Linking human islet structural heterogeneity to beta cell state

项目摘要/摘要 胰岛的具体结构，以及胰岛的一般结构，仍然知之甚少； 人类的小岛尤其如此。胰岛结构在整个胰腺中表现出异质性，并且 胰岛之间是否存在保守的结构特征尚不清楚。详细了解 胰岛的组织原则将提高我们将干细胞来源的胰岛重建为 治愈1型糖尿病(T1D)，并阻止导致β细胞丧失的事件的进展 糖尿病的进展。因此，这项提议的目标有两个：第一，通过实验确定 总体上验证胰岛的关键组织原则，特别是β细胞利基，以及 其次，利用这些组织原则设计更多功能更强的胰岛，以治愈T1D。 对于第一个目标，我们已经开发了定制的、半自动的、3D成像和分析 允许在亚微米分辨率下量化β细胞生态位的统计特性的管道 以及数百个单独的β细胞。健康小鼠和人胰岛的初步分析 (1)在两种物种中，β细胞和德尔塔细胞至少与基底源保持一次物理接触 (2)工程化胰岛中接触血管来源的β细胞。 基底膜显著增加胰岛素的表达。我们假设贝塔细胞与 基底膜是胰岛结构中的一种保守成分，必须整合到工程化的胰岛中 以优化β细胞功能。对于第二个目标，我们已经证明了重建干细胞-- 以最大限度地与基底膜接触的方式将衍生的β细胞转化为假性胰岛 在体外将它们对葡萄糖的反应提高至少两倍，并在体内进一步扩展它们的功能。 在这些初步发现的基础上，我们首先的目标是将这一分析大幅扩展到数十个 人类和小鼠胰岛中数以千计的单个细胞，整合了所有内分泌细胞类型以及 免疫细胞、血管细胞和神经。这将导致对内分泌的第一次定量评估 承认胰岛结构异质性的细胞结构生态位，旨在识别保守的 结构主题。第二，我们的目标是确定β细胞生态位的保守特征是否必要，以及 足以实现最佳的β细胞功能。我们将使用体外重组的原代胰岛来验证这一假说。 体外培养人胰岛，体内移植工程化人胰岛。最后，我们将使用 测试特定周细胞衍生基底膜分子必要性的基因组编辑技术 基因工程胰岛的葡萄糖平衡功能。总而言之，我们的研究将提供第一个 胰岛的定量结构蓝图，将确定胰岛β细胞生态位的特征 在人类和小鼠中保守和分化，并将展示一种重建更多 来自干细胞的功能性人体组织，它使用结构蓝图来指导组织工程。

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.