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Abstract: FR-PO984

CRISPR-Mutant Organoids Identify Cystogenic Signal Pathways in the Early and Late Stages of Polycystic Kidney Disease

Session Information

Category: Genetic Diseases of the Kidney

  • 1001 Genetic Diseases of the Kidney: Cystic


  • 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
  • Valerius, M. Todd, Brigham & Women’s Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Morizane, Ryuji, Brigham & Women’s Hospital/Harvard Medical School, Boston, Massachusetts, United States

Human pluripotent stem cell (hPSC)-derived nephron organoids have significant potential for the development of targeted therapies for polycystic kidney disease (PKD). Previously, we generated heterozygous and homozygous PKHD1 mutant hPSCs by CRISPR/Cas9 genome editing to establish autosomal recessive PKD (ARPKD) models in human cells. Following standardization to isogenic controls, here we identify cystogenic signal pathways of ARPKD for the potential development of novel therapeutics.


Following an identical differentiation protocol, nephron organoids were generated from heterozygous (PKHD1+/-, isogenic control) and homozygous (PKHD1-/-) mutants in 3D culture. Organoids were treated with and without forskolin, a cAMP inducer, and cyst formation was evaluated by bright field and fluorescent microscopy. Cell proliferation was assessed by Ki67 positivity in proximal (LTL+) and distal (CDH1+) tubule cells. RNA was isolated from organoids on differentiation day 35. RNA was amplified, fluorescently labeled, hybridized to a microarray (Toray, 3D-Gene® Human mRNA Oligo chip 25k), and signal pathway analysis was conducted using Metacore™.


Gene expression analysis identified 1056 genes that differed by > 2-fold between heterozygous and homozygous mutants cultured without forskolin, the latter manifesting tubular dilatation (early cystic phase). Pathway analysis identified 50 signal pathways specific to the homozygous mutant in this early cystic phase (p<0.01), including the Rho kinase pathway known to induce loss of cellular polarity in PKD. Treatment with forskolin induced large cysts (late cystic phase) limited to homozygous mutants, which display 1876 genes differing by > 2-fold compared to similarly treated isogenic controls. 96 signal pathways were identified as specific in the late cystic phase of the disease and included proliferative transcriptional phenotypes.


The CRISPR approach enabled generation of isogenic controls which differentiated into nephron organoids following an identical protocol. Global gene expression analyses identified 50 and 96 specific signal pathways in the early and late cystic phases of PKD, respectively, in comparison to an isogenic control. These signals may identify novel therapeutic targets for PKD at different stages of the disease.


  • NIDDK Support