A multidisciplinary approach towards cell therapy for Diabetes

描述（由申请人提供）：越来越多的人认识到成人胰腺可能是胰岛祖细胞的真正来源，因此该组织代表了糖尿病细胞治疗的可行选择。然而，缺乏对胚胎后期器官中祖细胞/干细胞壁龛的理解，阻碍了实验操作的实现。我们已经获得了成人胰腺中两个特定群体的证据，它们满足以下标准：1)胰腺祖细胞群体，表现出多谱系效力；2)胰腺间充质细胞群体，当与多能上皮细胞一起培养时，允许离体扩增。这两个种群的特点都是产生维甲酸。我们在这里组建了一个多研究者小组，研究与维甲酸产生上皮细胞的存在、功能和谱系效力以及控制分化有关的具体问题。这将涵盖成人胰腺组织的基因工程研究，使用慢病毒载体设计表达自切割多蛋白，模仿最近成功的体细胞重编程工作。我们展示了一种多基因/单载体方法的成功开发，该方法允许胰岛素基因在非胰腺细胞中激活。我们的具体目标如下：Leach组：表征小鼠胰腺中两种醛氟阳性细胞群的正向分化和持续生长。Oberholzer组：鉴定成人内分泌胰腺中aldeflue阳性细胞与小鼠细胞的比较特征。Jensen小组：补充Leach和Oberholzer小组对氟蝇种群的研究，使用生物信息学和功能测试并开发改良的多基因/单载体传递载体。鲑鱼组：向其他成员提供一系列慢病毒载体，编码单个蛋白质或可切割多蛋白，用于成年鼠和人类胰腺祖细胞的编程测试。

DESCRIPTION (provided by applicant): There is a growing understanding that adult pancreas may be a true source for islet progenitor cells, and that this tissue therefore represents a viable option for cell therapy in diabetes. However, a lacking understanding of progenitor/stem cell niches in the organ at post-embryonic stages, hinders experimental manipulation towards this end. We have obtained evidence for two specific populations in adult pancreas, that fulfill criteria of 1) a pancreatic progenitor epithelial population, exhibiting multi-lineage potency, and 2) a pancreatic mesenchymal cell population, that when cultured together with the multipotent epithelial cells, allows for ex-vivo expansion. Both populations are characterized by retinoic acid production. We have here formed a multi-investigator team to address specific issues related to the existence, the function and lineage potency, as well as the controlled differentiation of such retinoic acid producing epithelial cells. This will cover studies of adult human pancreatic tissue employing genetic engineering using lentiviral vectors designed to express self-cleavable polyproteins modeled over recent successful work in somatic cell reprogramming by others. We demonstrate the successful development of a multiple-gene/single vector method that allows insulin gene activation in non-pancreatic cells. Our specific goals are as follows: Leach group: to characterize the two aldefluor-positive cell populations from the murine pancreas with respect to forward differentiation and continuous growth. Oberholzer group: to identify and characterize Aldefluor-positive cells in the adult human endocrine pancreas comparatively to the murine cells. Jensen group: Complement the Leach and Oberholzer groups in studies of the Aldefluor populations, using bioinformatics and to functionally test and develop modified multiple gene/single-vector delivery vectors. Salmon group: to provide to other members a series of lentiviral vectors encoding individual proteins, or cleavable polyproteins, for testing in programming of adult murine, and human, pancreatic progenitors. RELEVANCE: This project is relevant for the advance of cell-based therapy in Diabetes, the project integrates efforts to understand the isolation and culture of pancreatic retinoic-acid producing progenitor cells, and seeks to differentiate such into fully function pancreatic insulin producing cells. The furthermore employs advanced gene delivery systems incorporating self-cleavable polyproteins capable of endocrine cellular programming.