Extracellular Vesicles Released by Human Induced-Pluripotent Stem Cell-Derived Cardiomyocytes Promote Angiogenesis
Extracellular Vesicles Released by Human Induced-Pluripotent Stem Cell-Derived Cardiomyocytes Promote Angiogenesis
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DOI：
10.3389/fphys.2018.01794
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发表时间：
2018-12-14
影响因子：
4
通讯作者：
Khan, Mahmood
中科院分区：
文献类型：
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作者：
Dougherty, Julie A.;Kumar, Naresh;Khan, Mahmood
虽然移植后的细胞存活率很低，但使用干细胞治疗心肌修复的新证据表明，旁分泌信号机制是改善心功能、减少纤维化和增加血管生成的基础。最近的研究表明，干细胞分泌的细胞外囊泡（EVs）如外泌体可以刺激血管生成，提供细胞保护，并调节细胞凋亡。然而，从人诱导多能干细胞来源的心肌细胞（hiPSC-CM）分泌的ev的血管生成潜能尚未被阐明。因此，本研究的主要目的是分离、表征和评估从hiPSC-CM条件培养基中收集的ev的体外血管生成潜力。hiPSC-CM培养2周，从细胞培养基中分离出ev。通过透射电子显微镜（TEM）、纳米颗粒跟踪分析和免疫印迹技术对分离的ev进行了表征。此外，通过牛主动脉内皮细胞（BAEC）的血管形成、伤口愈合和细胞增殖试验来评估这些ev的血管生成潜力。此外，用hiPSC-CM-derived EV （cm -EV）处理hiPSC-derived内皮细胞（hiPSC-EC），评估生长因子的基因表达水平，以评估其在促进血管生成中的作用。cm -EV的透射电镜成像显示存在双膜结合结构，这是EV的特征。纳米颗粒跟踪分析进一步证实了分泌颗粒的大小和形状与电动汽车一致。此外，免疫印迹显示，这些颗粒中富含ev特异性标志物（CD63和HSP70）。最重要的是，与对照组（无ev）相比，100 μ g/ml cm - ev处理的BAEC管形成、伤口闭合和细胞增殖显著增加。最后，用cm - ev处理hiPSC-EC诱导内皮细胞增加促血管生成生长因子的表达。总之，我们的研究结果表明，从hiPSC-CM中分离的ev可以促进内皮细胞的血管生成。这种脱细胞/无细胞方法是一种潜在的转化治疗方法，可诱导心肌梗死患者血管生成。
Although cell survival post-transplantation is very low, emerging evidence using stem cell therapy for myocardial repair points toward a primary role of paracrine signaling mechanisms as the basis for improved cardiac function, decreased fibrosis, and increased angiogenesis. Recent studies have demonstrated that extracellular vesicles (EVs) such as exosomes secreted by stem cells stimulate angiogenesis, provide cytoprotection, and modulate apoptosis. However, the angiogenic potential of EVs secreted from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), a terminally differentiated cell type, has not been elucidated yet. Therefore, the main objective of this study is to isolate, characterize, and evaluate the in vitro angiogenic potential of EVs collected from hiPSC-CM conditioned media. The hiPSC-CM were cultured for 2 weeks and EVs were isolated from cell culture medium. Isolated EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis, and immunoblotting. Furthermore, the angiogenic potential of these EVs was evaluated by tube formation, wound-healing, and cell-proliferation assays in bovine aortic endothelial cells (BAEC). In addition, gene expression levels of growth factors was evaluated in hiPSC-derived endothelial cells (hiPSC-EC) treated with hiPSC-CM-derived EV (CM-EVs) to assess their role in promoting angiogenesis. TEM imaging of CM-EVs showed a presence of a double-membrane bound structure, which is a characteristic of EV. Nanoparticle tracking analysis further confirmed the size and shape of the secreted particles to be consistent with EVs. Furthermore, EV-specific markers (CD63 and HSP70) were enriched in these particles as illustrated by immunoblotting. Most importantly, BAEC treated with 100 mu g/mlof CM-EVs showed significant increases in tube formation, wound closure, and cell proliferation as compared to control (no-EVs). Finally, treatment of hiPSC-EC with CM-EVs induced increased expression of pro-angiogenic growth factors by the endothelial cells. Overall, our results demonstrated that EVs isolated from hiPSC-CM enhance angiogenesis in endothelial cells. This acellular/cell-free approach constitutes a potential translational therapeutic to induce angiogenesis in patients with myocardial infarction.