岛素，从而使这些细胞能够减轻糖尿病SCID-米色小鼠的高血糖。这些数据描述了一种在成人胰腺细胞中生成具有逆转糖尿病潜力的临床相关数量新β细胞的新的、可靠且快速的方法。
