Linking human islet structural heterogeneity to beta cell state

项目总结/摘要 β细胞龛的详细结构以及胰岛的一般结构仍然知之甚少; 对于人胰岛尤其如此。胰岛结构在整个胰腺中呈现异质性， 在胰岛中是否存在保守的结构特征是未知的。详细了解 胰岛的组织原则将提高我们的能力，既重建干细胞衍生的胰岛， 治疗1型糖尿病（T1 D），并阻止导致β细胞损失的事件的进展， 糖尿病的进展。因此，该提案的目标是双重的：首先，确定和实验 验证胰岛的关键组织原则，特别是β细胞龛，以及 第二，利用这些组织原则来设计更多功能性胰岛，作为T1 D的治疗方法。 为了实现第一个目标，我们开发了一种定制的、半自动化的3D成像和分析系统， 允许以亚微米分辨率量化β细胞龛的统计特性的管道 以及数百个单独的β细胞。对健康小鼠和人类胰岛的初步分析显示， （1）在两个物种中，β细胞和δ细胞与基底源保持至少一次物理接触， 膜，而α细胞没有，和（2）β细胞在工程胰岛，接触来源的血管 基底膜的胰岛素表达显著升高。我们假设β细胞与 基底膜是胰岛结构的保守元件，其必须被并入工程胰岛中 来优化β细胞功能对于第二个目标，我们已经证明了重建干细胞- 将β细胞衍生为假胰岛，使其与基底膜接触最大化 在体外将它们对葡萄糖的反应提高至少两倍，并进一步扩展它们在体内的功能。 在这些初步发现的基础上，我们的第一个目标是在数十个国家中大幅扩展这一分析。 人类和小鼠胰岛中的数千个单个细胞，将所有内分泌细胞类型沿着 免疫细胞血管细胞和神经这将导致内分泌的第一次定量评估 细胞结构小生境，承认胰岛的结构异质性，旨在识别保守的 结构图案其次，我们的目标是确定β细胞龛的保守特征是否是必要的， 足以维持最佳的β细胞功能。我们将使用体外重建的胰岛、原代胰岛、 离体培养的人胰岛和体内移植到小鼠中的工程化人胰岛。最后，我们将使用 基因组编辑技术来测试特定周细胞衍生的基底膜分子的必要性 在工程胰岛中的葡萄糖稳态功能。总的来说，我们的研究将提供第一个 胰岛的定量结构蓝图，将确定β细胞小生境的特征， 在人类和小鼠中保守和不同，并将展示一种重建更多 从干细胞中提取功能性人体组织，利用结构蓝图指导组织工程。

Project Summary/Abstract The detailed structure of the beta cell niche, and that of the islet in general, remains poorly understood; this is particularly the case for human islets. Islet structure appears heterogeneous across the pancreas, and whether conserved structural features exist among islets is unknown. A detailed understanding of the organizational principles of islets would advance our ability both to reconstitute stem-cell derived islets as a cure for type 1 diabetes (T1D) and to block the progression of events that lead to the loss of beta cells during the progression of diabetes. Therefore, the goal of this proposal is twofold: first, to identify and experimentally validate the critical organizational principles of the islet in general and the beta cell niche in particular, and second, to leverage these organizational principles to engineer more functional islets as a cure for T1D. Towards the first goal, we have developed a custom, semi-automated, 3D imaging and analysis pipeline that permits quantification of the statistical properties of the beta cell niche at sub-micron resolution and across hundreds of individual beta cells. Preliminary analyses of healthy mouse and human islets revealed that (1) in both species beta and delta cells maintain at least one physical contact with a source of basement membrane, whereas alpha cells do not, and (2) beta cells in engineered islets that contact sources of vascular basement membrane have dramatically elevated insulin expression. We hypothesize that beta cell contact with basement membrane is a conserved element of islet structure that must be incorporated into engineered islets to optimize beta cell function. Towards the second goal, we have demonstrated that reconstituting stem cell- derived beta cells into pseudo-islets in a manner that maximizes their contact with basement membrane improves their response to glucose by at least two-fold in vitro and further extends their functionality in vivo. Building on these preliminary findings, we first aim to dramatically expand this analysis across tens of thousands of individual cells in human and mouse islets, incorporating all endocrine cell types along with immune cells, vascular cells, and nerves. This will result in the first quantitative assessment of the endocrine cell structural niche that acknowledges the structural heterogeneity of islets and aims to identify conserved structural motifs. Second, we aim to determine if conserved features of the beta cell niche are necessary and sufficient for optimal beta cell function. We will test this hypothesis using in vitro reconstituted islets, primary human islets cultured ex vivo, and engineered human islets transplanted into mice in vivo. Finally, we will use genome editing techniques to test the necessity of specific pericyte-derived basement membrane molecules for glucose homeostatic function in engineered islets. Taken together, our study will provide the first quantitative structural blueprint for the pancreatic islet, will identify features of the beta cell niche that are conserved and divergent across humans and mice, and will demonstrate a strategy for reconstituting more functional human tissues from stem cells that uses a structural blueprint to guide tissue engineering.