Large-scale organoid-derived cyst cultures as a drug discovery platform for polycystic kidney disease

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INTRODUCTION: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow, progressive formation of fluid-filled cysts within the kidneys, ultimately leading to kidney failure. Despite being the most common genetic kidney disease, therapies for ADPKD are limited. Ureteric bud (UB) organoids derived from human induced pluripotent stem cells (hiPSC) have been developed to model ADPKD. Here, we describe a protocol to generate UB organoids in large-scale suspension culture amenable to drug discovery. METHODS: Day 12 'nephron' organoids derived from wildtype and PKD2-deficient hiPSC lines were dissociated into single cells and exposed to UB-propagating growth conditions in suspension culture, leading to formation of organoids with UB-like identity (UB organoids). Wildtype and cyst-forming PKD2-deficient UB organoids were characterized by immunohistochemistry, RT-qPCR and single-cell RNA-sequencing. Compound screening for cyst reduction was performed over a ten-day period, with cyst number and diameter quantified from brightfield images. RESULTS: UB organoids generated by our protocol are readily scalable, yielding up to 2000 organoids from approximately 50 nephron organoids within seven days. They exhibit tubular extension alongside the expression of UB-specific markers, including AQP2, WNT9B, and KRT8. PKD2-deficient UB organoids initiate cystogenesis within seven days, achieving very high cyst-forming efficiency by about 20 days. The cysts express ADPKD markers and respond to cyst-reducing compounds, including the Bromodomain inhibitor JQ1 and the NF-κB inhibitor QNZ, but not tolvaptan, consistent with low AVPR2 expression. To validate the platform as a drug discovery tool, we screened a small-molecule library and identified M1, a putative Toll-like receptor 4 (TLR4) antagonist, that reduces cyst size. Consistent with TLR4-driven cystogenesis, the investigational TLR4 inhibitor TAK-242 reduced, whereas lipopolysaccharide increased, cyst formation. CONCLUSION: We have developed a scalable method to generate cystic UB organoids, providing a simple and efficient platform for ADPKD drug discovery.

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