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Abstract: TH-PO634

Generation of Induced-Pluripotent Stem Cells and Kidney Organoids from Pediatric Urine Specimens

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 501 Development, Stem Cells, and Regenerative Medicine: Basic

Authors

  • Mulder, Jaap, The Hospital for Sick Children, Toronto, Ontario, Canada
  • Sharmin, Sazia, University Of Queensland, Brisbane, New South Wales, Australia
  • Chow, Theresa, University of Toronto, Toronto, Ontario, Canada
  • Rodrigues`, Deivid, The Hospital for Sick Children, Toronto, Ontario, Canada
  • D'Cruz, Robert, Sickkids Research Institiute, Toronto, Ontario, Canada
  • Rogers, Ian M., Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada
  • Ellis, James, The Hospital for Sick Children, Toronto, Ontario, Canada
  • Rosenblum, Norman D., The Hospital for Sick Children, Toronto, Ontario, Canada
Background

Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) are the leading cause of renal failure in children worldwide. Mouse models are widely used to explore underlying mechanisms in CAKUT, but important differences exist between human and murine renal development. Induced-pluripotent stem cells (iPSCs) and kidney organoids generated from actual CAKUT-patients, and closely related, healthy controls, can serve as complementary tools to study processes as nephrogenesis.
Objective: Generate patient-specific iPSCs and kidney organoids in an efficient, non-invasive manner.

Methods

Previously published protocols (Zhou et al., Nat Protoc, 2012; Takasato et al., Nat Protoc, 2016) were adapted. Urine cells (UCs) were grown from remnant urine samples routinely obtained in a nephrology outpatient clinic (incl. bag-collected urine). UCs were reprogrammed to urinary iPSCs (UiPSCs) using non-integrating episomal reprogramming vectors (Oct4/Sox2/Lin28/L-Myc/Klf4). UiPSC pluripotency was confirmed by qRT-PCR, immunostaining and embryoid body assays, and euploidy by karyotyping. Kidney organoids were generated from UiPSCs and control blood-derived iPSCs (BiPSCs).

Results

Remnant urine specimens were obtained from 17 patients (1mo-17yr old). UC cultures were established in 71% (12/17) of single urine collections (urine bag: 4/7, 57%, p=0.59 vs. mid-stream specimens) with median urine volume 26ml (range 10-75ml). Sufficient UC numbers for reprogramming were obtained within 16-25 days. Bacterial contamination was not observed. Episome-free UiPSC clones were obtained within 2-3 passages after reprogramming three UC cultures. Differentiated UiPSCs showed mRNA expression patterns (HOXD11/GATA3) matching those of differentiated BiPSCs and published data of differentiated fibroblast iPSCs. Organoids generated from UiPSCs closely resembled BiPSC-kidney organoid morphology and mRNA/protein expression of glomerular, tubular and stromal markers.

Conclusion

A rapid and non-invasive protocol was established to generate iPSCs/kidney organoids from pediatric urine samples. These results provide a readily applicable new platform for CAKUT-research with a low threshold for participation of patients (including infants) and indispensable, patient-specific controls.

Funding

  • Government Support - Non-U.S.