Periodic Reporting for period 1 - GECKO (Generation of cartilage-free kidney organoids: a small molecule strategy)
Período documentado: 2022-09-01 hasta 2024-08-31
Chronic kidney disease (CKD), a progressive loss of kidney function affecting up to 17.3% of Europeans, is becoming increasingly prevalent worldwide, with an estimated 9.7 million people needing kidney replacement therapy. However, due to donor shortages, only a fraction receives treatment, underscoring the need for alternative therapies. Recent advances in generating kidney organoids from human induced pluripotent stem cells (hiPSCs) offer a promising regenerative solution, as these organoids can mimic human kidney structures and exhibit functional properties, such as glomerular filtration. Such organoids hold potential as functional kidney substitutes, providing relief from dialysis and improving patient quality of life.
The GECKO project aimed to address a key challenge in organoid development: the unintended formation of cartilage within kidney organoids, which hinders their functional application. The primary objective was to create a protocol for cartilage-free kidney organoids by identifying and modulating the pathways responsible for off-target chondrocyte formation, thus optimizing the organoids for therapeutic use.
GECKO followed a strategic approach to achieve these goals:
Assessing Cartilage in Organoids: Cartilage presence was evaluated at key developmental stages (days 7+18 to 7+25) using molecular markers and staining techniques, identifying when off-target chondrocytes emerge and providing a foundation for improved organoid protocols.
Understanding Cartilage-Related Pathways: Key pathways, including EGFR, SOX9, and Notch, were analyzed, with findings indicating that early signaling events influence cartilage development. Notch and protein kinase A (PKA) modulation proved effective in reducing off-target cell populations.
Inhibiting Cartilage Formation with Small Molecules: Small molecules were screened to prevent cartilage formation, with FGF9 and time-dependent Notch modulation successfully reducing chondrocytes. These results underscore a viable approach for developing functional kidney organoids free from off-target cells.
This work lays the groundwork for kidney organoid applications in regenerative medicine, ultimately aiming to improve therapy options for CKD patients and reduce dependence on donor kidneys and dialysis.
The GECKO project aimed to address a key challenge in organoid development: the unintended formation of cartilage within kidney organoids, which hinders their functional application. The primary objective was to create a protocol for cartilage-free kidney organoids by identifying and modulating the pathways responsible for off-target chondrocyte formation, thus optimizing the organoids for therapeutic use.
GECKO followed a strategic approach to achieve these goals:
Assessing Cartilage in Organoids: Cartilage presence was evaluated at key developmental stages (days 7+18 to 7+25) using molecular markers and staining techniques, identifying when off-target chondrocytes emerge and providing a foundation for improved organoid protocols.
Understanding Cartilage-Related Pathways: Key pathways, including EGFR, SOX9, and Notch, were analyzed, with findings indicating that early signaling events influence cartilage development. Notch and protein kinase A (PKA) modulation proved effective in reducing off-target cell populations.
Inhibiting Cartilage Formation with Small Molecules: Small molecules were screened to prevent cartilage formation, with FGF9 and time-dependent Notch modulation successfully reducing chondrocytes. These results underscore a viable approach for developing functional kidney organoids free from off-target cells.
This work lays the groundwork for kidney organoid applications in regenerative medicine, ultimately aiming to improve therapy options for CKD patients and reduce dependence on donor kidneys and dialysis.
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.
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.
The GECKO project showed that a one-week extension of FGF9 supplementation in iPSC-derived kidney organoids leads to a reduction of off-target cartilage. The renal structures are not impacted by FGF9 treatment and EMT markers are reduced. This modified organoid protocol will enable longer culture periods, a benefit for the use of organoids for screening or therapies.