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Abstract: PO0890

Generation of Branching Ureteric Bud Organoids from Human Pluripotent Stem Cells

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Mccracken, Kyle, Boston Children's Hospital, Division of Nephrology, Boston, Massachusetts, United States
  • Shi, Min, Brigham and Women's Hospital Department of Renal Medicine, Boston, Massachusetts, United States
  • Zhang, Weitao, Brigham and Women's Hospital Department of Renal Medicine, Boston, Massachusetts, United States
  • Ester, Lioba, Brigham and Women's Hospital Department of Renal Medicine, Boston, Massachusetts, United States
  • Bonventre, Joseph V., Brigham and Women's Hospital Department of Renal Medicine, Boston, Massachusetts, United States
Background

Directed differentiation of human pluripotent stem cells (hPSCs) to kidney organoids has been well established; however, the generation of hPSC-derived ureteric bud (UB), which undergoes branching morphogenesis to generate collecting duct (CD) epithelia, has remained a significant challenge. Here we describe a highly efficient method for deriving UB/CD organoids from hPSCs, which form unprecedented branching structures. This method provides a new platform for studying human CD development, physiology, and disease modeling. Moreover, this will provide the opportunity to markedly enhance existing kidney organoids by providing a collecting system and importantly, introducing an iterative branching component that is essential to driving metanephric kidney development.

Methods

First, we modified existing methods to efficiently direct hPSCs into anterior intermediate mesoderm cells in monolayer format. From that point, we optimized a process of 3-D development that included forced aggregation followed by spontaneous budding and then branching of the UB epithelia.

Results

We generated GATA3/PAX2 AIM with >80% efficiency within 5 days of induction, which was then aggregated into 3-D spheres. Over the subsequent days, the cells underwent a spontaneous process of organization and maturation that parallels normal development of the embryonic nephric duct (ND). Nearly synchronously, each aggregate then formed a single epithelial outgrowth that exhibited expression of UB markers PAX2, GATA3, RET, SOX9 and CALB1. Next, we embedded the UB-like buds into a hydrogel matrix, and they underwent a complex branching morphogenetic program driven by growth factor signals. At later stages, we identified culture conditions to stimulate differentiation of the ureteric epithelia into CD principal cells, identified by expression of AQP2 and SCNN1A/B. Additionally, GATA3+ UB progenitor cells were maintained and expanded over several passages in our 3-D culture system.

Conclusion

We have developed a novel strategy to generate branching UB tissues from hPSCs, which are also competent to differentiate into CD epithelia. Efforts are ongoing to investigate the functional and physiologic properties of these tissues, as well as to model genetic diseases that impact morphologic development of the collecting system.

Funding

  • NIDDK Support