Characterization of extracellular vesicles derived from two populations of human placenta derived mesenchymal stem/stromal cells

Abstract

Background & Aim Growing evidence shows that extracellular vesicles (EVs) are responsible for the therapeutic effect of stem cell therapy. EVs are a promising, potential cell-free therapy in regenerative medicine. The human placenta is a rich source of mesenchymal stem/stromal cells (MSCs). The first aim of this study was to isolate and characterize EVs derived from two well-characterised placental hTERT-transformed MSC lines; chorionic MSC line (CMSC29) and decidual MSC line (DMSC23). The second aim was to analyse the protein, lipid and small RNA content of isolated EVs from both cell lines, and to assess their biological activities. Methods, Results & Conclusion EVs were isolated and manufactured using the proprietary LEAP technology of Exopharm Ltd. Isolated EVs were then characterized at the biophysical level by microfluidic resistive pulse sensing (MRPS, particle size and number), transmission electron microscopy (morphology), and Western blot (EVs markers). The protein and lipid contents were analysed by mass spectrometry and the nucleic acid content by RNA sequencing. In addition, the biological activities of purified EVs were evaluated in a BioMAP® Diversity PLUS® screen and in vitro cell proliferation and migration assays. Spectradyne analysis showed that LEAP-purified EVs from CMSC29 had higher concentration of particles compared with DMSC23-derived EVs. Protein mass spectrometry analysis identified a large number of proteins which were mainly associated with binding. Traditional EV marker proteins were identified, including CD9 (DMSC-derived EV) and CD81 (CMSC-derived EV), and CD44 a marker for MSC was also identified on the DMSC23-derived EVs. There were several growth factors identified in both DMSC23- and CMSC29-derived EVs including fibroblast growth factor 2 (FGF2), insulin, and connective tissue growth factor. Lipid analysis identified the major species to be phosphatidylcholine, triacylglycerides and diacylglycerides in both DMSC23- and CMSC29-derived EVs. The nucleic acid content of both groups has been sequenced and bioinformatic analysis is currently underway. The EVs from both cell sources demonstrated biological activity in the BioMAP® Diversity PLUS® screen as well as by stimulating the proliferation and migration of dermal fibroblasts in in vitro assays. In conclusion, these data revealed differences between LEAP-purified EVs from human placenta-derived MSC cell lines DMSC23 and CMSC29, which may be potentially useful for different disease models.