ABSTRACT
Allogeneic regenerative cell therapy has shown surprising results despite lack of engraftment of the transplanted cells. Their efficacy was so far considered to be mostly due to secreted trophic factors. We hypothesized that extracellular vesicles (EVs) can also contribute to their mode of action. Here we provide evidence that EVs derived from therapeutic placental-expanded (PLX) stromal cells are potent inducers of angiogenesis and modulate immune cell proliferation in a dose-dependent manner.
Crude EVs were enriched >100-fold from large volume PLX conditioned media via tangential flow filtration (TFF) as determined by tunable resistive pulse sensing (TRPS). Additional TFF purification was devised to separate EVs from cell-secreted soluble factors. EV identity was confirmed by western blot, calcein-based flow cytometry and electron microscopy. Surface marker profiling of tetraspanin-positive EVs identified expression of cell-and matrix-interacting adhesion molecules. Differential tandem mass tag proteomics comparing PLX-EVs to PLX-derived soluble factors revealed significant differential enrichment of 258 proteins in purified PLX-EVs involved in angiogenesis, cell movement and immune system signaling. At the functional level, PLX-EVs and cells inhibited T cell mitogenesis. PLX-EVs and soluble factors displayed dose-dependent proangiogenic potential by enhancing tube-like structure formation in vitro.
Our findings indicate that the mode of PLX action involves an EV-mediated proangiogenic function and immune response modulation that may help explaining clinical efficacy beyond presence of the transplanted allogeneic cells.
Footnotes
Authorship Contributions: Conception and design: M Wolf and D Strunk.
Development of methodology: M Wolf, B Vari, AM Raninger, A Spittler, D Strunk
Acquisition of data: M Wolf, B Vari, C Blöchl, AM Raninger, CM Beez, A Hoog, E Eminger, G Brachtl, T Heuser, M Oeller, A Spittler
Analysis and interpretation of data: M Wolf, B Vari, C Blöchl, R Poupardin, HM Binder, M Seifert, G Brachtl, D Strunk
Writing of the manuscript: M Wolf and D Strunk
Review and/or revision of the manuscript: K Schallmoser, M Seifert, CG Huber, H-D Volk
Administrative, technical, or material support: R Ofir Study supervision: Dirk Strunk
Conflict of interest and funding: The authors have declared no conflict of interest. This work was supported by funding from the European Union’s Horizon 2020 research and innovation program (grant no. 668724 to DS and no. 731377 to KS) and a technology transfer funding EV-TT (grant no. P1812596) of the Government of the region of Salzburg (to DS & KS).
