Anti‐SARS‐CoV‐2 effect of extracellular vesicles released from mesenchymal stem cells

As of 10 December 2021, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 accounted for 267 million people with up to 5.3 million deaths worldwide (https://covid19.who.int). Since late 2019, much progress has been made in response to the COVID-19 pandemic, including the rapid developments of effective vaccines and the treatment guidelines consisting of antiviral drugs, immunomodulators, and critical care support (https://covid19.who.int). However, SARS-CoV-2 evolves over time as its genome has a highmutation rate that leads to reasonable concerns of breakthrough infection due to immune escape and resistant strain emergence under antiviral pressure (Lipsitch et al., 2021; Szemiel et al., 2021). A newly emergingOmicron (B.1.1.529) variant rings alarms around the globe that, perhaps, the COVID-19 war has just begun. Relentless efforts should be made to advance our knowledge and treatment regimens against COVID-19. These included studies of mesenchymal stem cell (MSC) therapy that aimed to mitigate cytokine storm and promote tissue repair in severely ill patients with COVID-19 pneumonia and acute respiratory distress syndrome (ARDS) (Hashemian et al., 2021; Meng et al., 2020; Zhu et al., 2021). Nevertheless, as extensively discussed in a recent review by Dr. Phillip W. Askenase of Yale University School of Medicine, the immunomodulatory and regenerative effects ofMSC therapy aremediated throughMSC-derived extracellular vesicles (MSC-EVs) (Askenase, 2020), while the use of MSC-EVs has less safety concerns of thromboembolism, arrhythmia and malignant transformation. In this direction, MSC-EV investigations for COVID-19 treatment would be more appealing and undeniable if MSC-EVs also exhibit anti-SARSCoV-2 effects. A previous study revealed that MSC-EVs pertained antiviral activity against influenza virus in a preclinical model (Khatri et al., 2018). It is known that MSCs are highly resistant to viral infections (Wu et al., 2018), including SARS-CoV-2 (Avanzini et al., 2021). We, therefore, hypothesized that the EVs released fromMSCs could inhibit SARS-CoV-2 infection. Accordingly, we applied in vitro anti-SARS-CoV-2 assays, which were previously developed by our group (Kanjanasirirat et al., 2020; Kongsomros et al., 2021; Sa-Ngiamsuntorn et al., 2021), to determine the dose dependent anti-SARS-CoV-2 effect of MSCEVs. In this study, MSC-EV was referred to as the exosome (or small EV) subpopulation of extracellular vesicles. Full details of the methods used in this study were provided in Supplementary material. MSC-EVs were isolated from 100 ml culture media (supplemented with exosome-depleted foetal bovine serum) of human umbilical cord-derived MSCs (ATCC®PCS-500-010) by the combination of step-wise centrifugation and 0.2 μm filtration (to remove microvesicles and apoptotic bodies), the 100-kDa cutoff ultrafiltration (to concentrate the culture medium), and the Izon qEV size exclusion chromatography (to separate extracellular vesicles from soluble proteins) (Figure 1a). The final volume of the MSC-EV isolate was 500 μl. Ten aliquots (50 μl each) were made before −80C storage, and one freeze-thaw cycle was allowable for each aliquot. The particle and protein evidence were validated to confirm MSC-EV presence in the isolate as per the International Society of Extracellular Vesicles (ISEV) guideline (Théry et al., 2018), with the extended validation of functional evidence. Transmission electronmicroscopy with negative staining showedMSC-EVs as cup-shaped vesicles with approximately 100 nm in diameter (Figure 1b). Nanoparticle tracking analysis demonstrated the enrichment of small extracellular vesicles with the diameter of 96.7 ± 44.4 nm in the isolate, whereas the amount of large extracellular vesicles with the diameter > 200 nm was minute (Figure 1c). Western blot analysis confirmed the presence of exosome markers (i.e., Alix, Tsg101, Hsp70, CD9) and the MSC-specific surface marker CD105, with the absence of EV negative control markers including GM130 (a Golgi marker), calnexin (an ER marker), and cytochrome C (Cyt-C, a mitochondrial marker), in the MSC-EV isolate compared to its original cell lysate (Figure 1d; the full-length blot images were provided in Supplementary Figure 1). Since the regeneration and repair capabilities of MSC-EVs are well documented (Hade et al., 2021), we then performed the wound healing assay to confirm that MSC-EVs in the isolate retained bioactivities. At 24-h, MSC-EVs promoted HEK293 fibroblast cell migration to close the scratch wound in a dose-dependentmanner (Figure 1e,f). Validations of particle, protein, and functional evidence ofMSC-EVs supported the use of this MSC-EV isolate to evaluate their potential activity against SARS-CoV-2 infection. Calu-3 cells (ATCC®HTB-55), a legitimate model of human lung epithelial cells for SARS-CoV-2 study (Hoffmann et al., 2020), were infected with live SARS-CoV-2 virus (SARS-CoV-2/01/human/Jan2020/Thailand; GenBank ID: QYZ85362.1) at