In Work Package 1, we focused on identifying the presence and characteristics of the chondrogenic population within kidney organoids. Transcriptomics analysis identified markers such as COL2A1, SOX9, and ACAN in a cluster of cartilage-like cells. These markers guided subsequent molecular assessments, which demonstrated that cartilage begins to appear between day 7+18 and day 7+25 of development. Additional visualization techniques, including qPCR and western blot, revealed increased marker expression by day 7+32. An optimized clearing protocol allowed for enhanced imaging of cartilage structures across the entire organoid, providing a comprehensive view of chondrogenic progression.
In Work Package 2, efforts focused on understanding pathways contributing to cartilage formation. Analysis of EGFR expression and phosphorylation at different stages (days 7+5, +10, +14, +18, and +25) suggested a correlation between EGFR activity, organoid deterioration, and chondrogenesis, though EGFR inhibition with Gefitinib led to organoid viability issues due to suppressed survival pathways. SOX9-mediated expression of collagen II was also observed to increase from day 7+18 to day 7+25, with evidence that treatments reducing cartilage also lowered SOX9 expression, suggesting a central role of SOX9 in cartilage development within organoids.
Work Package 3 focused on preventing cartilage formation using small molecules. Prolonged FGF9 exposure post-aggregation was hypothesized to influence organoid phenotype positively; indeed, extending FGF9 treatment by one week delayed cartilage formation until day 7+32 and decreased cartilage markers at both gene and protein levels. Small-molecule testing revealed that specific treatments like Notch and PKA modulation impacted cartilage formation. Inhibiting Notch with DAPT early in development led to increased cartilage, while later Notch1-specific inhibition significantly reduced cartilage presence without impairing renal structures. Additionally, activating PKA via dBcAMP increased nephron formation and reduced cartilage, while sequential inhibition and later activation of PKA yielded organoids with balanced nephron-tubule structures and no cartilage at day 7+25. Preliminary functionality assessments showed that FGF9 and dBcAMP treatments elevated aquaporin-2 expression and BSA uptake, indicating improved renal functionality in treated organoids. This work advances the potential for creating stable, cartilage-free kidney organoids with enhanced function and structure for regenerative applications.
