Introduction: Due to the relative poor regenerative properties of cardiac tissue, acute ischemic events can lead to progressive terminal heart failure over time unless cardiac regeneration can be affected. Recent evidence using stem cell therapy in the heart demonstrates a greater role of their paracrine signaling potential, as cell survival post-transplantation is very low. A growing body of evidence demonstrates that exosomes secreted by stem cells stimulate angiogenesis, provides cytoprotection, and modulates apoptosis.
Hypothesis: The main objective of this study was to determine the angiogenic potential of exosomes isolated from human-inducible pluripotent stem cell -derived cardiomyocytes (hiPSC-CMs).
Methods: hiPSC-CMs were cultured for 48 hours (21% O2, 5% CO2) with exosome-free serum (10%). At 48 hours after the culture, media was collected and exosomes were isolated using ExoQuick-TC reagent (System Biosciences kit). Isolated exosomes were characterized by Nanosight (size and concentration), western blotting and transmission electron microscopy (TEM). Angiogenesis was assessed by tube formation assay using culturing bovine aortic endothelial cells (BAECs) on GelTrex coated plates. BAECs were treated with 50 μg and 100 μg of exosomes. Sixteen hours after the treatment, phase-contrast images were captured and analyzed for tube formation via ImageJ analysis software.
Results: The average particle size of exosomes was determined to be 76 nm (mode), compatible with the established exosomes range (40-100 nm). Western blot analysis of isolated exosomes lysate from hiPSC-CMSs demonstrated enrichment of known exosome-specific cell-surface markers (CD63 and HSP70). TEM analysis further confirmed the size and shape of the isolated exosomes. Most importantly, BAECs treated with 100 μg of exosomes showed significant (p<0.05) increases in total tube length (1.5-fold) and the number of nodes (1.4-fold), compared to an un-treated control group.
Conclusions: Overall, our results demonstrated that BAECs treated with exosomes isolated from hiPSC-CMs media enhances angiogenesis. This acellular/cell-free approach constitutes a potential new innovative future clinical therapy to induce angiogenesis in the post- ischemic heart.