Abstract 21281: Cardiosphere-Derived Cell and Mesenchymal Stem Cell Extracellular Vesicles Contain Distinct RNA Cargo

Abstract

Introduction: Cardiosphere-derived cells (CDCs) possess regenerative, immunomodulatory and cardioprotective characteristics when delivered to the heart post-myocardial infarction (MI). CDCs derived from different donors possess variable regenerative potency in a MI model. We previously demonstrated that human CDC-derived extracellular vesicles (CDC-EVs) recapitulate the effects of CDCs in acute and chronic in vivo MI models. We are testing the hypothesis that a distinct cargo profile defines the functional efficacy between CDC-EVs and mesenchymal stem cell-derived EVs (MSC-EVs). Also, we assessed the immunomodulatory role of CDC-EVs derived from potent and non-potent CDCs in comparison with MSC-EVs. Methods: CDCs or MSCs were cultured in serum-free medium for 15 days and the conditioned media was concentrated by ultrafiltration to isolate EVs. The miRNeasy Serum/Plasma kit was used to extract total RNA from EVs followed by small RNA sequencing (NextSeq 500, Illumina) (CDC-EVs, n =12; MSC-EVs, n =4). We performed an in vitro and in vivo macrophage assay to assess the immunomodulatory role of EVs. Results: RNA sequencing analysis of CDC-EVs and MSC-EVs revealed a greater overall abundance of Y-RNA fragments and miRNA in CDC-EVs. Examining the specific Y-RNA classes, we found that MSC-EVs contained an expression profile with lower Y4 (p<0.05) but higher Y5 (p<0.05) Y-RNA fragments. Four miRNAs (miR-146a, miR-151, miR-409, miR-423) were highly enriched in CDC-EVs while miR-10a and miR-4792 were more abundant in MSC-EVs. EVs from potent CDCs showed a stronger upregulation of anti-inflammatory genes in activated macrophages ( in vitro ) and prevented accumulation of macrophages ( in vivo ) when compared with EVs obtained from non-potent CDCs or MSCs. Conclusions: We demonstrated that CDC-EVs contain a unique RNA cargo set that can differentiate CDC-EVs from MSC-EVs. The higher presence of Y-RNA in CDC-EVs as compared to MSC-EVs could be responsible for the ability of CDCs to regulate stem cell activation and tissue regeneration. The highly enriched panel of miRNAs observed in CDC-EVs and the ability to reduce the inflammatory reaction by macrophages, in contrast to MSC-EVs, suggest that they may support some of the functional benefits observed post-MI.