Human induced pluripotent stem cell derived nanovesicles for cardiomyocyte protection and proliferation.

It remains a significant challenge to reactivate the cell cycle activity of adult mammalian cardiomyocytes (CMs). This study created a hypo-immunogenic human induced pluripotent stem cell (hiPSC) line using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing to knockout β2-microglobulin in hiPSCs ((B2MKO)hiPSCs) for manufacturing nanovesicles ((B2MKO)hiPSC-NVs). Approximately 9500 (B2MKO)hiPSC-NVs were produced from a single (B2MKO)hiPSC. Proteomic analyses indicated that, compared to (B2MKO)hiPSCs, the cargos of (B2MKO)hiPSC-NVs were enriched in spindle and chromosomal proteins, as well as proteins that regulate the cell cycle and scavenge reactive oxygen species (ROS). When administrated to hiPSCs derived CMs (hiPSC-CMs), (B2MKO)hiPSC-NVs reduced lactate dehydrogenase leakage and apoptosis in hypoxia-cultured hiPSC-CMs through activating the AKT pathway, protected hiPSC-CMs from H(2)O(2)-induced damage by ROS scavengers in the NV cargo, increased hiPSC-CM proliferation via the YAP pathway, and were hypoimmunogenic when co-cultured with human CD8(+) T cells or delivered to mice. Furthermore, when (B2MKO)hiPSC-NVs or 0.9 % NaCl were intramyocardially injected into mice after cardiac ischemia/reperfusion injury, cardiac function and infarct size, assessed 4 weeks later, were significantly improved in the (B2MKO)hiPSC-NV group, with increased mouse CM survival and cell cycle activity. Thus, the proteins in the (B2MKO)hiPSC-NV cargos convergently activated the AKT pathway, scavenged ROS to protect CMs, and upregulated YAP signaling to induce CM cell cycle activity. Thus, (B2MKO)hiPSC-NVs hold great potential for cardiac protection and regeneration.