SAFETY AND EFFICIENCY OF INHALATION METHOD FOR ADMINISTRATION OF SMALL EXTRACELLULAR VESICLES DERIVED FROM MULTIPOTENT MESENCHYMAL STROMAL CELLS OF HUMAN UMBILICAL CORD IN SARS-COV-2 ASSOCIATED PNEUMONIA.

The coronavirus infection (COVID-19), an acute viral disease with predominant affection of the upper respiratory tract, is a challenge for modern medicine. Considering the fact that in the patho-genesis of coronavirus pneumonia there is violation of the im-mune response (hyper-response, cytokine storm) the drugs that locally regulate it may be promising in the pneumonia treatment. Biological activity of exosomes is widely investigated in the world. Small extracellular vesicles of mesenchymal cells have the following effects: anti-apoptotic, proliferation stimulation, anti-inflammatory and immunomodulatory. Objective(s): to evaluate the safety and efficacy of the method of inhalation administration of small extracellular vesicles in bilateral pneumonia caused by a new SARS-CoV-2 coronavirus infection. To study these effects an interventional, prospective, random-ized, double-blind, placebo-controlled study has been conducted to evaluate the safety and efficacy of inhaled small extracellular vesicles administration to the patients with bilateral pneumonia caused by the new coronavirus infection SARS-CoV-2. Altogether 36 patients with confirmed new coronavirus infection COVID-19, complicated by bilateral pneumonia of moderate severity (12 patients each in study groups 1 and 2, depending on the type of given small extracellular vesicles, and the control group) participated in the study. Small extracellular vesicles were inhaled twice a day in the dose of 2-10x1010 particles. The efficacy and safety of the method were assessed judging by the patients' general state, as-sessment of the disease severity, general and biochemical blood tests, coagulogram, saturation, CT scan of the lungs before and after 10 days of treatment. The observation period was 30 days after hospitalization. During the study the safety of the method was proved, all the patients recovered. Reliable differences of the blood CRP index, which normalized by day 10 of treatment in groups 1 and 2, but remained elevated in the control group. No significant differences were found in other assessed parameters.Copyright © 2022, Human Stem Cell Institute. All rights reserved.

miRNAs derived from milk small extracellular vesicles inhibit porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration, and high mortality in neonatal piglets. It has caused huge economic losses to animal husbandry worldwide. Current commercial PEDV vaccines do not provide enough protection against variant and evolved virus strains. No specific drugs are available to treat PEDV infection. The development of more effective therapeutic anti-PEDV agents is urgently needed. Our previous study suggested that porcine milk small extracellular vesicles (sEV) facilitate intestinal tract development and prevent lipopolysaccharide-induced intestinal injury. However, the effects of milk sEV during viral infection remain unclear. Our study found that porcine milk sEV, which was isolated and purified by differential ultracentrifugation, could inhibit PEDV replication in IPEC-J2 and Vero cells. Simultaneously, we constructed a PEDV infection model for piglet intestinal organoids and found that milk sEV also inhibited PEDV infection. Subsequently, in vivo experiments showed that milk sEV pre-feeding exerted robust protection of piglets from PEDV-induced diarrhea and mortality. Strikingly, we found that the miRNAs extracted from milk sEV inhibited PEDV infection. miRNA-seq, bioinformatics analysis, and experimental verification demonstrated that miR-let-7e and miR-27b, which were identified in milk sEV targeted PEDV N and host HMGB1, suppressed viral replication. Taken together, we revealed the biological function of milk sEV in resisting PEDV infection and proved its cargo miRNAs, miR-let-7e and miR-27b, possess antiviral functions. This study is the first description of the novel function of porcine milk sEV in regulating PEDV infection. It provides a better understanding of milk sEV resistance to coronavirus infection, warranting further studies to develop sEV as an attractive antiviral.

Nanopore membrane chip-based isolation method for metabolomic analysis of plasma small extracellular vesicles from COVID-19 survivors.

Multiple studies showed that metabolic disorders play a critical role in respiratory infectious diseases, including COVID-19. Metabolites contained in small extracellular vesicles (sEVs) are different from those in plasma at the acute stage, while the metabolic features of plasma sEVs of COVID-19 survivors remain unknown. Here, we used a nanopore membrane-based microfluidic chip for plasma sEVs separation, termed ExoSEC, and compared the sEVs obtained by UC, REG, and ExoSEC in terms the time, cost, purity, and metabolic features. The results indicated the ExoSEC was much less costly, provided higher purity by particles/proteins ratio, and achieved 205-fold and 2-fold higher sEVs yield, than UC and REG, respectively. Moreover, more metabolites were identified and several signaling pathways were significantly enriched in ExoSEC-sEVs compared to UC-sEVs and REG-sEVs. Furthermore, we detected 306 metabolites in plasma sEVs using ExoSEC from recovered asymptomatic (RA), moderate (RM), and severe/critical COVID-19 (RS) patients without underlying diseases 3 months after discharge. Our study demonstrated that COVID-19 survivors, especially RS, experienced significant metabolic alteration and the dysregulated pathways mainly involved fatty acid biosynthesis, phenylalanine metabolism, etc. Metabolites of the fatty acid biosynthesis pathway bore a significantly negative association with red blood cell counts and hemoglobin, which might be ascribed to hypoxia or respiratory failure in RM and RS but not in RA at the acute stage. Our study confirmed that ExoSEC could provide a practical and economical alternative for high throughput sEVs metabolomic study.

SAFETY AND EFFICIENCY OF INHALATION METHOD FOR ADMINISTRATION OF SMALL EXTRACELLULAR VESICLES DERIVED FROM MULTIPOTENT MESENCHYMAL STROMAL CELLS OF HUMAN UMBILICAL CORD IN SARS-COV-2 ASSOCIATED PNEUMONIA.

The coronavirus infection (COVID-19), an acute viral disease with predominant affection of the upper respiratory tract, is a challenge for modern medicine. Considering the fact that in the patho-genesis of coronavirus pneumonia there is violation of the im-mune response (hyper-response, cytokine storm) the drugs that locally regulate it may be promising in the pneumonia treatment. Biological activity of exosomes is widely investigated in the world. Small extracellular vesicles of mesenchymal cells have the following effects: anti-apoptotic, proliferation stimulation, anti-inflammatory and immunomodulatory. Objective(s): to evaluate the safety and efficacy of the method of inhalation administration of small extracellular vesicles in bilateral pneumonia caused by a new SARS-CoV-2 coronavirus infection. To study these effects an interventional, prospective, random-ized, double-blind, placebo-controlled study has been conducted to evaluate the safety and efficacy of inhaled small extracellular vesicles administration to the patients with bilateral pneumonia caused by the new coronavirus infection SARS-CoV-2. Altogether 36 patients with confirmed new coronavirus infection COVID-19, complicated by bilateral pneumonia of moderate severity (12 patients each in study groups 1 and 2, depending on the type of given small extracellular vesicles, and the control group) participated in the study. Small extracellular vesicles were inhaled twice a day in the dose of 2-10x1010 particles. The efficacy and safety of the method were assessed judging by the patients' general state, as-sessment of the disease severity, general and biochemical blood tests, coagulogram, saturation, CT scan of the lungs before and after 10 days of treatment. The observation period was 30 days after hospitalization. During the study the safety of the method was proved, all the patients recovered. Reliable differences of the blood CRP index, which normalized by day 10 of treatment in groups 1 and 2, but remained elevated in the control group. No significant differences were found in other assessed parameters. Copyright © 2022, Human Stem Cell Institute. All rights reserved.

SAFETY AND EFFICIENCY OF INHALATION METHOD FOR ADMINISTRATION OF SMALL EXTRACELLULAR VESICLES DERIVED FROM MULTIPOTENT MESENCHYMAL STROMAL CELLS OF HUMAN UMBILICAL CORD IN SARS-COV-2 ASSOCIATED PNEUMONIA

The coronavirus infection (COVID-19), an acute viral disease with predominant affection of the upper respiratory tract, is a challenge for modern medicine. Considering the fact that in the patho-genesis of coronavirus pneumonia there is violation of the im-mune response (hyper-response, cytokine storm) the drugs that locally regulate it may be promising in the pneumonia treatment. Biological activity of exosomes is widely investigated in the world. Small extracellular vesicles of mesenchymal cells have the following effects: anti-apoptotic, proliferation stimulation, anti-inflammatory and immunomodulatory. Objective: to evaluate the safety and efficacy of the method of inhalation administration of small extracellular vesicles in bilateral pneumonia caused by a new SARS-CoV-2 coronavirus infection. To study these effects an interventional, prospective, random-ized, double-blind, placebo-controlled study has been conducted to evaluate the safety and efficacy of inhaled small extracellular vesicles administration to the patients with bilateral pneumonia caused by the new coronavirus infection SARS-CoV-2. Altogether 36 patients with confirmed new coronavirus infection COVID-19, complicated by bilateral pneumonia of moderate severity (12 patients each in study groups 1 and 2, depending on the type of given small extracellular vesicles, and the control group) participated in the study. Small extracellular vesicles were inhaled twice a day in the dose of 2–10×1010 particles. The efficacy and safety of the method were assessed judging by the patients' general state, as-sessment of the disease severity, general and biochemical blood tests, coagulogram, saturation, CT scan of the lungs before and after 10 days of treatment. The observation period was 30 days after hospitalization. During the study the safety of the method was proved, all the patients recovered. Reliable differences of the blood CRP index, which normalized by day 10 of treatment in groups 1 and 2, but remained elevated in the control group. No significant differences were found in other assessed parameters. © 2022, Human Stem Cell Institute. All rights reserved.

Comparison of viral inactivation methods on the characteristics of extracellular vesicles from SARS-CoV-2 infected human lung epithelial cells.

The interaction of SARS-CoV-2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS-CoV-2 contaminated-EV isolates in instruments located outside of the biosafety level-3 (BSL-3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet-C (UVC; 1350 mJ/cm2 ), ß-propiolactone (BPL; 0.005%), heat (56°C, 45 min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS-CoV-2-infected human lung epithelial Calu-3 cells by stepwise centrifugation, ultrafiltration and qEV size-exclusion chromatography. The EV isolates contained SARS-CoV-2. UVC, BPL and heat completely abolished SARS-CoV-2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS-CoV-2 spike detectability by quantitative RT-PCR and slightly altered EV-derived ß-actin detection. Fibroblast migration-wound healing activity of the SARS-CoV-2 contaminated-EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS-CoV-2 contaminated-EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS-CoV-2 infected cells.

Proviral role of human respiratory epithelial cell-derived small extracellular vesicles in SARS-CoV-2 infection.

Small Extracellular Vesicles (sEVs) are 50-200 nm in diameter vesicles delimited by a lipid bilayer, formed within the endosomal network or derived from the plasma membrane. They are secreted in various biological fluids, including airway nasal mucus. The goal of this work was to understand the role of sEVs present in the mucus (mu-sEVs) produced by human nasal epithelial cells (HNECs) in SARS-CoV-2 infection. We show that uninfected HNECs produce mu-sEVs containing SARS-CoV-2 receptor ACE2 and activated protease TMPRSS2. mu-sEVs cleave prefusion viral Spike proteins at the S1/S2 boundary, resulting in higher proportions of prefusion S proteins exposing their receptor binding domain in an 'open' conformation, thereby facilitating receptor binding at the cell surface. We show that the role of nasal mu-sEVs is to complete prefusion Spike priming performed by intracellular furin during viral egress from infected cells. This effect is mediated by vesicular TMPRSS2 activity, rendering SARS-CoV-2 virions prone to entry into target cells using the 'early', TMPRSS2-dependent pathway instead of the 'late', cathepsin-dependent route. These results indicate that prefusion Spike priming by mu-sEVs in the nasal cavity plays a role in viral tropism. They also show that nasal mucus does not protect from SARS-CoV-2 infection, but instead facilitates it.

Mesenchymal stem cells and their derived small extracellular vesicles for COVID-19 treatment.

Since December 2019, the coronavirus (COVID-19) pandemic has imposed huge burdens to the whole world, seriously affecting global economic growth, and threatening people's lives and health. At present, some therapeutic regimens are available for treatment of COVID-19 pneumonia, including antiviral therapy, immunity therapy, anticoagulant therapy, and others. Among them, injection of mesenchymal stem cells (MSCs) is currently a promising therapy. The preclinical studies and clinical trials using MSCs and small extracellular vesicles derived from MSCs (MSC-sEVs) in treating COVID-19 were summarized. Then, the molecular mechanism, feasibility, and safety of treating COVID-19 with MSCs and MSC-sEVs were also discussed.

Anti-Viral Activities of Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Against Human Respiratory Viruses.

The endemic and pandemic caused by respiratory virus infection are a major cause of mortality and morbidity globally. Thus, broadly effective antiviral drugs are needed to treat respiratory viral diseases. Small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (U-exo) have recently gained attention as a cell-free therapeutic strategy due to their potential for safety and efficacy. Anti-viral activities of U-exo to countermeasure respiratory virus-associated diseases are currently unknown. Here, we tested the antiviral activities of U-exo following influenza A/B virus (IFV) and human seasonal coronavirus (HCoV) infections in vitro. Cells were subject to IFV or HCoV infection followed by U-exo treatment. U-exo treatment significantly reduced IFV or HCoV replication and combined treatment with recombinant human interferon-alpha protein (IFN-α) exerted synergistically enhanced antiviral effects against IFV or HCoV. Interestingly, microRNA (miR)-125b, which is one of the most abundantly expressed small RNAs in U-exo, was found to suppress IFV replication possibly via the induction of IFN-stimulated genes (ISGs). Furthermore, U-exo markedly enhanced RNA virus-triggered IFN signaling and ISGs production. Similarly, human nasal epithelial cells cultured at the air-liquid interface (ALI) studies broadly effective anti-viral and anti-inflammatory activities of U-exo against IFV and HCoV, suggesting the potential role of U-exo as a promising intervention for respiratory virus-associated diseases.

Inhalation of MSC-EVs is a noninvasive strategy for ameliorating acute lung injury.

Mesenchymal stem cell-derived small extracellular vesicles (MSC-EVs) are promising nanotherapeutic agent for pneumonia (bacterial origin, COVID-19), but the optimal administration route and potential mechanisms of action remain poorly understood. This study compared the administration of MSC-EVs via inhalation and tail vein injection for the treatment of acute lung injury (ALI) and determined the host-derived mechanisms that may contribute to the therapeutic effects of MSC-EVs in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (macrophage cell line) and animal models. Luminex liquid chip and hematoxylin and eosin (HE) staining revealed that, compared with the vehicle control, inhaled MSC-EVs outperformed those injected via the tail vein, by reducing the expression of pro-inflammatory cytokines, increasing the expression of anti-inflammatory cytokine, and decreasing pathological scores in ALI. MSC-EV administration promoted the polarization of macrophages towards a M2 phenotype in vitro and in vivo (via inhalation). RNA sequencing revealed that immune and redox mediators, including TLR4, Arg1, and HO-1, were associated with the activity MSC-EVs against ALI mice. Western blotting and immunofluorescence revealed that correlative inflammatory and oxidative mediators were involved in the therapeutic effects of MSC-EVs in LPS-stimulated cells and mice. Moreover, variable expression of Nrf2 was observed following treatment with MSC-EVs in cell and animal models, and knockdown of Nrf2 attenuated the anti-inflammatory and antioxidant activities of MSC-EVs in LPS-stimulated macrophages. Together, these data suggest that inhalation of MSC-EVs as a noninvasive strategy for attenuation of ALI, and the adaptive regulation of Nrf2 may contribute to their anti-inflammatory and anti-oxidant activity in mice.