Abstract: FR-OR103

Defining the Cellular Composition of Human Kidney Organoids Using High Throughput Single Cell Sequencing

Session Information

Category: Developmental Biology and Inherited Kidney Diseases

  • 402 Stem Cells

Authors

  • Combes, Alexander N., University of Melbourne, Parkville, Victoria, Australia
  • Zappia, Luke, Murdoch Children's Research Institute, Melbourne, Victoria, Australia
  • Phipson, Belinda, Murdoch Children's Research Institute, Melbourne, Victoria, Australia
  • Er, Pei Xuan, Murdoch Children's Research Institute, Melbourne, Victoria, Australia
  • Oshlack, Alicia, Murdoch Children's Research Institute, Melbourne, Victoria, Australia
  • Little, Melissa H., Murdoch Children's Research Institute, Melbourne, Victoria, Australia
Background

Single cell sequencing is a rapidly developing method for transcriptional analysis that has resulted in new insight into the cellular composition of complex tissues. Our lab and others have recently established protocols to direct the differentiation of human pluripotent cells to a kidney fate. This resulted in the formation of self-organizing structures that contain the basic components of the developing kidney. Understanding the cellular composition of these organoids is important to clarify the extent of tissue-specific diseases that may be modelled in this system, and to determine whether all known renal progenitors have been generated.

Methods

Kidney organoids were generated as previously described and harvested after 18 days of culture. We used the 10x Genomics Single Cell platform to profile ~7000 cells from three independent organoids.

Results

Clustering the single cell data identified multiple cell types enriched for markers of early proximal tubule, podocyte, pre-podocyte, collecting duct, ureter, vasculature, neural cells, and multiple interstitial populations. Cell types such as the vasculature and podocytes expressed multiple markers that enable unequivocal identification and give an insight into the state of maturity and fidelity of those cell types. Two distinct populations were identified which were enriched for podocyte markers such as PODXL, NPHS1, NPHS2, PTPRO, and SYNPO. While these both suggest podocyte identity, this may infer distinctions between early visceral and parietal epithelial cell types. Nephron segments such as the loop of Henle and distal tubule were not clearly identified. However, remaining epithelial clusters may represent progenitors of mature nephron segments. This is consistent with organoid morphology, which suggests nephron segmentation equivalent to Stage III/capillary-loop formation.

Conclusion

The analysis of organoid cellular composition using single cell transcriptional profiling has enabled a rapid survey of cellular diversity and fidelity of the cell types generated. The major cell types previously identified in kidney organoids were confirmed and extended with gene expression profiles for each. This data suggests that our kidney organoids will be useful for modelling development and disease of the early proximal tubule, collecting duct, and podocytes.

Funding

  • Government Support - Non-U.S.