Abstract Body (Do not enter title and authors here): Introduction: It is well-known that reactive oxygen species (ROS) play important roles in myocardial ischemia reperfusion (I/R) injury. Exosomes have shown great potential and application in mitigating I/R induced cell and tissue damage. However, the clinical translation of exosomes has been limited by deficiencies in the yield of exosomes from cultured cells. Instead, nanovesicles (NVs), products manufactured directly from cells, can be easily scaled-up which facilitates translation into clinic.
Methods: NVs were manufactured from human induced pluripotent stem cells (hiPSCs) using an extruder and with a 0.2-μm filter membrane. The size and number of hiPSCs derived NVs (hiPSC-NVs) were measured by a Nanosight, and morphology of NVs were visualized with an electron microscope. The potency of hiPSC-NVs on protection of hiPSCs derived cardiomyocytes (hiPSC-CMs) against hypoxia and H₂O₂ were assessed in culture and in a mouse model of I/R injury. Proteomic analysis was performed to identify proteins that have ROS scavenger properties in hiPSC-NVs.
Results: About 9,500 NVs can be manufactured per one hiPSC. hiPSC-NVs starting 6.25x10⁸ /mL concentration effectively reduced lactate dehydrogenase leakage and apoptosis in hiPSC-CMs cultured in HBSS for 24 h in a hypoxia incubator, and protected cultured hiPSC-CMs from 200 µM H₂O₂ induced damage. When hiPSC-NVs or 0.9% NaCl was intramyocardially injected into C57BL mice hearts after cardiac I/R, measurements of cardiac function and infarct size conducted 4 weeks later were significantly better in the hiPSC-NV treatment group and were accompanied by increases in recipient mouse CM survival. Notably, proteomic analyses indicated that compared to hiPSCs, hiPSC-NVs were enriched in numerous proteins that scavenge ROS, including NUDT2, PRDX2, PRDX4, PRDX5, PRDX6, and GSR. Thus, through all these proteins in NV cargos, hiPSC-NVs exert CM protection activity against ROS injury after I/R.
Conclusions: The use of hiPSCs as parental cells for manufacturing NVs, can easily scale-up production, and have great potential for cardiac protection after I/R.