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

摘要 在 1 型糖尿病 (T1D) 中，胰岛朗格汉斯 (Langerhans) 产生胰岛素的  细胞丢失，并且 α细胞分泌的升糖激素胰高血糖素失调，导致 高血糖和反调节受损。最近的研究表明存在明显的异质性 在体外和原位的  细胞和  细胞群中。新兴的单细胞方法 建立了  细胞亚群，其 Ca2 信号传导和转录组特征不同，可能代表 “起搏器”细胞或复制生态位。证据也在积累，包括初步数据 本申请表明胰腺 α 细胞既具有异质性又具有可塑性—— 1 型糖尿病 (T1D) 中人类 α 细胞功能的改变与向 β 细胞的转变一致 表型。这可能导致胰高血糖素分泌失调。其他人已经展示了 T1D 中“抵抗”或存活的  细胞在胰岛内和整个胰腺中持续存在，尽管 它们的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，以及其与胰岛微环境成分（例如脉管系统或 神经，很重要，因为这可能提供纠正胰高血糖素分泌功能障碍的途径， 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组、蛋白质组、功能表型分析，以了解潜在的 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相关的原位图谱 以及当地环境的组成部分。目的是 (1) 检查人类胰岛细胞功能 表型以及表型变异与单细胞基因表达的联系； (2) 绘制标记 定义健康和 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； (3) 连接胰岛细胞功能，单细胞 基因表达、单细胞代谢和原位单细胞蛋白质组学以了解胰岛细胞 病理生理学。整合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组分析 分离细胞并在原位使用活人胰腺切片将有助于实现获得高水平的目标 分辨率了解健康和糖尿病中局部组织结构内的胰岛细胞。

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.