Strategies to Identify In Vitro Islet Progenitors

描述（由申请人提供）：虽然β细胞替代作为糖尿病治疗的概念似乎很简单，但该治疗的主要障碍是可用于移植的胰岛组织数量有限。一种方法是在体外操作成人胰腺组织。从这些实验中获得了2个一般结果：1)胰岛缺失组织的扩张有限，但功能β细胞数量增加；2)胰岛富集制剂在进一步操作后具有低水平胰岛标志物的多次传代广泛扩张。成人胰腺的胰腺祖细胞尚未得到严格的证明。如果体外祖细胞能够被识别、扩增和分化，从而提供功能性β细胞的可靠来源，那么这些细胞是否是培养产物和体内不存在就无关紧要了。我们建议建立一种基于报告慢病毒的体外遗传谱系追踪系统，该系统可以通过Cre-lox系统特异性标记特定的初始细胞类型及其所有后代。在体外人体组织中进行遗传谱系追踪的系统将是探索细胞分化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，然后按照体外生成新的胰岛素生成细胞的方案，我们应该能够识别出在体外产生胰岛素生成细胞的原始细胞或细胞类型。鉴定出这样的细胞将会把重点放在该细胞类型的扩展和分化上，从而成为新的β细胞的可持续来源。

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.