Extracellular Vesicle Isolation and Characterization

EV isolation was performed as previously described^{72 (link)}. Briefly, human bone marrow MSCs (BM-MSCs) (ATCC PCS-500-012) and human lung carcinoma cell line A549 (ATCC CCL-185) were cultured in the presence of exosome-depleted FBS (Thermo Fisher Scientific) prior to collection of conditioned medium. Human iPSCs (ATCC ACS-1019), which were cultured in serum-free/xeno-free medium, received full media changes every day and the conditioned medium was collected and stored at 4 °C throughout expansion. Bulk conditioned medium was first spun at 2000×g and then filtered with a 0.22 µm filter to remove cellular debris and larger vesicles. Filtered medium then underwent concentration and buffer exchange via TFF (Spectrum) to isolate EVs. Samples were subsequently aliquoted and frozen at − 20 °C for downstream analysis. The size distribution and concentration of each EV sample was assessed via nanoparticle tracking analysis (NTA) using the NanoSight NS300 (Malvern Panalytical). Prior to analysis the equipment was calibrated per the manufacturer’s protocols. EV samples were diluted in sterile PBS immediately before measurement. The acquired data was analyzed by the instrument’s built-in software.
For isolation of the iPSC EVs for repeat RNA sequencing experiments, EVs were isolated following the methods described above for HIV-1 (U1) EVs.