源自人类多能干细胞（hPSC）的肾脏类器官现在被用作肾脏疾病和肾毒性筛查的模型。然而，负责大多数肾脏重吸收功能的近端小管（PT）在肾脏类器官中仍然不成熟，对肾单位功能至关重要的关键转运蛋白的表达有限。在这里，我们描述了一种改进 hPSC 肾单位祖细胞规格的方案，与使用标准肾类器官方案生成的肾类器官相比，该方案产生具有细长近端肾单位的肾类器官，显示出改善的 PT 成熟度。我们还描述了一种评估类器官内 PT 功能并通过免疫荧光可视化成熟标记的方法。使用这些测定，PT 增强的类器官显示一系列关键转运蛋白的表达增加，转化为通过底物摄取和转运测量的功能的改善。该协议由延长的 (13 d) 单层分化阶段组成，在此期间 hPSC 暴露于肾单位祖细胞维持介质 (CDBLY2)，更好地模拟体内人后肾祖细胞规范。肾单位祖细胞规格化后，细胞在气液界面上聚集并培养为三维微团，以促进进一步分化和分割成近端肾单位。执行该方案需要培养 hPSC 的经验，并且肾类器官生成方面的专业知识是有利的。© 2023。Springer Nature Limited。

Kidney organoids derived from human pluripotent stem cells (hPSCs) are now being used as models of renal disease and nephrotoxicity screening. However, the proximal tubules (PTs), which are responsible for most kidney reabsorption functions, remain immature in kidney organoids with limited expression of critical transporters essential for nephron functionality. Here, we describe a protocol for improved specification of nephron progenitors from hPSCs that results in kidney organoids with elongated proximalized nephrons displaying improved PT maturity compared with those generated using standard kidney organoid protocols. We also describe a methodology for assessing the functionality of the PTs within the organoids and visualizing maturation markers via immunofluorescence. Using these assays, PT-enhanced organoids display increased expression of a range of critical transporters, translating to improved functionality measured by substrate uptake and transport. This protocol consists of an extended (13 d) monolayer differentiation phase, during which time hPSCs are exposed to nephron progenitor maintenance media (CDBLY2), better emulating human metanephric progenitor specification in vivo. Following nephron progenitor specification, the cells are aggregated and cultured as a three-dimensional micromass on an air-liquid interface to facilitate further differentiation and segmentation into proximalized nephrons. Experience in culturing hPSCs is required to conduct this protocol and expertise in kidney organoid generation is advantageous.© 2023. Springer Nature Limited.