VHL-Deficient Human Kidney Organoids to Model Clear Cell Renal Cell Carcinoma

Background: Renal cancer ranks among the top ten most prevalent cancers worldwide, with clear cell renal cell carcinoma (ccRCC) emerging as the predominant subtype, accounting for 70-80% of cases and displaying a relatively aggressive phenotype. The inactivation of Von Hippel-Lindau (VHL) tumor suppressor gene, found in approximately 90% of cases, was identified as the primary driver event of ccRCC development. Existing genetic engineered mouse models cannot fully capture the distinctive features of ccRCC, limiting our understanding of the role of VHL mutation in ccRCC initiation. Human pluripotent stem cell derived 3D human kidney organoid comprises of segmentally patterned nephron structures and vascular network, manifesting higher physiological relevance with the human kidney. In this study, we aim to develop VHL knockout human kidney organoids to better understand the role of VHL in tumor initiation. Methods: CRISPR/Cas9 was utilized to generate VHL biallelic loss-of-function mutant human embryonic stem cells (ESCs). WT and VHL-/- human ESCs were differentiated into kidney organoids which were characterized via histology, immunofluorescence, PAS staining, Oil Red O staining, and qRT-PCR, etc. Results: Immunostaining confirmed that patterned nephron segments, including glomeruli, proximal tubules, and distal tubules were successfully generated from VHL-/- human ESCs. Histological analysis revealed the emergence of a 'clear cell-like' phenotype in kidney tubular epithelium upon VHL loss. PAS and Oil Red O staining detected accumulation of glycogen and lipids in VHL-/- tubular epithelial cells. qRT-PCR analysis demonstrated metabolic alterations akin to those observed in human ccRCC patients, characterized by the upregulation of genes associated with glycolysis and lipid synthesis, alongside downregulation of genes involved in beta-oxidation. Additionally, VHL-/- kidney organoids acquired higher proliferation competency upon long-term culture. Conclusion: Human ESC-derived VHL-/- kidney organoids effectively replicate critical morphological and metabolic features reminiscent of human ccRCC patients, thereby offering a novel in vitro platform for studying ccRCC tumorigenesis.