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Kidney Week

Abstract: FR-OR049

Improved Human Pluripotent Stem Cell-Derived Kidney Organoids for Modeling Collecting Duct Biology and Tubular Injury

Session Information

  • Development and Stem Cells
    November 08, 2019 | Location: 152, Walter E. Washington Convention Center
    Abstract Time: 06:06 PM - 06:18 PM

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Uchimura, Kohei, Washington University School of Medicine, Saint Louis, Missouri, United States
  • Wu, Haojia, Washington University School of Medicine, Saint Louis, Missouri, United States
  • Humphreys, Benjamin D., Washington University School of Medicine, Saint Louis, Missouri, United States
Background

Maximizing the potential of human kidney organoids for drug testing, regenerative medicine and to model development and disease requires addressing cell immaturity, the lack of a branching collecting system and off target cell types.

Methods

We separately induced posterior intermediate mesoderm and anterior intermediate mesoderm from human iPS and ES cells and combined them on day 7. For the next five days, the combined organoids were incubated in a cocktail including FGF9, heparin, GDNF, retinoic acid and EGF. At day 12, we compared organoids left to mature in basal medium vs. medium supplemented with aldosterone, vasopressin and the NTRK2 inhibitor K252a. Organoid cell diversity and maturation state was assessed by scRNA-seq, immunofluorescence, AQP2 trafficking assays and nephrotoxicity responses.

Results


Our new protocol induced a definitive ureteric bud-derived branching collecting duct system interconnected to more proximal nephron segments. The hormones aldosterone and vasopressin were critical to promote maturation of collecting duct cell types including both principal and intercalated cells. The resulting principal cells express aquaporin-2 protein which undergoes translocation to the apical membrane after vasopressin or forskolin stimulation. By scRNA-seq (35,954 cells total), we define all cell types present, define their maturity and demonstrate superior proximal tubule maturation. Compared to organoids differentiated with existing protocols generated in parallel, this new protocol results in superior downregulation of progenitor cell types, substantially reduced off-target cell types and improved modeling of tubular injury.

Conclusion

We developed a new protocol for the separate induction of both metanephric mesenchyme and ureteric bud from hPSC. This results in robust collecting system development. We report that aldosterone and vasopressin drive the maturation of collecting duct cell types, including principal cells complete with vasopressin-stimulated AQP2 trafficking and the emergence of intercalated cells for the first time. Our scRNA-seq analysis demonstrates that the protocol also results in more mature nephron segments, appropriate downregulation of progentors and improved injury modeling.