Abstract: FR-OR101

Nephron Organoids Derived from Patients with ARPKD Model Polycystic Kidney Disease Respond to a cAMP Inducer and an Src Inhibitor and Provide a Platform for Drug Screening In Vitro

Session Information

Category: Developmental Biology and Inherited Kidney Diseases

  • 402 Stem Cells

Authors

  • Morizane, Ryuji, Brigham & Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Miyoshi, Tomoya, Brigham & Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Gupta, Navin R., Brigham & Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Susa, Koichiro, Brigham & Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Garcia, Edgar, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Lam, Albert Q., Brigham & Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Zhou, Jing, Brigham & Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Valerius, M. Todd, Brigham & Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Bonventre, Joseph V., Brigham & Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
Background

Nephron organoids, derived from human pluripotent stem cells (hPSCs), represent a method to study inherited kidney diseases and perform drug screening in vitro. Previously, we established proof of concept by modeling autosomal dominant PKD (ADPKD), an adult-onset form, using PKD1 or PKD2 CRISPR-mutant hPSC lines that generated 6% cystic organoids. Here, we sought to model autosomal recessive PKD (ARPKD), an early-onset form, using patient derived induced pluripotent stem cell lines (hiPSCs) to generate cystic organoids with greater efficiency, establishing a suitable platform for studying cystogenic mechanisms and therapeutic screens in vitro.

Methods

Nephron organoids were generated in 96-well culture plates from a human embryonic stem cell (hESC) line and a hiPSC line derived from subjects without cystic kidney disease (controls), and 2 ARPKD patient-derived hiPSC lines using our established protocol. Cyst formation was evaluated by bright field imaging and staining for LTL and CDH1. Organoids were treated with forskolin, a cAMP inducer, and tubular cell proliferation assessed by Ki67 staining. Abnormal polarization of cystic epithelia was determined by NaK-ATPase staining. The effect of Src inhibition on cystogenesis was assessed by organoid size.

Results

Patient-derived ARPKD nephron organoids spontaneously formed cysts and demonstrated enhanced cystogenesis with forskolin treatment. Bright field imaging detected cysts in >90% of ARPKD organoids, while control hESC and hiPSC organoids formed very few cysts. Forskolin increased the number of Ki67+ tubular epithelial cells in ARPKD organoids, but not in controls. Mislocalization of the NaK-ATPase to the apical membrane was observed in ARPKD organoids, while control organoids demonstrated NaK-ATPase basolateral restriction. Src inhibition decreased the size of ARPKD organoids.

Conclusion

Patient-derived ARPKD organoids efficiently demonstrated cystic phenotypes that retain known pathophysiology: proliferative response to cAMP and loss of tubular epithelial cell polarity. Src treatment suppressed cystogenesis in a 96-well format, indicating the feasibility of drug screening in vitro.

Funding

  • NIDDK Support