PROJECT SUMMARY Studies have demonstrated that adult stem cells such as mesenchymal stem cells (MSCs) repair myocardial infarction (MI) or ischemia/reperfusion (I/R) injury by indirect paracrine mechanisms rather than by differentiation and tissue replacement. In the past decade, extracellular vesicles (EVs) have emerged as promising cell-free agents for treating ischemic injury. Several exosome-based therapeutic companies have launched early phase clinical trials. However, the large-scale production of EVs remains a challenge. We seek to develop a scale-up friendly method to generate a large number of therapeutic nanovesicles (NVs) from MSCs by extrusion. Passing through a series of extrusion microfilters, the MSCs can be converted into billions of NVs. Those engineered cell vesicles serve as “cell-drones” to deliver biological information to the recipient cells. Companies such as MDImune and SQZ Biotech are conducting clinical trials using extruded nanovesicles in the area of oncology and infectious diseases. We wonder whether those cell-drones would carry the regenerative materials from MSCs and can promote tissue repair in a way similar or superior to naturally secreted EVs. In this proposed study, we plan to investigate the fabrication, characterization and toxicity of NVs (Preliminary study). After that, we will test the therapeutic effect of NVs on a mouse model (AIM 1) and a porcine model (AIM 2) with cardiac I/R injury. Then, we will elucidate the mechanisms underlying the therapeutic benefits of MSC NVs and create “super” NVs with enhancement cargos (AIM 3). This leads to our central hypothesis that extrusion is a highly efficient method to generate a large quantity of therapeutic NVs that can potentially be manipulated, and may replace EVs in regenerative medicine applications.