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Abstract: TH-PO628

Xenogeneic Kidney Generation with Human Nephron Progenitor Cells in Zebrafish

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 501 Development, Stem Cells, and Regenerative Medicine: Basic

Authors

  • Yin, Wenqing, Brigham and Women's Hospital , Boston, Massachusetts, United States
  • Gupta, Navin R., Brigham & Women''s Hospital/Massachusetts General Hospital, Brighton, Massachusetts, United States
  • Miyoshi, Tomoya, Brigham and Women's Hospital , Boston, Massachusetts, United States
  • Valerius, M. Todd, Brigham and Women's Hospital , Boston, Massachusetts, United States
  • Bonventre, Joseph V., Brigham and Women's Hospital , Boston, Massachusetts, United States
  • Morizane, Ryuji, Brigham and Women's Hospital , Boston, Massachusetts, United States
Background

Generating human stem cell-based tissue in animals may provide replacement human organs, establish novel disease models, and aid in drug development through toxicity screening and therapeutic testing. Human nephron progenitor cells (hNPCs) derived from human pluripotent stem cells (hPSCs) are attractive sources for human kidney generation in animals; however, techniques to replace host kidney tissue with hNPCs are yet to be developed. Here we employ transparent zebrafish (Casper) to enable real-time monitoring of xenotransplanted, fluorescent hNPCs and visualize human nephron formation.

Methods

hNPCs were differentiated from hPSCs by our previously established protocol. Cas9 mRNA and gRNAs targeting Lhx1a or Wt1b, essential transcriptional factors for kidney development in zebrafish, were injected into single cell stage embryos. Cell Tracker- or GFP-labeled hNPCs were transplanted to blastoderm stage embryos and nephrogenesis visualized overtime. Nephrons were evaluated by qPCR with human-specific primers and immunostaining for human solute transporters and segment-specific proteins.

Results

Zebrafish with targeted disruption of either Lhx1a or Wt1b died by 24 days post treatment (dpt), while transplantation of hNPCs rescued 14.5% of similarly treated zebrafish (p<0.00001). All zebrafish that underwent gRNA treatment exhibited renal insufficiency with evidence of edema at 3 dpt, however, hNPC transplantation resolved renal insufficiency by 20 dpt. Transplanted hNPCs migrated to the pronephric area, expressed human SIX2 in early nephrogenesis (9 dpt), and formed glomerular and tubular structures that connected to DBA+ zebrafish collecting ducts at a later stage (26 dpt). Notably, hNPCs contributed to Podocalyxin+ cells in membrane-enclosed structures that contained human CD31+ capillary loops, indicating human glomerular development in zebrafish. qPCR using human-specific primers validated human nephron formation in zebrafish.

Conclusion

We developed novel methods for generating human kidney tissue in animals. After eliminating NPCs in early zebrafish embryos using CRISPR/Cas9 genome editing, xenotransplantation of hNPCs generated human nephron structures in zebrafish. Our results serve as a proof-of-concept that isogenic patient-specific kidney tissue may be generated in animals for translational applications.

Funding

  • Private Foundation Support