Abstract: FR-OR050
An Approach for Improving iPSC-Derived Ureteric Epithelial Identity In Vitro
Session Information
- Development and Stem Cells
November 08, 2019 | Location: 152, Walter E. Washington Convention Center
Abstract Time: 06:18 PM - 06:30 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 500 Development, Stem Cells, and Regenerative Medicine
Authors
- Howden, Sara E., Murdoch Childrens Research Institute, Parkville, Victoria, Australia
- Wilson, Sean, Murdoch Childrens Research Institute, Parkville, Victoria, Australia
- Tan, Ker sin, Murdoch Children''s Research Institute, Parkville, New South Wales, Australia
- Starks, Lakshi T., Murdoch Childrens Research Institute, Parkville, Victoria, Australia
- Lawlor, Kynan T., Murdoch Children''s Research Institute, Parkville, New South Wales, Australia
- Vanslambrouck, Jessica May, Murdoch Childrens Research Institute, Parkville, Victoria, Australia
- Little, Melissa H., Murdoch Childrens Research Institute, Parkville, Victoria, Australia
Background
We previously described a protocol for generating iPSC-derived human kidney organoids containing segmented nephrons, renal interstitium, endothelium and a CDH1+/GATA3+/PAX2+/CALB1+ ureteric epithelium (UE). Recent single cell analyses of mouse and human fetal kidney have identified expression of many presumed UE marker genes, including GATA3 and HOXB7, within the distal nephron and connecting segment. This has raised questions about the true identity of GATA3+ epithelium within kidney organoids. Here we describe the transcriptional profile of GATA3+ epithelium isolated from organoids both prior to and after in vitro culture.
Methods
Kidney organoids were generated from iPSCs harboring an mCherry fluorescent reporter gene within the endogenous GATA3 locus. We used fluorescent activated cell sorting (FACS) to isolate GATA3+/mCherry+ epithelium for subsequent culture in conditions previously shown to promote expansion of mouse UE. Immunofluorescence and RNAseq-based transcriptional profiling was performed to evaluate cell identity pre- and post-culture.
Results
We observed extensive proliferation and branching of FACs-isolated GATA3+/mCherry+ cells cultured in conditions known to promote propagation of mouse UE. GATA3/mCherry expression was maintained even after several rounds of dissociation and re-plating. Additional UE markers (KRT8, PAX2, SOX9, CALB1) were detected by immunofluorescence. Although global RNAseq of GATA3+/mCherry+ epithelium at the time of isolation suggested a cellular identity more akin to distal tubule, cultured GATA3+/mCherry cells showed loss of KCNJ1 and induction of WNT11 and RET expression. Single cell transcriptional analysis of cultured cells revealed 3 clusters representative of tip (RET+/GFRα1+/ETV4+), stalk (AQP2+) and a putative medullary compartment (UPK1A+). Notably, WNT9B expression was evident at a level consistent with that observed in human fetal kidney.
Conclusion
Our results redefine the identity of the GATA3+/CDH1+ epithelium within kidney organoids to early connecting segment/distal tubule. However, when cultured in appropriate conditions, this can transition to a more recognizable collecting duct fate. The capacity to expand and propagate a branching epithelial compartment with ureteric identity may represent a useful approach for the generation of collecting duct tissue.
Funding
- NIDDK Support