Department of Medicine, Division of Nephrology, University of Washington School of Medicine, Seattle, WA 98109, USA; Kidney Research Institute, University of Washington School of Medicine, Seattle, WA 98109, USA; Institute for Stem Cell and Regenerative Medicine and Quellos High Throughput Screening Core, University of Washington School of Medicine, Seattle, WA 98109, USA.
Department of Internal Medicine, Division of Nephrology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Cell Stem Cell. 2018 Jun 1;22(6):929-940.e4. doi: 10.1016/j.stem.2018.04.022. Epub 2018 May 17.
Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening.
由人类多能干细胞衍生的类器官是高通量筛选（HTS）的一种有潜力的强大工具，但类器官培养的复杂性给小型化和自动化带来了重大挑战。在这里，我们提出了一种全自动的、适用于高通量筛选的平台，用于增强人类肾脏类器官的分化和表型分析。整个 21 天的方案，从种植到分化再到分析，都可以通过液体处理机器人自动进行，也可以通过手动移液进行。高内涵成像分析揭示了类器官分化过程中的剂量依赖性和阈值效应。免疫荧光和单细胞 RNA 测序鉴定了类器官内以前未检测到的壁层、间质和部分分化区室，并定义了大大扩大血管内皮的条件。可以对毒性和疾病表型的化学调节进行定量，以预测安全性和疗效。在该系统中对基因编辑的类器官进行筛选揭示了肌球蛋白在多囊肾病中的意外作用。因此，HTS 格式的类器官为多维表型筛选建立了一个有吸引力的平台。
