Therapeutic effects of hypoimmunogenic universal human iPSC-derived endothelial cells in a humanized mouse model of peripheral artery disease.

BACKGROUND: Peripheral arterial disease (PAD) is a leading cause of limb disability due to ischemia caused by atherosclerotic plaques. Cell-based therapies using endothelial cells (ECs) have shown promise in promoting angiogenesis for PAD, but challenges remain in obtaining sufficient ECs from human tissues. Induced pluripotent stem cells (iPSCs) provide a potential solution, though immune rejection issues arise due to human leukocyte antigen (HLA) mismatches. The depletion of HLA class I and II through gene editing aims to broadly avoid lymphocyte recognition and can be achieved by inactivating β2-microglobulin (B2M) and class II transactivator (CIITA). However, B2M inactivation can lead to a 'missing self' killing response by NK cells and macrophages. To overcome this, we proposed universal iPSCs by knocking out B2M and CIITA and over-expressing CD24 to reduce immune rejection. METHODS: Universal iPSCs were tested for their pluripotency and ability to differentiate into ECs. The stimulation of universal iPSC-derived endothelial cells (U-ECs) to T and NK cells was evaluated by activation marker using flow cytometry. We generated a humanized mouse model by intravenously injecting CD34(+) hematopoietic stem cells isolated from umbilical cord blood into NSG mice. Finally, we induced a humanized PAD mouse model by removing the femoral artery of the left hindlimb. Then we injected U-ECs, demonstrating the therapeutic efficacy of U-ECs. RESULTS: We generated hypoimmunogenic universal iPSC by knocking out B2M and CIITA, along with over-expressing CD24, and confirmed that their pluripotency was maintained. We demonstrated that U-ECs exhibit functional endothelial properties and reduced immunogenicity, effectively mitigating immune recognition from both adaptive and innate immune responses. U-ECs survived in significantly greater numbers after transplantation and elicited a weaker immune response in humanized mice. Then we induced hindlimb ischemia in humanized mice to establish a humanized PAD model. U-ECs induced effective angiogenic capabilities, leading to significant blood flow restoration in ischemic limbs. CONCLUSIONS: This study demonstrates the feasibility of creating hypoimmunogenic iPSCs and their derivatives that can reduce immune response and function effectively in vivo.