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

摘要 在1型糖尿病(T1D)中，朗格汉斯胰岛分泌胰岛素的细胞丢失，并 细胞分泌的升糖激素胰高血糖素失调，导致 高血糖和反调节功能受损。最近的研究显示出明显的异质性 在体外和原位的细胞和细胞群体中。新兴的单细胞方法已经 已建立的细胞亚群，它们的钙信号和转录图谱不同，可能代表着 “起搏器”细胞或复制利基。证据也在积累，包括 目前的应用是为了表明胰腺细胞既是异质性的，也是可塑性的- 1型糖尿病(T1D)患者细胞功能改变与向细胞转变一致 表型。这可能会导致胰高血糖素分泌失调。其他人则展示了 在T1D中，无论是在胰岛内还是在整个胰腺中，细胞都持续存在，尽管 这些机构的性质和功能仍不清楚。了解人类胰岛的可变性和延展性 细胞功能及其与胰岛微环境成分的关系，如血管系统或 神经是重要的，因为这可能为纠正胰高血糖素分泌功能障碍提供途径， 保护细胞，或细胞团的再生。本提案将深入结合 在逐个细胞的基础上进行转录、蛋白质组学和功能表型分析，以了解潜在的 对胰岛细胞功能异质性的调节，并将这些与其他胰岛细胞类型的关系进行原位定位 以及当地环境的组成部分。其目的是(1)检测人类胰岛细胞的功能 表型，以及表型变异与单细胞基因表达的联系；(2)标记作图 其定义了健康和健康的3D胰岛微环境中的胰岛细胞异质性和亚群 T1D使用跨越一系列分辨率和尺度的方法；以及(3)链接胰岛细胞功能，单细胞 基因表达、单细胞代谢和单细胞蛋白质组学原位研究胰岛细胞 病理生理学。集成了内部人类胰岛分离程序、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图谱分析 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 了解健康和糖尿病患者局部组织结构内胰岛细胞的分辨率。

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.