. Seoul National University Seoul National University, United States
Abstract: Despite the increase in the number of peripheral artery disease (PAD) patients, cultivating primary endothelial cells (ECs) from patients for therapeutic angiogenesis is difficult due to time and cost constraints. Human-induced pluripotent stem cells (hiPSCs) can be an attractive substitute because they can be easily induced in vitro while maintaining in vivo efficiency. However allogeneic transplantation of hiPSCs derivatives has the potential to elicit the patient’s immune response leading to graft rejection. To address these challenges, we diminished the immunogenicity of hiPSCs through the inactivation of major histocompatibility complex (MHC) class I and II genes to evade T cells. As MHC class knockout renders cells susceptible to NK cell attack, we over-expressed the ‘don’t eat me signal’ CD24. We conducted T cell proliferation assay and activation assay using CD3+, CD4+, and CD8+ T cells. To verify the abilities to evade NK cell attack, we co-cultured NK cells with hiPSC-derived ECs. Based on these in vitro data, we transplanted hiPSC-derived ECs into humanized mouse models generated by injecting CD34+ hematopoietic stem cells from human umbilical cord blood into immune-deficient mice. Using luminescence of luciferase-expressing In Vivo Imaging Systems, we observed that U-ECs survived for longer periods after transplantation compared to WT-ECs, while reducing immune response. We induced humanized PAD model by ligating and excising the femoral artery of the left hindlimb. Then we injected U-ECs, demonstrating the therapeutic efficacy of U-ECs. The U-ECs demonstrated improved survival and contributed to improvement in blood perfusion. We also observed that the alleviation of muscle degeneration was most significant in the U-EC group. These findings suggest that human-engineered U-ECs can be utilized as an “off-the-shelf” cell therapy in PAD patients.
Funding Source: This study was partially supported by the Research Institute for Veterinary Science, Seoul National University, and Korean Fund for Regenerative Medicine(KFRM) grant (22A0101L1-11).
