Charting human islet maturation via combined soft nanoelectronics and single-cell spatial transcriptomics

胰岛依赖于异种细胞之间时空协调的相互作用来维持 血糖动态平衡。在1型糖尿病中，胰岛导向的自身免疫攻击会导致功能性β的丧失 在其他类型的胰岛细胞中伴有缺陷。糖尿病患者患有慢性并发症 葡萄糖调节失调，最终会减少预期寿命。注射胰岛素本身可以治疗1型糖尿病 糖尿病。然而，每天注射胰岛素是昂贵的，繁重的，并且有副作用，包括 酮症酸中毒和昏迷。人类干细胞来源的胰岛类器官(SC-ISlet)提供了一个产生无限 人胰岛通过移植提供潜在的治疗方法。然而，SC小岛缺乏精确度， 自然胰岛在成年期间表现出的胰岛素/胰高血糖素分泌的动力学和数量。不管是这些 局限性反映了干细胞-胰岛细胞类型的群体之间(或群体内)的时空协调不良，或者 内在的三维(3D)异质性在发育和成熟过程中，仍是未知的。 在这里，我们建议通过实验捕获细胞的轨迹来解决这些基本问题 通过集成新颖的方式跨3D体积发展中的SC-胰岛的活动和相互作用 干细胞生物学、软薄膜纳米电子学、组织清除和单细胞空间技术 转录学、计算生物学和系统生物学。具体地说，我们已经(1)利用了可伸缩单元 分化纯化方法构建具有定制α和β组成的“设计型”SC-胰岛；(2)全球 在SC岛中嵌入软可伸展传感器阵列，构建用于长期稳定跟踪的“半机械人岛” 体外和体内单细胞分辨率下的全胰岛α和β细胞型特异性电活动；(3) 实施3D组织清除、染色、成像和原位单细胞RNA测序，以绘制空间地图 亚细胞分辨率下完整SC-胰岛的激素、生物标志物、基因表达和细胞类型；以及(4) 使用荧光标记的电子条形码来识别已清除的SC-胰岛内的传感器位置 将慢性电子记录与荷尔蒙、生物标记物和基因表达数据在 单细胞水平。 我们建议整合和使用这些发明来解决干细胞-胰岛成熟过程中的主要挑战。 具体地说，我们的目标是利用SC-胰岛发育的这种多模式特征来解决(1)角色 DEC1在昼夜夹带介导的胰岛成熟中的作用：(2)SC-胰岛成熟的三维异质性； (3)神经支配和血管形成在移植的SC-胰岛成熟中的作用。成功之路 这一建议的结果将导致一个平台，可以监测现场单细胞活动的SC-胰岛在一个长期的 稳定的方式，提供了对干细胞胰岛发育和成熟过程中3D异质性的理解。 我们设想，它最终将使我们能够为人类建立功能专门的和成熟的SC-胰岛 治疗学。

Pancreatic islets rely on spatiotemporally orchestrated interactions between heterogenous cells to maintain blood glucose homeostasis. In type 1 diabetes, an islet-directed autoimmune attack leads to loss of functional β cells, which is accompanied by defects in the other islet cell types. Diabetics suffer complications from chronic glucose misregulation, which ultimately reduce life expectancy. Administering insulin itself can treat type 1 diabetes. However, daily insulin injection is expensive, onerous, and carries side effects including risk of ketoacidosis and coma. Human stem cell-derived islet organoids (SC-islets) offer a chance to generate a limitless human islet supply as potential therapeutics through transplantation. However, SC-islets lack the precision, kinetics, and magnitude of insulin/glucagon secretion that natural islets show during adult life. Whether these limitations reflect poor spatiotemporal coordination between (or within) populations of SC-islet cell types, or intrinsic three-dimensional (3D) heterogeneity in development and maturation, is still unknown. Here, we propose to address these fundamental questions by experimentally capturing the trajectories of cellular activity and interaction across the 3D volume of developing SC-islets through the integration of novel technologies from stem cell biology, soft thin-film nanoelectronics, tissue clearing and single-cell spatial transcriptomics, and computational and system biology. Specifically, we have (1) exploited scalable cell differentiation and purification methods to build “designer” SC-islets with custom α and β composition; (2) globally embedded soft stretchable sensor arrays within SC-islets, building “cyborg islets” for chronically-stable tracing of islet-wide α- and β-cell type specific electrical activities at single-cell resolution in vitro and in vivo; (3) implemented 3D tissue clearing, staining, imaging, and in situ single-cell RNA sequencing to spatially map hormones, biomarkers, gene expression, and cell types in the intact SC-islets at subcellular resolution; and (4) used fluorescently-labeled electronic barcodes to identify sensor positions within cleared SC-islets and computationally integrate chronic electrical recording with hormones, biomarker and gene expression data at the single-cell level. We propose to integrate and use these inventions to address major challenges in SC-islet maturation. Specifically, we aim to employ such multimodal characterization of SC-islet development to address (1) the role of Dec1 in islet maturation mediated by circadian entrainment; (2) the 3D heterogeneity in SC-islet maturation; and (3) the role of nerve innervation and vascularization in the maturation of transplanted SC-islets. The success of this proposal will result in a platform that can monitor the in situ single-cell activity of SC-islets in a chronically stable manner, provide an understanding of the 3D heterogeneity during SC-islet development and maturation. We envision that it will ultimately enable us to build functionally specialized and mature SC-islets for human therapeutics.