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

胰岛依赖于异质细胞之间时空协调的相互作用来维持 血糖稳态在1型糖尿病中，胰岛定向的自身免疫攻击导致功能性β 细胞，这是伴随着其他类型的胰岛细胞的缺陷。糖尿病患者患有慢性 葡萄糖失调，最终降低预期寿命。胰岛素本身可以治疗1型糖尿病 糖尿病然而，每日胰岛素注射是昂贵的、繁重的，并且具有副作用，包括糖尿病风险。 酮症酸中毒和昏迷人类干细胞衍生的胰岛类器官（SC胰岛）提供了一个机会， 通过移植作为潜在治疗剂提供人胰岛。然而，SC-胰岛缺乏精确性， 动力学和胰岛素/胰高血糖素分泌的幅度，天然胰岛在成年期显示。是否这些 局限性反映了SC-胰岛细胞类型群体之间（或内部）的时空协调性差，或 发育和成熟中内在的三维（3D）异质性仍然是未知的。 在这里，我们建议通过实验捕捉细胞的轨迹来解决这些基本问题。 通过整合新的活性和相互作用， 干细胞生物学、软薄膜纳米电子学、组织清除和单细胞空间 转录组学，计算和系统生物学。具体而言，我们（1）利用可扩展单元 分化和纯化方法，以构建具有定制α和β组成的“设计师”SC-胰岛;（2）全球 在SC-胰岛内嵌入软可拉伸传感器阵列，构建用于长期稳定跟踪的“半机械胰岛” 体外和体内单细胞分辨率下胰岛全α和β细胞类型特异性电活动;（3） 实施了3D组织透明化、染色、成像和原位单细胞RNA测序， 亚细胞分辨率下完整SC-胰岛中的激素、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识别清除的SC胰岛内的传感器位置， 计算整合慢性电记录与激素，生物标志物和基因表达数据， 单细胞水平。 我们提出整合和使用这些发明来解决SC-胰岛成熟中的主要挑战。 具体来说，我们的目标是采用这种多模式表征SC胰岛发展，以解决（1）作用 （2）SC-胰岛成熟的三维异质性; （3）神经支配和血管化在移植SC-胰岛成熟中的作用。成功 这一建议的结果将是一个平台，可以监测原位单细胞活性的SC胰岛在长期 稳定的方式，提供了SC胰岛发育和成熟过程中的三维异质性的理解。 我们设想，它最终将使我们能够为人类构建功能特化和成熟的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.