Doctoral Student The Jikei University School of Medicine, United States
Abstract:
Background: Organ transplantation is the definitive treatment for end-stage organ failure. However, the global rise in kidney failure and severe organ shortages necessitate alternative approaches. While organ regeneration using pluripotent stem cells holds promise, generating fully functional three-dimensional organs remains a major challenge. This study focuses on regenerating human nephrons using fetal animal kidneys as scaffolds.
Previous Work: We previously achieved interspecies nephron regeneration by eliminating host Six2-positive nephron progenitor cells (NPCs) and Foxd1-positive stromal progenitor cells (SPCs) using a diphtheria toxin-based Cre-loxP system, followed by transplanting rat progenitor cells. However, this method was unsuitable for human applications due to the non-specific toxicity of diphtheria toxin to human cells.
Methods: To address this issue, we developed a mouse model incorporating an inducible caspase-9 (iC9) system to selectively eliminate fetal kidney progenitor cells without harming human cells. Foxd1-inducible caspase-9 mice (Foxd1-iC9) were created using CRISPR-Cas9 to knock in iC9 and a Venus reporter at the Foxd1 C-terminus. Homozygous mice were bred, and E13.5 fetal kidneys were harvested and cultured in transwell plates. AP20187 was administered daily to induce cell elimination. Immunostaining was performed to evaluate SPC elimination efficiency after four days.
Results: Cell death began within 24 hours of AP20187 administration. By 48 hours, fluorescence microscopy showed a significant reduction in Venus-positive SPCs. On day 4, immunostaining confirmed successful elimination of mouse SPCs. This elimination caused hyperproliferation of NPCs at the ureteric bud tips, consistent with previous reports of NPC dysregulation after SPC removal.
Conclusions: We developed a novel mouse model using the iC9 system for selective elimination of kidney progenitor cells, compatible with human applications. We plan to create double-knock-in mice that eliminate both NPCs and SPCs. This will allow transplantation of human iPSC-derived NPCs and SPCs, potentially enabling the generation of advanced three-dimensional human nephron structures within mouse fetal kidneys. This represents a major step toward organ regeneration using human stem cells.
