Identification of a specific marker panel for human mesenchymal stem cell‐derived small extracellular vesicles

Abstract Human mesenchymal stem cell‐derived small extracellular vesicles (MSC‐sEVs) have demonstrated significant immunomodulatory and pro‐regenerative potentials. However, the lack of specific markers to define MSC‐sEVs presents a major challenge for their clinical application. Here, the proteomic datasets of MSC‐sEVs from three cell sources were synchronously analyzed, and several surface antigens commonly found on MSCs were selected as candidate markers due to their high abundances in MSC‐sEVs. Next, MSC‐sEVs from three cell sources (adipose tissue, umbilical cord, and induced pluripotent stem cells) were stained with fluorescein‐conjugated antibodies and analyzed by NanoFCM at single‐vesicle resolution. The positive rates of CD13, CD29, and CD90 all exceeded 60% across sEVs derived from three MSCs sources, whereas other candidates generally exhibited lower positive rates. The high positive rates of them were further verified in MSC‐sEVs purified via other methods. Moreover, high‐resolution microscopy, as an orthogonal method, visually validated their high presence in MSC‐sEVs. Meanwhile, none of the non‐MSC‐sEVs showed concurrent positive rates for CD13, CD29, and CD90 exceeding 40%, suggesting that this marker panel (with a positive rate threshold of 50% for all three markers) could specifically distinguish MSC‐sEVs from non‐MSC‐sEVs. Finally, the positive rates of this panel of markers were assessed in sEVs derived from MSCs at successive passages. The results revealed a progressive diminution of the positive rates of CD29 and CD90 in the sEVs secreted by MSCs with successive passages, accompanied by a reduction in the pro‐proliferative activity of these sEVs. Taken together, we have identified a specific and quantifiable marker panel for MSC‐sEVs characterization, which facilitates the development of standardized assays for defining MSC‐sEVs and ultimately accelerates the clinical translation of MSC‐sEVs.