Rethinking miRNAs in MSC-sEV therapeutics: implications for manufacture, mechanism of action, and development of...

Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) have emerged as a promising cell-free alternative to MSC-based therapies, offering superior safety, scalability, and stability profiles. These nanosized vesicles are now widely regarded as the principal therapeutic effectors of MSCs, capable of recapitulating many of the benefits attributed to their parental cells. However, their successful clinical translation depends on overcoming key challenges, particularly those related to product variability, viral safety, and the definition of mechanistically relevant potency-associated critical quality attributes (CQAs). This review explores the sources of MSC-sEV variability, including MSC tissue origin, manufacturing parameters, and limitations associated with primary and pluripotent stem cell-derived MSCs. The use of immortalized monoclonal MSC lines is discussed as a potential solution to improve batch consistency. Regulatory frameworks such as the International Council for Harmonisation (ICH) guideline Q5A(R2) are also highlighted for ensuring viral safety in sEV manufacturing processes. A major focus is the critical evaluation of microRNAs (miRNAs) - long regarded as leading candidates for potency CQAs in MSC-sEV products. Despite their prevalence in the extracellular vesicle literature, mounting evidence challenges their functional relevance in therapeutic contexts. Studies consistently show that miRNAs are underrepresented in sEVs, occur at very low copy numbers, and lack essential components (e.g., Argonaute proteins) required for canonical RNA interference. Moreover, the efficiency of EV internalization and endosomal escape remains exceedingly low, rendering miRNA-based gene regulation mechanistically implausible at physiologically relevant doses. These findings call into question the widespread assumption that miRNAs are primary effectors of MSC-sEV activity.