Abstract: FR-PO362
Utilizing a Patient-Specific iPSC Platform for the Study of Rare Genetic Kidney and Vascular Diseases
Session Information
- Genetics, Development, Regeneration
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Development‚ Stem Cells‚ and Regenerative Medicine
- 500 Development‚ Stem Cells‚ and Regenerative Medicine
Authors
- Diatlov, Daniel, University of Toronto Institute of Medical Science, Toronto, Ontario, Canada
- Melzer, Franz Leonard, The Hospital for Sick Children Research Institute Cell Biology Program, Toronto, Ontario, Canada
- Bohorquez, Arlette, The Hospital for Sick Children Research Institute Cell Biology Program, Toronto, Ontario, Canada
- Ma, Alison Lap Tak, Hong Kong Children's Hospital Department of Paediatric Nephrology, Kowloon City, Hong Kong
- Licht, Christoph, The Hospital for Sick Children Division of Nephrology, Toronto, Ontario, Canada
Background
The increasing use of induced pluripotent stem cells (iPSCs) to model human genetic diseases in vitro has allowed researchers to better understand the pathology of countless disorders, and subsequently develop better treatments for patients. Currently in our lab we use blood-outgrowth endothelial cells (BOECs) to study endothelial cell (EC) dysfunction in kidney diseases and thrombotic microangiopathies. However, isolating patient BOECs is often challenging, as it requires a significant sample of fresh blood which is not always readily available. As such, we are working to utilize a platform that uses patient skin fibroblasts to create patient-specific iPSCs, that can then be differentiated into ECs which capture the genetic conditions of patients.
Methods
Patient skin fibroblast-derived iPSCs and healthy control iPSCs were created using a commercially available Sendai virus CytoTune-iPS 2.0 reprogramming kit (Thermo Fisher). These iPSCs were then differentiated into ECs using a directed differentiation protocol in factor-defined media. Following differentiation, CD31 (PECAM1) positive cells were sorted using fluorescence-activated cell sorting (FACS), to isolate a pure population. Isolated ECs were then characterized using immunofluorescence (IF) staining for CD31 and VE-Cadherin, as well as a tubule formation assay.
Results
We were able to successfully create the iPSCs and differentiate them into ECs that express the appropriate markers (CD31 and VE-Cadherin) and are able to successfully form robust capillary-like tubes over a 48 hour period.
Conclusion
Our iPSC differentiation protocol provides a verified and valuable tool for the study of genetic kidney disorders involving endothelial cells when BOEC isolation is not possible.
Figure 1. (A) Patient iPSC ECs sorted for CD31 using FACS express CD31 and VE-Cadherin (20x). (B) Patient iPSC ECs form robust capillary-like tubes over 48 hours (4x).
Funding
- Government Support – Non-U.S.