High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping

• Published in: Cell stem cell Vol. 22; no. 6; pp. 929 - 940.e4
• Main Authors: Czerniecki, Stefan M., Cruz, Nelly M., Harder, Jennifer L., Menon, Rajasree, Annis, James, Otto, Edgar A., Gulieva, Ramila E., Islas, Laura V., Kim, Yong Kyun, Tran, Linh M., Martins, Timothy J., Pippin, Jeffrey W., Fu, Hongxia, Kretzler, Matthias, Shankland, Stuart J., Himmelfarb, Jonathan, Moon, Randall T., Paragas, Neal, Freedman, Benjamin S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2018
• Subjects: Automation; Cell Culture Techniques; Cell Differentiation; High-Throughput Screening Assays; Humans; Kidney - cytology; Organoids - cytology; Phenotype; Pluripotent Stem Cells - cytology; Sequence Analysis, RNA
• ISSN: 1934-5909; 1875-9777; 1875-9777
• DOI: 10.1016/j.stem.2018.04.022

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. [Display omitted] •Liquid-handling robots generate and analyze kidney organoids in microwell arrays•Single-cell RNA-seq reveals that organoid cell types recapitulate human kidney complexity•Growth factor addition greatly increases vascular endothelial cells in organoids•A phenotypic drug screen discovers a role for myosin in polycystic kidney disease Organoids derived from human iPSCs 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.