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Marria Khalid Hawa Patel Shaza Syed Sabiha Lakhi

Abstract

Introduction: Diabetic retinopathy (DR), the most common complication of diabetes, is characterized by vision loss due to vascular endothelial cell damage of the retina. Notably, induced pluripotent stem cell (iPSC) therapy has shown promise in the regeneration of the retina after damage. A limitation of using these cells includes the risk of immune-rejection. The knockout of human leukocyte antigen (HLA) proteins prevents a host immune response to non-native cells; however, HLA depletion introduces natural killer (NK) cell-mediated responses. Overexpression of cluster of differentiation 47 (CD47) inhibits the activity of NK cells. This project aims to create a universal DR patient-derived iPSC platform whose immunogenicity is limited through genetic alterations.


Methods: iPSCs will be reprogrammed from DR patient-derived fibroblasts and CRISPR-Cas9 will be used to knock out HLA and overexpress CD47. Alterations will be validated through Sanger sequencing, Western Blot and Immunofluorescence (IF) analysis. DR patient-derived iPSCs will be differentiated into endothelial cells to mimic the DR afflicted endothelial cells. These differentiated cells will then be co-cultured with NK cells and a cytotoxicity assay will be performed. Specifically, a chromium-release assay will be used by loading radioactive chromium into the genetically modified and unmodified endothelial cells and the chromium released by dying cells was monitored.


Results: Compared to the unmodified cells, we expect less NK-mediated cell death for the genetically altered endothelial cells.


Discussion: Effectively limiting the immunogenicity of the donor-derived iPSCs can establish a universal platform for future studies in DR therapy.


Conclusion: The resulting donor-derived iPSCs can be used to test drug therapies for DR or new methods to repair blood vessel damage, among a multitude of new research.

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Section
Research Protocol