Surface marker expression in small and medium/large mesenchymal stromal cell-derived extracellular vesicles in naive or apoptotic condition using orthogonal techniques

Extracellular vesicles released by mesenchymal stromal cells (MSC EVs) are a promising resource for regenerative medicine. In particular, small MSC EVs represent the active EV fraction for therapeutic applications. A bulk analysis is applied to characterize MSC EVs identity and purity, coupled with the assessment of single EV morphology, size and integrity using electron microscopy. We here applied different orthogonal methods to provide a quantitative analysis of size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC EVs obtained by sequential ultracentrifugations. Bone marrow, adipose tissue, and umbilical cord MSC EVs were compared, in naive and apoptotic conditions. The 100k EV size <100 nm, as detected by electron microscopy, was confirmed by super-resolution microscopy and ExoView. Quantitative single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanin expressions, being all MSC EV fractions single, double and triple positive, in variable proportions, for CD63, CD81 and CD9. Moreover, ExoView analysis allowed a comparative multiplex screening of single MSC EV tetraspanin and mesenchymal marker levels. Finally, a semiquantitative bead based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC EV fraction. Apoptotic MSC EVs were released in higher number, without significant differences from the naive fractions in surface marker expression. These results indicate that a consistent profile of MSC EV fractions among the different MSC sources, and a safer profile of the 100k MSC EV population for clinical application. Finally, our study identified suitable applications for different EV analytical techniques.