High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping.
Stefan M Czerniecki, Nelly M Cruz, Jennifer L Harder, Rajasree Menon, James Annis, Edgar A Otto, Ramila E Gulieva, Laura V Islas, Yong Kyun Kim, Linh M Tran, Timothy J Martins, Jeffrey W Pippin, Hongxia Fu, Matthias Kretzler, Stuart J Shankland, Jonathan Himmelfarb, Randall T Moon, Neal Paragas, Benjamin S Freedman
Author Information
Stefan M Czerniecki: 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.
Nelly M Cruz: 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.
Jennifer L Harder: Department of Internal Medicine, Division of Nephrology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Rajasree Menon: Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
James Annis: Institute for Stem Cell and Regenerative Medicine and Quellos High Throughput Screening Core, University of Washington School of Medicine, Seattle, WA 98109, USA.
Edgar A Otto: Department of Internal Medicine, Division of Nephrology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Ramila E Gulieva: 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.
Laura V Islas: 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.
Yong Kyun Kim: 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.
Linh M Tran: 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 Medicine, Division of Hematology, University of Washington School of Medicine, Seattle, WA 98109, USA.
Timothy J Martins: Institute for Stem Cell and Regenerative Medicine and Quellos High Throughput Screening Core, University of Washington School of Medicine, Seattle, WA 98109, USA.
Jeffrey W Pippin: Department of Medicine, Division of Nephrology, University of Washington School of Medicine, Seattle, WA 98109, USA.
Hongxia Fu: 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 Medicine, Division of Hematology, University of Washington School of Medicine, Seattle, WA 98109, USA.
Matthias Kretzler: Department of Internal Medicine, Division of Nephrology, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Stuart J Shankland: Department of Medicine, Division of Nephrology, University of Washington School of Medicine, Seattle, WA 98109, USA.
Jonathan Himmelfarb: 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.
Randall T Moon: 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 Pharmacology, University of Washington School of Medicine and Howard Hughes Medical Institute, Seattle, WA 98109, USA.
Neal Paragas: Department of Medicine, Division of Nephrology, University of Washington School of Medicine, Seattle, WA 98109, USA.
Benjamin S Freedman: 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 Pathology, University of Washington School of Medicine, Seattle, WA 98109, USA. Electronic address: benof@uw.edu.
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.
