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Abstract: FR-PO765

Generation of Kidney Branching Ureteric Bud Organoid from Human Pluripotent Stem Cells

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Li, Zhongwei, University of Southern California, Los Angeles, California, United States
  • Zeng, Zipeng, University of Southern California, Los Angeles, California, United States
  • Huang, Biao, University of Southern California, Los Angeles, California, United States
Background

The repetitive reciprocal interactions between metanephric mesenchyme (MM) and ureteric bud (UB) lay the basis for kidney organogenesis. Nephron progenitor cell (NPC) within the MM generates the nephrons, while UB branches to generate the collecting duct network. Human pluripotent stem cell (hPSC)-derived NPCs and nephron organoids have been well established. However, currently available hPSC-derived UB still lack their definitive features – they had very limited branching ability and did not interact with NPCs. Here we present a novel method to generate a branching UB organoid from hPSCs. Our platform will advance the kidney regeneration field and it will also provide a novel platform for modeling diseases in the collecting duct.

Methods

We first developed a robust culture medium, named UB culture medium (UBCM), to support the long-term expansion of mouse UB as three-dimensional (3D) branching organoid. Meanwhile, we employed SOX9-GFP reporter hPSC line to monitor and optimize our UB differentiation protocol. After differentiation, the SOX9-GFP+ UB progenitor-like cells were purified via flow cytometry. Aggregated SOX9-GFP+ cells branched out in UBCM and can be passaged as 3D organoid. The identity of the UB organoids were confirmed by gene expression, ability to interact with NPCs and the potential to generate mature collecting duct.

Results

The expandable mouse UB organoid cultured in UBCM showed homogenous expression of broad UB markers (Lhx1, Gata3, Pax2, Pax8) and UB progenitor markers (Ret, Etv5, Sox9). By qPCR, our hPSC-derived UB showed the expression of various UB markers at similar levels to that in the primary UB obtained from human fetal kidney. Immunostaining also confirmed the homogeneous expression of RET, GATA3, SOX9, PAX2, CDH1 and KRT8 in the hPSC-derived UB organoids. More importantly, when the hPSC-derived UB were reconstructed with hPSC-derived NPCs, kidney organogenesis was restored, generating nephrons from the NPCs and collecting duct from the UB.

Conclusion

1. Novel UBCM supports the long-term expansion of mouse UB
2. Novel directed differentiation protocol generates UB progenitor-like cells from hPSCs
3. Aggregated hPSC-derived UB branches in UBCM and can be passaged as 3D organoids
4. Restored kidney organogenesis in vitro when hPSC-derived NPC and UB are reconstructed

Funding

  • Private Foundation Support