High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping.
High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping.
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DOI：
10.1016/j.stem.2018.04.022
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发表时间：
2018-06-01
期刊：
影响因子：
23.9
通讯作者：
Freedman BS
中科院分区：
文献类型：
--
作者：
Czerniecki SM;Cruz NM;Harder JL;Menon R;Annis J;Otto EA;Gulieva RE;Islas LV;Kim YK;Tran LM;Martins TJ;Pippin JW;Fu H;Kretzler M;Shankland SJ;Himmelfarb J;Moon RT;Paragas N;Freedman BS
来源于人类多能干细胞的类器官是高通量筛选（HTS）的潜在强大工具，但类器官培养的复杂性对小型化和自动化提出了重大挑战。在这里，我们提出了一个全自动的，HTS兼容的平台，用于增强人类肾脏类器官的分化和表型。整个21天的方案，从平板接种到分化到分析，可以通过液体处理机器人自动执行，或者通过手动移液。高含量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测序鉴定了以前未检测到的类器官内的壁层、间质和部分分化的隔室，并确定了大大扩展血管内皮的条件。毒性和疾病表型的化学调节可以量化，用于安全性和有效性预测。在该系统中筛选基因编辑的类器官揭示了肌球蛋白在多囊肾疾病中的意想不到的作用。HTS形式的类器官因此建立了多维表型筛选的有吸引力的平台。来源于人类iPS细胞的类器官具有巨大的药物筛选潜力，但其复杂性对小型化和自动化提出了挑战。Freedman及其同事建立了一个机器人管道来制造和分析微孔阵列中的肾脏类器官。他们应用这个系统来改善分化，测量毒性和理解疾病。
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. Organoids derived from human iPS cells have great potential for drug screening, but their complexity poses a challenge for miniaturization and automation. Freedman and colleagues establish a robotic pipeline to manufacture and analyze kidney organoids in microwell arrays. They apply this system to improve differentiation, measure toxicity, and comprehend disease.
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作者：
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