Nanosized UCMSC-derived extracellular vesicles exclusively inhibit the inflammatory response of stimulated T cells

Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data.

Here, researchers from the Health Science Research Institute Germans Trias i Pujol used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC’s paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, these results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect.

EVs were successfully isolated by Size-Exclusion Chromatography

A: Scheme of the methodological procedure followed for the generation of the different study fractions obtained from UCMSC 48h-culture: supernatant was cleared of debris by centrifugation to obtain conditioned media (CM); concentrated CM (CCM) and eluted CM (ECM) were collected after ultrafiltration; CCM was loaded to the SEC column and fractions collected. B-C: UCMSC-EVs were found on fractions 5-7 while protein eluted after fraction 8 on both samples coming from unconditioned (B) and IFNγ-conditioned UCMSCs (C). SEC eluted fractions were checked for EV markers (CD9 and CD63), MSC markers (CD73 and CD90) by bead-based flow cytometry (left axis). Protein elution was monitored by absorption at 280nm (right axis). D-E: Cryo-EM images confirmed UCMSC-EVs presence in pooled EV fractions (F5-7) of unconditioned (D) and IFNγ-conditioned SEC preparations (E). Images of 20,000x and 30,000x magnifications are shown, with 200nm and 100nm scale bars, respectively. F: Box plot of the fold increase in protein content, CD9 and CD63 MFI of pooled EV fractions obtained from IFNγ-primed UCMSCs relative to unconditioned UCMSCs. Medians of ten independent experiments are depicted as horizontal bars, outliers as points. G: CD73, CD90, HLA-ABC (MHC-I) and HLA-DR (MHC-II) expression on EVs from unconditioned (white dots) and corresponding IFNγ-primed MSCs (black dots) are shown normalized to their CD9 MFI. Each dot corresponds to an independent experiment (n=10 and 6).