Sox9-iCaspase9 Mouse Model for Inducible Ureteric Bud Ablation to Generate Chimeric Scaffolds for Kidney Regeneration

Background: The shortage of organs for transplantation is severe, and the development of new supply sources presents a difficult yet crucial challenge. Our research utilizes animal developmental programs to create chimeric kidneys by transplanting progenitor cells into fetal kidney niches. Previous work successfully replaced rat nephron progenitor cells (NPCs) and stromal progenitor cells (SPCs) in developing mouse kidneys, but replacement of the ureteric bud (UB), which forms the collecting duct system, has not yet been achieved. The transcription factor Sox9 is highly expressed in UB tips and is vital for its branching morphogenesis. We aimed to develop a mouse model for inducible ablation of Sox9-expressing UB cells to try UB replacement. Methods: We developed a mouse model with an inducible caspase-9 system for selective cell elimination. Sox9-inducible caspase-9 mice (Sox9-iC9) were generated using CRISPR-Cas9 to knock in iC9 and a Venus reporter at the Sox9 C-terminus. Heterozygous mice were bred and E13.5 fetal kidneys were harvested and cultured in transwell plates. AP20187 and AT406 were daily added to the culture medium to eliminate Sox9-positive cells. Those fetal kidneys were fixed after four days of culture and were immunostained to evaluate cell elimination. Results: Fluorescence microscopy four days after AP20187 and AT406 administration revealed a reduction of Venus-positive cells, suggesting Sox9-positive cell death. We also confirmed it by immunostaining. Destruction of the Sox9-expressing UB tips prevented NPC aggregation around the UB and cap mesenchyme formation was consequently impaired. Conclusion: We successfully developed a Sox9-iC9 mouse model which enables inducible ablation of Sox9-expressing cells. In the developing kidney, this system disrupted cap mesenchyme organization, indicating targeted UB tip cell death. The future goal is to create an optimal niche that permits the connection of donor UB to the host UB. Achieving this would allow donor NPCs and SPCs to interact with allogeneic donor-derived UB system, enabling efficient nephron formation via same-species inductive signals. This approach could be crucial for overcoming species-specific barriers in the generation of kidney chimeras. This Sox9-iC9 mouse model is a promising platform for these critical next steps. Funding: Government Support – Non-U.S.