key: cord-0730053-af2aso4s
authors: Chutipongtanate, Somchai; Kongsomros, Supasek; Pongsakul, Nutkridta; Panachan, Jirawan; Khowawisetsut, Ladawan; Pattanapanyasat, Kovit; Hongeng, Suradej; Thitithanyanont, Arunee
title: Anti‐SARS‐CoV‐2 effect of extracellular vesicles released from mesenchymal stem cells
date: 2022-03-14
journal: J Extracell Vesicles
DOI: 10.1002/jev2.12201
sha: 96f99820b0e4ce48edf9bf6461e25f3b2ff0b9a5
doc_id: 730053
cord_uid: af2aso4s

nan

25TCID50 for 2 h and then treated with MSC-EVs at different concentrations of 100, 1000 or 10,000 particles per cell for 48 h ( Figure 1g ; the detailed methods in Supplementary material). The inhibitory effect of MSC-EVs against the viral replication was measured by reducing SARS-CoV-2 nucleoprotein expression. The Cytation 7 cell imaging system (Biotek) was applied to detect the immunofluorescent signals of SARS-CoV-2 nucleoprotein-positive infected cells. Interestingly, the result showed that MSC-EVs significantly suppressed SARS-CoV-2 replication in a dose-dependent manner (Figure 1h ,i). To confirm the anti-SARS-CoV-2 activity of MSC-EVs, the culture supernatant of SARS-CoV-2 infected cells was harvested to determine the levels of infectious virion by the foci forming assay (Figure 1j ), and the copy numbers of SARS-CoV-2 spike gene by a quantitative RT-PCR ( Figure 1k ). As expected, MSC-EVs suppressed the release of SARS-CoV-2 infectious virions and their genetic materials with the dose-response relationship. Heat treatment (65 • C for 2.5 h; inactivating thermolabile molecules in the isolate) (Hettich et al., 2020) and the centrifugal filtration (100-kDa cutoff; depleting EVs from the isolate) caused a significant reduction of anti-SARS-CoV-2 effect of MSC-EVs compared to the untreated condition (Figure 1l-n; the detailed methods in Supplementary material). This evidence suggested that anti-SARS-CoV-2 activity was predominantly associated with MSC-EVs rather than soluble mediators in the isolate.

Since human umbilical cord-derived MSCs naturally do not express surface ACE2 protein (Avanzini et al., 2021; Hernandez et al., 2021) , MSC-EVs should not have the receptor decoy mode of action (to prevent the viral entry) or the promoting effect of viral infectivity (through transferring ACE2 receptors to the recipient cells). To address this issue, SARS-CoV-2 at 25TCID50 was preincubated with various dosages of MSC-EVs for 2 h, then adsorbed by Calu-3 cells for 2 h, washing and replacing the culture with the fresh medium, and maintaining the culture for a further 48 h (Supplementary Figure 2a Figure 2d) .

Taken together, we communicated that the anti-SARS-CoV-2 effect of MSC-EVs was mediated through EV-cell interaction. MSC-EVs induced SARS-CoV-2 infected lung epithelial cells to suppress viral replication and mitigate the production/release of infectious virions without the ability to modulate the viral entry process. For a mechanistic insight, we foresee that MSC-EVs released the functional cargoes to induce the antiviral defence state of SARS-CoV-2 infected lung epithelial cells, at least in part, through the interferon-stimulated genes (ISGs)-related innate immune signalling pathways (Wu et al., 2018) . From a clinical perspective, MSC-EVs inhalation therapy would deliver the anti-SARS-CoV-2 effect of MSC-EVs directly to the infected respiratory epithelial cells, together with anti-inflammatory and regenerative effects to ameliorate the outcomes in patients with COVID-19 pneumonia and ARDS. It should be acknowledged that this study focuses on one source of EVs. Future research should be conducted to systematically examine the antiviral effects of EVs from other sources, e.g., human plasma, breastmilk, or ACE2-expressed cells (Civra et al., 2021; Wang et al., 2021; Yao et al., 2018; Zhang et al., 2021) . Molecular mechanisms behind the anti-SARS-CoV-2 effects of EVs should also be determined. Preclinical and clinical studies, especially the ongoing trials of MSC-EV treatment in COVID-19 pneumonia and ARDS (ClinicalTrials.gov identifiers NCT04798716, NCT04602442), should include SARS-CoV-2 viral titters in addition to anti-inflammation and tissue regeneration endpoints. This study provided evidence to support MSC-EV investigations in the context of stem cell-free therapy for COVID-19. 

The authors report no conflict of interest. (h) The fluorescent images of SARS-CoV-2 infected Calu-3 cells. Scale bar, 2 mm. (i) The fluorescent intensity of SARS-CoV-2 nucleoprotein-positive cells (n = 3 biological replicates). The culture supernatant was subjected to viral output study and qRT-PCR. (j) Viral output was determined by the percentage of foci reduction (n = 3 biological replicates). (k) The copy number of SARS-CoV-2 spike gene normalized to 10 6 copies of β-actin was measured by qRT-PCR (n = 3 biological replicates). (l) The fluorescent images of SARS-CoV-2 infected Calu-3 cells (scale bar, 2 mm), (m) the fluorescent intensity of SARS-CoV-2 nucleoprotein-positive cells (n = 3 biological replicates), and (n) the percentage of foci reduction by viral output study (n = 3 biological replicates) after treatments with heated MSC-EVs (65 • C for 2.5 h), depleted MSC-EVs (100 kDa-cutoff centrifugal filtration) or the untreated MSC-EVs using the same starting dosage of 10,000 EVs per Calu-3 cell. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001; ns, not significant

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