A multidisciplinary approach towards cell therapy for Diabetes

描述(申请人提供)：越来越多的人认识到成人胰腺可能是胰岛前体细胞的真正来源，因此该组织代表了糖尿病细胞治疗的可行选择。然而，对胚胎后阶段器官中的祖细胞/干细胞生态位缺乏了解，阻碍了朝着这一目标进行的实验操作。我们已经获得了成人胰腺中两种特定细胞群的证据，这两种细胞群满足以下条件：1)具有多向分化潜能的胰腺前体上皮细胞群，以及2)胰腺间充质细胞群，当与多潜能上皮细胞一起培养时，允许体外扩增。这两个种群的特征都是维甲酸的生产。我们在这里组建了一个由多名研究人员组成的团队，以解决与这种产生维甲酸的上皮细胞的存在、功能和谱系潜力以及受控分化相关的具体问题。这将包括对成人胰腺组织的研究，使用使用慢病毒载体的基因工程，该载体旨在表达可自我切割的多蛋白，模仿其他人最近在体细胞重新编程方面的成功工作。我们展示了一种多基因/单载体方法的成功开发，该方法允许在非胰腺细胞中激活胰岛素基因。我们的具体目标如下：LEACH组：从小鼠胰腺中鉴定两个ALDFOR阳性细胞群在向前分化和持续生长方面的特征。Oberholzer小组：鉴定和鉴定成人内分泌胰腺中Aldeflor阳性细胞，并与小鼠细胞进行比较。Jensen小组：利用生物信息学补充Leach和Oberholzer小组对Aldeflor种群的研究，并从功能上测试和开发改良的多基因/单载体递送载体。鲑鱼组：为其他成员提供编码单个蛋白或可切割多蛋白的一系列慢病毒载体，用于成年小鼠、人类和胰腺祖细胞的编程测试。 相关性：该项目与糖尿病基于细胞的治疗的进展相关，该项目整合了了解胰腺维甲酸产生前体细胞的分离和培养的努力，并试图将其区分为全功能的胰腺胰岛素产生细胞。此外，该系统还采用了先进的基因传递系统，结合了能够进行内分泌细胞编程的可自我切割的多蛋白。

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.