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Kidney Week

Abstract: PO0892

Delaying Nephrogenesis In Vitro Results in Enhanced Proximal Tubule Alignment and Maturity in Kidney Organoids

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Vanslambrouck, Jessica May, Murdoch Children's Research Institute, Parkville, Victoria, Australia
  • Tan, Ker sin, Murdoch Children's Research Institute, Parkville, Victoria, Australia
  • Wilson, Sean, Murdoch Children's Research Institute, Parkville, Victoria, Australia
  • Howden, Sara E., Murdoch Children's Research Institute, Parkville, Victoria, Australia
  • Lawlor, Kynan T., Murdoch Children's Research Institute, Parkville, Victoria, Australia
  • Scurr, Michelle, Murdoch Children's Research Institute, Parkville, Victoria, Australia
  • Little, Melissa H., Murdoch Children's Research Institute, Parkville, Victoria, Australia

Group or Team Name

  • Kidney Development, Disease and Regeneration
Background

Stem cell-derived organoids represent a promising model for complex organs such as the kidney, with studies of disease, physiology and drug interactions relying mostly on simplistic cell cultures or animals that do not completely recapitulate the complex human environment. However, kidney organoids are still less mature than the in vivo organ, a limitation arguably most apparent in the proximal tubule (PT) compartment. The critical role of the PT in performing the bulk of solute reabsorption makes it a key requirement for drug screening and bioengineering. By inhibiting promiscuous epithelialisation, we report the development of PT-enhanced kidney organoids with improved PT alignment, maturity, functionality and suitability for therapeutic applications.

Methods

Standard and fluorescent reporter iPSC lines were subjected to prolonged monolayer differentiations, with 4 - 5 day initial CHIR exposure durations (modified from: Howden et al, EMBO Rep, 2019; Vanslambrouck et al. JASN, 2019) and precisely-timed modifications to signalling pathways such as canonical WNT, BMP and NOTCH. Organoids were generated as previously published (Takasato et al, Nat Protoc, 2016) and analysed using confocal immunofluorescence, live imaging of fluorescent reporters, transcriptional profiling (single cell RNAseq) and functional transport assays.

Results

PT-enriched organoid could be generated in a highly reproducible manner from multiple cell lines. Proximal tubules showed mature protein and gene expression, as well as transport capacity in multiple assays. Nephron spatial arrangement/directionality, as well as shifts in proximo-distal nephron patterning, were influenced by these modified conditions.

Conclusion

Here, we describe PT-enhanced kidney organoids with improved PT maturity and functionality, with this approach providing a more stringent control over the spatial arrangement of nephrons. Such findings have significant implications for downstream applications including drug screening, toxicology assays and bioengineering of functional replacement cells or tissues.

Funding

  • NIDDK Support