Abstract: TH-OR102
Kidney Organoids without Cilia Establish a Human Model of Ciliopathy-Associated Polycystic Kidney Disease
Session Information
- PKD: Genetic, Mechanistic, and Clinical
October 25, 2018 | Location: 25A, San Diego Convention Center
Abstract Time: 04:42 PM - 04:54 PM
Category: Genetic Diseases of the Kidney
- 1001 Genetic Diseases of the Kidney: Cystic
Authors
- Cruz, Nelly M., University of Washington, Seattle, Washington, United States
- Himmelfarb, Jonathan, Kidney Research Institute, Seattle, Washington, United States
- Freedman, Benjamin S., University of Washington, Seattle, Washington, United States
Background
Primary cilia are specialized, antenna-like organelles at the plasma membrane associated with a spectrum of human syndromes called ciliopathies. Phenotypes of polycystic kidney disease (PKD) are commonly associated with ciliopathies, but how cilia function to prevent kidney cysts remains poorly understood. A major barrier to understanding ciliary function is the absence of cellular models that accurately reconstitute the complex human phenotypes of ciliopathies.
Methods
We used the CRISPR-Cas9 genome editing system to introduce mutations in two genes that are required for ciliogenesis, kinesin family member 3A (KIF3A) and KIF3B, in human pluripotent stem cells (hPSC). Genome modified KIF3A-/- or KIF3B-/- hPSC were differentiated in vitro into kidney organoids containing proximal tubules, distal tubules, and podocytes in nephron-like segments. Structure and composition of organoids were analyzed by immunofluorescence. To determine the mechanistic consequences of ciliary loss, a biochemical assay was used to measure ciliopathy-linked protein levels in both whole cell lysates and extracellular vesicles.
Results
We isolated four independent cell lines with indel mutations in the KIF3A gene and three cell lines with indels in KIF3B. Immunoblots confirmed that no full-length protein is produced in these mutants. The gene-edited hPSC completely lacked cilia, while ~50% of cells were ciliated in the isogenic controls. Importantly, the absence of cilia did not alter hPSC growth, pluripotency, self-renewal, or their ability to differentiate into kidney organoids. Upon differentiation, however, the cilia-deficient kidney organoids formed large cysts from tubular epithelial cells, which were absent in controls. Defects were furthermore observed in the expression of the disease-associated proteins polycystin-1 and polycystin-2 in kinesin mutants.
Conclusion
Genome modified KIF3A-/- or KIF3B-/- hPSC establish a general tool for studying the function of human cilia in diverse cell types and organoids. Our findings directly link the loss of ciliary kinesins to polycystin expression defects and kidney cyst formation in human cells. Using this new tool, it is now possible to perform phenotypic screens and mechanistic studies to reveal the functions of human cilia and test interventional strategies for ciliopathy syndromes.
Funding
- Private Foundation Support