Wei Yuhua, Geng Xiaoxiao, You Qing, Zhang Yu, Cao Fangfang, Narayanan Gunaseelan, Nguyen Thanh, Chen Xiaoyuan, Zhang Jianyi, Ye Lei
Department of Diagnostic Radiology, Yong Loo Lin School of Medicine, National University of Singapore, 1190742, Singapore.
Department of Chemical and Biomolecular Engineering, College of Design and Engineering, National University of Singapore, 1175753, Singapore.
Bioact Mater. 2025 May 2;50:585-602. doi: 10.1016/j.bioactmat.2025.04.017. eCollection 2025 Aug.
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 (hiPSCs) for manufacturing nanovesicles (hiPSC-NVs). Approximately 9500 hiPSC-NVs were produced from a single hiPSC. Proteomic analyses indicated that, compared to hiPSCs, the cargos of 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), hiPSC-NVs reduced lactate dehydrogenase leakage and apoptosis in hypoxia-cultured hiPSC-CMs through activating the AKT pathway, protected hiPSC-CMs from HO-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 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 hiPSC-NV group, with increased mouse CM survival and cell cycle activity. Thus, the proteins in the hiPSC-NV cargos convergently activated the AKT pathway, scavenged ROS to protect CMs, and upregulated YAP signaling to induce CM cell cycle activity. Thus, hiPSC-NVs hold great potential for cardiac protection and regeneration.
重新激活成年哺乳动物心肌细胞（CMs）的细胞周期活性仍然是一项重大挑战。本研究利用成簇规律间隔短回文重复序列（CRISPR）/Cas9基因编辑技术敲除人诱导多能干细胞（hiPSC）中的β2-微球蛋白，创建了一种低免疫原性的hiPSC系，用于制造纳米囊泡（hiPSC-NVs）。单个hiPSC可产生约9500个hiPSC-NVs。蛋白质组学分析表明，与hiPSC相比，hiPSC-NVs的货物富含纺锤体和染色体蛋白，以及调节细胞周期和清除活性氧（ROS）的蛋白。当将hiPSC-NVs应用于hiPSC衍生的心肌细胞（hiPSC-CMs）时，hiPSC-NVs通过激活AKT途径减少了缺氧培养的hiPSC-CMs中的乳酸脱氢酶泄漏和细胞凋亡，通过纳米囊泡货物中的ROS清除剂保护hiPSC-CMs免受HO诱导的损伤，通过YAP途径增加hiPSC-CM增殖，并且在与人CD8 T细胞共培养或递送至小鼠时具有低免疫原性。此外，在心脏缺血/再灌注损伤后将hiPSC-NVs或0.9% NaCl心肌内注射到小鼠体内，4周后评估心脏功能和梗死面积，hiPSC-NV组显著改善，小鼠CM存活率和细胞周期活性增加。因此，hiPSC-NV货物中的蛋白质共同激活AKT途径，清除ROS以保护CMs，并上调YAP信号以诱导CM细胞周期活性。因此，hiPSC-NVs在心脏保护和再生方面具有巨大潜力。
