Strategies to Identify In Vitro Islet Progenitors

描述(申请人提供)：尽管作为糖尿病治疗方法的β细胞替代疗法的概念看起来很简单，但这种疗法的一个主要障碍是可用于移植的胰岛组织数量有限。1体外操作成人胰腺组织的方法。这些实验获得了两个一般性的结果：1)胰岛枯竭组织有限扩增，但功能β细胞数量增加；2)进一步操作后，胰岛浓缩制剂广泛扩增，多代扩增，胰岛标志物水平较低。尚未显示来自成人胰腺的胰腺祖细胞的严格证明。如果体外祖细胞能够被鉴定、扩增和分化以提供可靠的功能β细胞来源，那么这些细胞是否是培养的产物并且不存在于体内将是无关紧要的。我们建议开发一种基于报告Lenti病毒的体外遗传谱系追踪系统，通过Cre-lox系统可以特异性地标记特定的初始细胞类型及其所有后代。一个在体外进行人类组织遗传谱系追踪的系统将是寻求分化β细胞以及任何其他体外分化模型的重要工具。通过基因标记不同的胰腺细胞类型，然后遵循体外产生新的胰岛素产生细胞的方案，我们应该能够识别在体外产生胰岛素产生细胞的原始细胞或细胞类型。确定这样的细胞(S)将把重点放在该细胞类型的扩增和分化为新的β细胞的可持续来源上。

DESCRIPTION (provided by applicant): While the concept of beta cell replacement as a therapy for diabetes seems straightforward, a major obstacle for this therapy has been the limited amount of available islet tissue for transplantation. 1 approach has been to manipulate adult human pancreatic tissue in vitro. There are 2 general results obtained from these experiments: 1) from islet depleted tissue limited expansion but with increased numbers of functional beta cells and 2) from islet enriched preparations extensive expansion with multiple passages with low levels of islet markers after further manipulation. A rigorous demonstration of pancreatic progenitor cells from the adult human pancreas has not yet been shown. It would be immaterial whether such cells were a product of culture and did not exist in vivo if in vitro progenitors could be identified, expanded and differentiated to provide a reliable source of functional beta cells. We propose to develop an in vitro genetic lineage tracing system based on a reporter lenti virus that through the Cre-lox system can specifically mark a particular initial cell type and all its progeny. A system to do genetic lineage tracing in human tissue in vitro would be an important tool in the quest for differentiating beta cells as well as for any other in vitro differentiation model. By genetically marking different pancreatic cell types and then following through the protocol for in vitro generation of new insulin-producing cells, we should be able to identify the original cell or cell types that give rise in vitro to the insulin-producing cells. Identifying such cell(s) would then direct focus on the expansion and differentiation of that cell type to a sustainable source of new beta cells.