Abstract We0080: Vascular Regeneration in a Murine Model of Peripheral Artery Disease using Human GMP Compatible Embryonic Vascular Progenitor Cell Exosomes

Introduction: Peripheral artery disease (PAD) affects over 8 million individuals in the US. Biological off-the-shelf therapies to treat PAD by augmenting vascular regeneration is promising. The objective was to characterize human embryonic stem cell (hESC)-derived progenitor-exosomes and to assess their therapeutic efficacy in a murine limb ischemia model. Methods: We developed GMP-grade protocols for exosome production and purification which combine methods of tangential filtration flow and size exclusion chromatography. The particle number and size of exosomes were measured by both tunable resistive pulse sensing as well as nanoparticle tracking analysis (NTA) for comparison. Purity was assessed by measuring particle number per ug of exosome protein. Exosomes were characterized by detection of exosome surface markers and absence of cellular markers. Exosome cargo contents including miRNAs and proteins were defined by small RNA-seq and proteomic mass spectrometry analysis. The angiogenic activity of a panel of vascular progenitor cell-exosomes was assessed in vitro using live-cell imaging scratch wound and vascular tube forming assays. We further assessed the most active exosomes in a mouse model of PAD by unilateral hindlimb ischemia induction, followed by the assessment of vascular regeneration in the ischemic animals using laser Doppler spectroscopy and histological analysis of capillary density. Results: The human vascular progenitor cell derived-exosomes were highly purified, resulting in the range of 1E10-5E10 particles/µg. Progenitor exosomes were directly taken up by HUVECs and promoted the migration, proliferation, and tube formation of endothelial cells. These progenitor exosomes showed higher angiogenic potency than primary MSC- derived exosomes. The progenitor-exosomes were enriched with angiogenic miRNAs (miR-126), anti-inflammatory miRNA (miR-146), and anti-apoptotic miRNAs (miR-21). Intramuscular injection of exosomes showed significant improvement in vascular perfusion recovery and capillary density, compared to saline-treated animals (P<0.05). Conclusion: These data demonstrate the potential for using embryonic vascular progenitor cell lines as a highly scalable source of therapeutic exosomes. Our methods resulted in highly pure exosomes that retain their angiogenic activity, based on enhanced endothelial migration and proliferation in vitro, as well as augmented vascular regeneration in a murine PAD model.