Size matters: Functional differences of small extracellular vesicle subpopulations in cardiac repair responses

Cardiac progenitor cell (CPC)‐derived small extracellular vesicles (sEVs) exhibit great potential to stimulate cardiac repair. However, the multifaceted nature of sEV heterogeneity presents a challenge in understanding the distinct mechanisms underlying their regenerative abilities. Here, a dual‐step multimodal flowthrough and size‐exclusion chromatography method was applied to isolate and separate CPC‐derived sEV subpopulations to study the functional differences related to cardiac repair responses. Three distinct sEV subpopulations were identified with unique protein profiles. Functional cell assays for cardiac repair‐related processes demonstrated that the middle‐sized and smallest‐sized sEV subpopulations exhibited the highest pro‐angiogenic and anti‐fibrotic activities. Proteasome activity was uniquely seen in the smallest‐sized subpopulation. The largest‐sized subpopulation showed no effect in any of the functional assays. This research uncovers the existence of sEV subpopulations, each characterized by a distinct composition and biological function. Enhancing our understanding of sEV heterogeneity will provide valuable insights into sEV mechanisms of action, ultimately accelerating the translation of sEV therapeutics.