Synergy effects of extracellular vesicles from human embryonic stem cells and their-derived cardiac lineage cells in cardiac repair

Introduction: Myocardial infarction (MI) and resulting heart failure (HF) are leading causes of death worldwide. Existing therapies are challenging to salvage the damaged myocardium and prevent the progression of HF following MI. Recent preclinical and clinical studies indicate that human pluripotent stem cells (hPSCs)-derived cardiovascular progenitor cells (CVPCs) and cardiomyocytes (CMs) hold great promise for promoting repair of infarcted hearts. Their beneficial effects are largely mediated by paracrine mechanisms, including the secreted extracellular vesicles (EVs). However, the characteristics of EVs from hPSCs, CVPCs, and CMs have not yet been fully clarified and whether they have synergistic effects remain unclear. This study aims to investigate the properties of the EVs from these cell types and their synergistic effects of mixed EVs for repair of infarcted hearts by a side-by-side comparison.
Methods: H9 human embryonic stem cells (hESCs) were cultured in mTeSR1 medium and differentiated into hCVPCs and hCMs using chemically defined protocols. The EVs were isolated from culture supernatants via differential ultracentrifugation with a sucrose cushion and characterized by NanoSight, transmission electron microscopy, Western blot, and LC-MS/MS for proteomics. MI was induced in C57BL/6 mice via left anterior descending artery ligation, followed by EV injections into infarct border zones. Cardiac function was assessed using echocardiography. Infarct sizes and histological changes were evaluated with Masson’s trichrome and immunohistochemical staining. Proteomic and phosphoproteomic analyses were performed using cardiac tissues.
Results: The EVs derived from hESCs, hCVPCs, and hCMs showed comparable cardioprotective effects in a murine MI model, characterized by improving cardiac function and reducing scar formation. Notably, these beneficial effects were significantly enhanced by the mixed EVs from the three type cells. Proteomic analysis revealed unique and shared protein cargo among the EVs, reflecting their cellular origins and roles in cardioprotective pathways, including angiogenesis, anti-inflammation, and anti-apoptosis. Comparison of proteomics and phosphoproteomics of the infarcted heart tissues identified dominant proteins and pathways involved in cell survival, angiogenesis, and immune regulation in the mixed EVs group. Further analysis, combined with gene manipulation, revealed a cardiac repair network and mediators of cardioprotective effects in the mixed EVs.
Conclusions: The EVs from hESCs, hCVPCs, and hCMs exhibit their unique properties and the mixed EVs derived from three of them demonstrate synergistic cardioprotective effects compared with these from the EVs of single type cells for repair of infarcted hearts. These findings suggest a therapeutic potential by complementation of different type EVs in ischemic heart disease.