Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids.
Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids.
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DOI：
10.3389/fcell.2022.978888
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发表时间：
2022
影响因子：
5.5
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中科院分区：
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从人造血干细胞衍生的肾类器官为临床前研究和免疫相容肾组织再生开辟了新的机会。类器官类似于天然肾单位，由过滤单位和小管组成，但对这些类器官结构的功能能力知之甚少。转录组学分析提供了对代表肾功能的成熟和转运蛋白活动的深入了解。然而，类器官的功能分析是必要的，以证明这些转运蛋白在活组织中的活性。三维（3D）结构增加了肾脏类器官实时分析的复杂性。在这里，我们开发了一种功能分析，使用实时成像来评估罗丹明123 (Rh123)， p糖蛋白（P-gp）的荧光底物，在贴在盖盖培养板上的类器官中经上皮运输，以进行准确的实时观察。利用与近端小管表面糖蛋白结合的荷花四角龙凝集素（LTL）对类器官结构进行了鉴定。将Rh123添加到培养基中20分钟内，Rh123在类器官的管腔中积累。药物抑制MDR1或OCT2可减少染料/标记物从基底侧到根尖的积累，OCT2抑制可减少Rh123的摄取。Rh123的转运程度是成熟依赖性的，与通过RNA-seq和免疫组织化学评估的MDR1表达水平一致。具体来说，与分化后第30天的类器官相比，第21天的类器官在管腔中表现出较少的Rh123积累。我们的工作建立了三维肾类器官的实时功能评估，使健康和疾病的类器官功能表型成为可能。
Kidney organoids derived from hPSCs have opened new opportunities to develop kidney models for preclinical studies and immunocompatible kidney tissues for regeneration. Organoids resemble native nephrons that consist of filtration units and tubules, yet little is known about the functional capacity of these organoid structures. Transcriptomic analyses provide insight into maturation and transporter activities that represent kidney functions. However, functional assays in organoids are necessary to demonstrate the activity of these transport proteins in live tissues. The three-dimensional (3D) architecture adds complexity to real-time assays in kidney organoids. Here, we develop a functional assay using live imaging to assess transepithelial transport of rhodamine 123 (Rh123), a fluorescent substrate of P-glycoprotein (P-gp), in organoids affixed to coverslip culture plates for accurate real-time observation. The identity of organoid structures was probed using Lotus Tetragonolobus Lectin (LTL), which binds to glycoproteins present on the surface of proximal tubules. Within 20 min of the addition of Rh123 to culture media, Rh123 accumulated in the tubular lumen of organoids. Basolateral-to-apical accumulation of the dye/marker was reduced by pharmacologic inhibition of MDR1 or OCT2, and OCT2 inhibition reduced the Rh123 uptake. The magnitude of Rh123 transport was maturation-dependent, consistent with MDR1 expression levels assessed by RNA-seq and immunohistochemistry. Specifically, organoids on day 21 exhibit less accumulation of Rh123 in the lumen unlike later-stage organoids from day 30 of differentiation. Our work establishes a live functional assessment in 3D kidney organoids, enabling the functional phenotyping of organoids in health and disease.