Linking islet cell function and identity from in vitro to in situ

摘要 在1型糖尿病（T1 D）中，胰岛的胰岛素产生β细胞丢失， 胰岛细胞分泌的升糖激素胰高血糖素失调， 高血糖和反调节受损。最近的研究显示出明显的异质性 在体外和原位的β-细胞和β-细胞群中。新兴的单细胞方法 已建立的细胞亚群在其Ca 2+信号传导和转录组学特征方面不同，并可能代表 “起搏器”细胞或复制壁龛。证据也在不断积累，包括 为了表明胰腺间充质细胞是异质的和可延展的， 1型糖尿病（T1 D）患者胰岛β细胞功能的改变与胰岛β细胞向胰岛β细胞的转变是一致的。 表型这可能导致胰高血糖素分泌失调。其他人已经表明， 在T1 D中，在胰岛内和整个胰腺中，“抗性”或存活的胰岛细胞的持续存在，尽管 其性质和功能仍不清楚。了解人类胰岛的变异性和可塑性 细胞功能，以及这与胰岛微环境的组分如脉管系统或 神经是重要的，因为这可以提供纠正胰高血糖素分泌功能障碍的途径， 保护胰岛细胞或胰岛细胞团的再生。本提案将联合收割机 转录组学，蛋白质组学，功能表型在细胞的基础上，以了解潜在的 调节胰岛细胞功能异质性，并将这些与其他胰岛细胞类型相关的原位作图 和当地环境的组成部分。目的：（1）检测人胰岛细胞功能 表型，以及表型变异与单细胞基因表达的联系;（2）绘制标记 定义健康状况下3D胰岛微环境内的胰岛细胞异质性和亚群， T1 D使用跨越一系列分辨率和尺度的方法;以及（3）将胰岛细胞功能，单细胞 基因表达，单细胞代谢和单细胞蛋白质组学原位了解胰岛细胞 病理生理学整合内部人体胰岛分离程序，多维细胞成像 专业知识，以及使用电生理学（Patch-Seq）的单细胞双功能和转录组学分析， 分离的细胞和原位使用活的人胰腺切片将有助于实现获得高表达的目标。 在健康和糖尿病的局部组织结构内的胰岛细胞的分辨率理解。

Abstract In type 1 diabetes (T1D) insulin producing -cells of the pancreatic islets of Langerhans are lost and secretion of the glucose-raising hormone glucagon from -cells is dysregulated, contributing to hyperglycemia and impaired counter-regulation. Recent studies demonstrate appreciable heterogeneity within the -cell and -cell populations both in vitro and in situ. Emerging single-cell approaches have established -cell sub-groups that differ in their Ca2+ signaling and transcriptomic profiles and may represent ‘pacemaker’ cells or replication niches. Evidence is also accumulating, including preliminary data in the present application, to suggest that the pancreatic -cells are both heterogeneous and malleable – the altered function of human -cells in type 1 diabetes (T1D) is consistent with a shift towards a -cell phenotype. This could contribute to the dysregulation of glucagon secretion. Others have shown the persistence of ‘resistant’ or surviving -cells in T1D, both within islets and throughout the pancreas, although the nature and function of these remain unclear. Understanding the variability and malleability of human islet cell function, and the relationship of this to components of the islet microenvironment such as vasculature or nerves, is important since this may provide avenues for correction of glucagon secretory dysfunction, protection of -cells, or the regeneration of -cell mass. The present proposal will combine in-depth transcriptomic, proteomic, functional phenotyping on a cell-by-cell basis to understand the underlying regulation of islet cell functional heterogeneity and will map these in situ in relation to other islet cells types and components of the local environment. The Aims are to (1) examine human islet cell functional phenotypes, and the linkage of phenotypic variability to single-cell gene expression; (2) map the markers that define islet cell heterogeneity and sub-populations within the 3D islet microenvironment in health and T1D using approaches that span a range of resolutions and scales; and (3) link islet cell function, single-cell gene expression, single-cell metabolism, and single-cell proteomics in situ to understand islet cell pathophysiology. Integration of an in-house human islet isolation program, multi-dimensional cell imaging expertise, and single-cell dual functional and transcriptomic profiling using electrophysiology (Patch-Seq) on isolated cells and in situ using live human pancreas slices will help accomplish the goal of obtaining a high resolution understanding of islet cells within the local tissue architecture in health and diabetes.