Stem Cell Extracellular Vesicles as Anti-SARS-CoV-2 Immunomodulatory Therapeutics: A Systematic Review of Clinical and Preclinical Studies

Background COVID-19 rapidly escalated into a worldwide pandemic with elevated infectivity even from asymptomatic patients. Complications can lead to severe pneumonia and acute respiratory distress syndrome (ARDS), which are the main contributors to death. Because of their regenerative and immunomodulatory capacities, stem cells and their derived extracellular vesicles (EVs) are perceived as promising therapies against severe pulmonary conditions, including those associated with COVID-19. Herein, we evaluate the safety and efficacy of stem cell EVs in treating COVID-19 and complicating pneumonia, acute lung injury, and ARDS. We also cover relevant preclinical studies to recapitulate the current progress in stem cell EV-based therapy. Methods Using PubMed, Cochrane Central Register of Controlled Trials, Scopus, and Web of Science, we searched for all English-language published studies (2000–2023) that used stem cell EVs as a therapy for COVID-19, ARDS, or pneumonia. The risk of bias (ROB) was assessed for all studies. Results Forty-eight studies met our inclusion criteria. Various-sized EVs derived from different types of stem cells were reported as a potentially safe and effective therapy to attenuate the cytokine storm induced by COVID-19. EVs alleviated inflammation and regenerated the alveolar epithelium by decreasing apoptosis, proinflammatory cytokines, neutrophil infiltration, and M2 macrophage polarization. They also prevented fibrin production and promoted the production of anti-inflammatory cytokines and endothelial cell junction proteins. Conclusion Similar to their parental cells, stem cell EVs mediate lung tissue regeneration by targeting multiple pathways and thus hold promise in promoting the recovery of COVID-19 patients and improving the survival rate of severely affected patients. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s12015-023-10675-2.

Upon infection with SARS-CoV-2, the patient's immune system induces an inflammatory "cytokine storm" to defeat the virus.This response can also result in damage and aggravate other conditions, including interstitial pneumonia, acute respiratory distress syndrome (ARDS), multiple organ failure, or even death, depending on many factors, including the strength of the patient's immune system [4,5].In an early study, ARDS was reported in 42% of COVID-19 patients, and 61-81% of the total cohort needed intensive care due to severe hypoxemia that required mechanical ventilation [6].Affected lung tissue displayed endothelial damage with severe inflammation, poor pulmonary oxygenation, increased vascular permeability, and pulmonary interstitial fibrosis [7].ARDS is thus considered more severe in COVID-19 patients and even results in post-COVID-19 pulmonary fibrosis in some survivors [8].
Multiple treatments for COVID-19 have been implemented or proposed.Some treatments, such as repurposed anti-malarial and anti-viral drugs, may improve recovery and survival rates but do not regenerate damaged lung tissue [9].In contrast, stem cell therapy was proposed as a COVID-19 approach specifically because of promising regenerative capacities for a plethora of cardiovascular [10], degenerative [11], and lung diseases [12].The therapeutic effects of stem cells are attributed to anti-inflammatory, immunomodulatory, regenerative, pro-angiogenic, and anti-fibrotic properties, along with a vast variety of potential sources [13].Stem cells may be especially attractive as COVID-19 treatments since early passages do not display the SARS-CoV-2 receptor (angiotensin-converting enzyme 2 (ACE2)), rendering them resistant to infection [14].Different stem cells and their secretomes have thus been tested in preclinical and clinical settings to combat COVID-19 complications [15,16].For example, umbilical cord, adipose and bone marrow-derived mesenchymal stem cells have successfully ameliorated the cytokine storm by regulating immune cells such as macrophages, neutrophils, B and T cells, DCs, and natural killer cells [17][18][19][20].
Stem cell therapy faces the challenges of finding proper tissue matching, the use of immune suppressive regimens, and the complications of graft rejection or graft vs. host disease.Extracellular vesicles (EVs) may thus present a viable alternative, as they provide many of the stem cell regenerative properties and avoid the complications of whole-cell therapy.EVs are small lipid bilayer nanovesicles of different sizes and origins that are released by live cells and possess the same immunomodulatory and regenerative properties as their parental cells [21,22].Since cell-based therapies also face application hurdles, including large-scale production and the limitations of reconstituting cryopreserved cells, EVs are shifting regenerative medicine from cellular to acellular therapy [23,24].In addition to advantages such as low immunogenic and teratogenic properties, EVs have been reported to trigger anti-inflammatory cytokine release and reduce inflammation [24,25].In different lung injury models, EVs are thought to function by shuttling microRNA, mRNA, DNA, proteins, and metabolites to and/or into recipient cells in injured tissue, thereby promoting repair and regeneration [26].At the time of this writing, at least 28 clinical trials had been registered worldwide to investigate the safety and/or efficacy of stem cell EVs for ARDS and/or pneumonia in patients critically ill with COVID-19 (ClinicalTrials.gov,Chinese Clinical Trial Register (ChiCTR), IRCT, ISRCTN Registry, EU Clinical Trials Register, last accessed: 9th of September 2022).Amidst this growing interest in EV therapies, we aim in this systematic review to assess the immunomodulatory effects and efficacy of stem cell EVs in treating severe pulmonary conditions associated with COVID-19, such as acute lung injury (ALI), ARDS, and severe pneumonia in humans.The review also covers preclinical studies in induced animal models and recapitulates the current progress in stem cell EV-based therapy.

Literature Search Strategy
A defined literature search was conducted by three authors independently using the following databases: PubMed, Scopus, Web of Science, and Cochrane Central Register of Controlled Trials.The following MeSH headings and keywords were used: "extracellular vesicles", "exosomes", "microvesicles", "COVID-19", "SARS-CoV-2", "coronavirus", "acute respiratory distress syndrome", "acute lung injury", "pneumonia", and "stem cells".Studies published from 2000 until May 31, 2023, were included.This review was reported using the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement [27] and was registered on the international prospective register of systematic reviews (PROSPERO; CRD42022335053 and CRD42022336501) [28].

Inclusion Criteria
We included all in vitro studies, preclinical in vivo animal studies, and clinical interventional studies of stem cell EVs of various designations (extracellular vesicles, microvesicles, and exosomes) from any tissue source (bone marrow, adipose, umbilical cord, dental pulp, placenta, etc.) used as an intervention to treat COVID-19, ARDS and/ or lung injury.We included studies that used syngeneic, allogeneic, or xenogeneic cells as the secretome source.Studies that were fully accessible and published in English were included.The PICO search strategy is provided as supplemental file 1.

Exclusion Criteria
We excluded studies that (1) administered only nonstem cell-based therapeutics to treat COVID-19, such as cell therapy using somatic cells other than stem cells, antiviral, immunomodulatory, and anti-cytokine drugs or a combination thereof; (2) did not report EV isolation methods; (3) were conducted in silico only; (4) were on pulmonary fibrosis, asthma or other respiratory conditions that were not directly related to COVID-19; (5) were review articles, meta-analyses, comments, notes, book chapters or surveys and theses, conference proceedings and editorials; and (6) used EVs to treat organs other than the lungs.

Study Selection and Data Extraction
Using the inclusion and exclusion criteria, the title and abstract were initially screened independently by SA, MA, and AG via Rayyan.ai(https:// rayyan.qcri.org/), an online platform for the study selection process.Differences in opinion or discrepancies were resolved by discussion and consultation with the NB.All duplicated studies were checked and removed before the study selection process.Full-text articles were retrieved by three authors independently to assess the final eligibility.Data extraction and subsequent full-text review were performed using an Excel data extraction form to search for data in the Results and Method sections as well as in tables, graphs, and figures.Cross-checking of the data retrieved for each article was performed by the other authors.Specific data extracted from studies included study characteristics (e.g., lead author, year of publication, country), study design, and intervention characteristics (e.g., sample size, source of stem cell, reported size and type of EV, EV separation and characterization methods, mode of administration, EV dosage, and time of assessment).All data about the primary and secondary outcomes (e.g., survival rate, lung injury score, oxygenation level, days in ICU, inflammatory cytokine concentrations, CT, laboratory and radiologic findings, and adverse events) were also recorded.

Quality Assessment
We used the Cochrane Bias Risk Assessment tools to assess the quality of the included studies, where the risk of bias was based on the following criteria: random sequence generation (selection bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), allocation concealment (selection bias), blinding of participants and researchers (performance bias), selective reporting (reporting bias) and other bias.The RoB-2 tool for randomized studies was used to assess the risk of bias for the interventional controlled studies [30].The ROBINS-I tool for nonrandomized studies was used to assess the risk of bias for the interventional controlled studies [31].Additionally, we used the systematic review center for laboratory animal experimentation (SYRCLE) risk of bias tool to evaluate the risk of bias in preclinical studies [32].The Confidence in the Evidence from the Reviews of Qualitative research (CERQual) tool was used to assess the evidence quality of each outcome in the systematic review [33].A PRISMA figure following the PRISMA checklist criteria was created [27].

Literature Search
A search on PubMed, Web of Science, Scopus, and Cochrane Central Register of Controlled Trials retrieved a total of 1351 studies.After removing duplicate publications, the title and/ or abstract of 700 studies were screened to include articles that assessed the efficacy and/or safety of stem cell EVs in combating COVID-19 or severe pulmonary conditions such as ARDS, ALI, and pneumonia.We excluded 625 studies that were review articles, meta-analyses, comments, news, book chapters, surveys, theses, conference papers, and/or editorials.These studies were also excluded because they did not match the outcome of interest (e.g., lung injury models not relevant to COVID-19, such as bronchopulmonary dysplasia, cystic fibrosis, and asthma) or the treatment criteria (e.g., stem cell EVs employed as biomarkers and not for treatment) or were reported in languages other than English.Thus, the remaining 75 articles were assessed for eligibility via full-text screening.Thirty-seven studies that did not characterize EVs by size and/ or at least one protein marker were excluded, and 48 studies were eventually retained for final analysis (Fig. 1) .The selected studies were published between 2000 and 2023 and included in vitro, ex vivo, in vivo, and human subjects and/ or human-origin materials.These studies targeted COVID-19, ALI/ARDS, pneumonia, or allergic airway inflammation.A list of excluded articles is provided in supplemental file 2.

Study Characteristics
The 48 studies remaining after the application of the inclusion and exclusion criteria included seven studies of human patients or tissues.The 48 studies were targeting ALI (75%), SARS-CoV-2 (20.8%),ARDS (14.6%), and pneumonia (4.2%) (Fig. 2A).Four studies targeted severely ill COVID-19 patients.Two studies examined mild to moderate COVID-19 patients or long-haul patients via two FDA-approved (Exoflo and Zofin) treatments.One study used an Escherichia coli-induced model of severe pneumonia in ex vivo perfused human lungs.To model COVID-19 or ALI/ARDS in experimental animals, inducing agents including lipopolysaccharide (LPS), Escherichia coli, endotoxin, Pseudomonas aeruginosa, histones, bleomycin, burn injury, influenza virus injection, ovalbumin, cytokine exposure, trauma or mechanical ventilation injury.These studies primarily evaluated the therapeutic efficacy and safety of EVs derived from various sources as a cell-free therapy for recovery from lung injury.The major sources for these EVs were MSCs derived from human or animal bone marrow, adipose tissue, umbilical cords, amniotic fluid, and Wharton's jelly.However, other EV sources, such as placenta, endothelial progenitor cells, neuronal stem cells, human neonatal fibroblasts, menstrual blood, and IPCS were included (Fig. 2B).All but one in vivo study (of commercial pigs as a large animal model) was carried out in mice or rats.Table 1 shows the reported characteristics of stem cell EVs in all selected studies.

Stem Cell-EV Intervention Characteristics
Twenty-two studies out of the forty-eight selected studies used EV terminology based on the guidelines of the International Society for Extracellular Vesicles (ISEV) [82].
Table 1 shows the separation, characterization, reported nomenclature and size, dosage, and mode of administration of EVs, along with the time of assessment, for all studies.Separation methods included ultracentrifugation (the most common procedure, 66.7%), commercially available kits based on precipitation reagents (14.6%), size exclusion or anion exchange chromatography (6.3%), ultrafiltration (UF) (2.8%), combined methods including UC with UF (4.2%), UC with sucrose cushion (4.2%), and UC with PEG (2.8%) (Fig. 2C).Likewise, as recommended by ISEV, various characterization methods were involved, including transmission (75%) or scanning electron microscopy (10.4%), particle tracking analysis (66.7%), western blotting (52.1%), flow cytometry (35.4%), dynamic light scattering (10.4%), resistive pulse sensing (4.2%), and atom force microscopy (4.2%) (Fig. 2D).It is noteworthy that all the included studies reported the methods used in EV characterization, while only four studies used a single characterization procedure [39,46,49,77].Regarding the route of EV administration, EVs are most commonly administered to humans by inhalation or intravenous administration, while injection into the tail or jugular veins or intratracheal or intraperitoneal administration is also used in animal models (Fig. 2E).EVs were administered to humans one to five times but in animal models in just one or two doses.Throughout this review, EV therapy was in the form of whole EVs or specific EV-derived molecular cargos (i.e., miRNA, mRNA, or protein) that were isolated from EVs and tested and/or evaluated for their potential antiviral and therapeutic effects (Fig. 3).

Stem Cell EVs as COVID-19 Therapeutics
The included studies were classified into seven clinical and forty-one preclinical studies (Fig. 4).The seven studies evaluated the safety and/or efficiency of EVs against SARS-COV-2 in acute or long-hauler patients.Among them, four studies targeted COVID-19 mild, moderate, severe, or longhauler patients using FDA-approved EV-based drugs (Zofin and Exoflo) derived from human amniotic fluid or bone marrow MSCs [35][36][37][38].Similarly, three other studies highlighted the feasibility, tolerance, and safety of human umbilical, menstrual, and adipose MSC-derived EVs in alleviating SARS-CoV-2 [34,39,40].Table 2 shows patient characteristics, EV source, effects, and outcomes.In these studies, EV-based drugs were shown to have no adverse events with improved oxygen saturation level, survival rates, SOFA and Glasgow scores, partial pressure of arterial oxygen to fraction of inspired oxygen (PaO2/FiO2), and absolute lymphocyte count (ALC).Moreover, they also improved immunocompetence by reducing neutrophil infiltration as well as the pro-inflammatory and anti-inflammatory cytokine storm, including tumor necrosis factor-alpha (TNF-α), interleukin-6

Coding mRNAs and Proteins
Several studies identified EV mRNAs or proteins as contributing to therapeutic effects.In five studies, EVs were reported to promote the healing of lung injuries via hepatocyte growth factor (HGF), angiopoietin-1 (Ang1), or keratinocyte growth factor (KGF) [46,49,53,70,72] (Table 5).HGF associated with EVs was reported to alleviate acute lung injury by reducing apoptosis, pro-and anti-inflammatory cytokines, neutrophil infiltration, and total protein content BALF [53].Additionally, it was reported that MSC-EVs play a role in regulating endothelial permeability partly by HGF, as evidenced by elevated levels of lung integrity VE-cadherin and occludin proteins [72].EV-mediated transfer of angiopoietin-1 mRNA to injured cells induced the secretion of anti-permeability factors and reduced white blood cells, total protein, and inflammatory TNF-α in BALF [46,70].Likewise, EV-associated KGF was reported to have protective effects as efficient as those of the parent MSCs, as indicated by reduced levels of TNF-α, neutrophils, protein, permeability, and extravascular lung water (EVLW), as well as elevated levels of MIP-2 and IL-10 [49].

Quality Assessment and Risk of Bias
Studies were subjected to quality assessment and categorized as having a "low", "high", or "unclear" risk of bias.
As shown in Fig. 5A, human clinical studies scored low and moderate risk of bias using ROBINS-I due to measurement of outcomes and confounding biases.The in vivo animal studies assessed using SYRCLE had an unclear risk of bias, as most of them did not report details of sequence generation, allocation concealment, or random housing details.The in vitro studies assessed using modified SYRCLE for in vitro models showed a low risk of bias across all domains (Fig. 5B).The tables of the risk of bias assessment are provided in supplemental file 3. The CERQual tool that was used to rate the outcomes showed that the overall rating for assessment of confidence was "high" for inflammatory response and recovery of lung injury in the alveolar epithelium and lower in the other domains (Supplemental file 4).Finally, the PRISMA checklist was completed with further details for the review scoring (Supplemental file 5).

Discussion
In this systematic review, we evaluate the reported role of stem cell EVs in targeting COVID-19 and its commonly induced complications, including ARDS, ALI, and pneumonia.Clinical studies showed that EVs derived from diverse stem cell sources could significantly ameliorate the clinical symptoms of lung injury induced by COVID-19 or complicating ARDS/ALI and reduce the time in the ICU or on mechanical ventilators.EV-treated patients had better survival rates, reversed hypoxia, and restored respiratory function and oxygenation index.Modulation of the cytokine storm was supported by downregulated proinflammatory cytokines, elevated anti-inflammatory cytokines, and decreased levels of immune cells, including neutrophils, lymphocytes, and macrophages.

Stem Cell EVs are as Efficient as their Parental Cells Against COVID-19, ARDS, and Pulmonary Lung Injuries
MSCs were recently employed in 122 phase I and II clinical trials as a cell-based therapy against COVID-19, as detected by the Cochrane Central Register of Controlled Trials.In moderate to severe COVID-19 patients, administration of MSCs led to a significant increase in survival rates by reducing lung inflammation and modulating the immune system toward an anti-inflammatory status, with no serious complications reported [18,85,86].In long-term follow-up, MSCs were shown to be safe and effective alternative therapeutic agents with a reliable recovery of lung lesions and COVID-19 symptoms.Minimal serious adverse effects during treatment or thereafter were reported [17].
Based on these data, MSCs were promoted to large-scale phase III clinical trials in subjects with varying severity profiles of COVID-19-induced ARDS and ALI to further evaluate their effect on mortality and long-term pulmonary disabilities [87].Given the important role of EVs in MSC mechanisms of action, the outcome of MSC-EV-based clinical trials might also predict the efficacy of MSC-EV-based therapy against COVID-19, ARDS, pneumonia, or ALI.In the included studies, EVs were employed against COVID-19, ARDS, and/or ALI with or without identification of the roles of specific molecular cargo, such as miRNA, mRNA, or protein.

Clinical Studies
In the included clinical studies, the whole EV cargo showed a significant capacity to maintain a reparative phenotype that restored lung vascular damage when used as a cell-free therapy against COVID-19, ARDS, pneumonia, and ALI.Sengupta et al. reported that administration of an MSC-derived EV preparation (Exoflo) in 24 patients diagnosed with severe COVID-19-induced ARDS restored their immunity and oxygenation capacity after the inflammation was ameliorated [38].Similarly, in a clinical trial of 11 moderately to severely ill COVID-19 patients and a single long hauler, EVs derived from amniotic fluid (Zofin) were reported to be an accessible, feasible, safe, and efficacious treatment for respiratory failure induced by COVID-19 infection [35][36][37][38].In another study by Zhu et al., aerosol inhalation of an EV preparation derived from human adipose-derived MSCs suggested safety and efficiency in 7 severe COVID-19 patients [34].Likewise, Fathi-Kazerooni et al. showed that a menstrual stem cell-derived secretome was an efficient and feasible therapeutic that improved hypoxia, restored immune function and controlled the cytokine storm in 15 severe COVID-19 patients [39].These findings were consistent with multiple systematic reviews that assessed the MSC therapeutic effect on severe COVID-19 patients [88][89][90].This finding supports the efficacy of stem cell EVs as a cell-free therapy against different respiratory disorders, including COVID-19.

Preclinical Studies
These findings were similar to those stated of experimental animal studies in which MSC-EVs were reported to significantly improve and mediate lung function and pathology via different pathways, including the TRPV4/Ca2, Nrf-2/ ARE, and NF-κB signaling pathways [44,65,75].Moreover, restoration of lung tissue function was mediated via mitochondrial transfer, resulting in reducing oxidative stress and promotion of an anti-inflammatory and highly phagocytic macrophage phenotype [64,76].Indeed, these findings aligned with similar mechanisms reported using stem cellbased therapy administration [91][92][93][94] reported that mitochondrial transfer to alveolar macrophages was mediated via tunneling nanotubes (TNTs), leading to enhanced macrophage oxidative phosphorylation and phagocytosis [94].Both MSCs and their EVs were found to be effective in diminishing inflammatory cytokines by inhibiting MMP-9 synthesis and upregulating SP-C [54].MSC-EVs possess anti-inflammatory and antiviral properties that inhibit influenza virus-induced apoptosis and propagation in animal lung epithelial cells [56].

EV RNAs and Proteins with COVID-19 Therapeutic Effects
MSC-EVs were shown to be superior, simpler, and clinically more convenient than their parental MSCs in COVID-19 therapy since EVs do not provoke immunological responses or lead toteratomas, and they protect their cargos against digestive circulating enzymes [95].Moreover, whole MSC therapy might lead to significant vascular insufficiency, as cells may tend to aggregate intravascularly, and could synergize with COVID-19-induced vascular clots [96].However, MSC-derived RNAs and proteins have been used as therapeutic targets in lung injuries, including COVID-19 [97,98].
In comparison, in the analyzed preclinical studies, EV-miRNAs were reported to suppress endothelial damage, inflammatory interleukins, and apoptosis or to promote autophagy and macrophage polarization by mediating the lncRNA-p21/miR-181/SIRT1, PI3K/Akt, TLR4/NF-κB, and MAPK signaling pathways [45,47,48,59,60,66].Similar findings were reported by Li et al., who suggested that parental MSCs attenuated lung injury by a KGFdependent PI3K/AKT/mTOR signaling pathway [99].Similarly, MSCs mediate a therapeutic effects, in part, by many proteins, such as hepatocyte growth factor (HGF), angiopoietin-1 (Ang1), and keratinocyte growth factor (KGF) [100].Perreau et al. and others reported that HGF expression levels could predict the severity of COVID-19 and that HGF could contribute to alleviating lung injury by suppressing the transforming growth factorbeta (TGF-β) signaling pathway [101][102][103].While Adas et al. showed that KGF secreted by MSCs can reduce lung injury [103], similar protective effects were reported using MSC-MVs through the transfer of KGF mRNA to injured alveolar cells [49].Lastly, Ang-1 mRNA transfer by MSC-EVs mediated the immunomodulatory properties of macrophages and was associated with a therapeutic effect on ALI [46,70].These findings were supported by a study by Lu et al., who reported that Ang-1-derived peptide inhibited apoptosis and improved endothelial cell survival, thus reducing inflammation induced by the SARS-CoV-2 virus [104].

EV Characteristics and Application Considerations for Clinical Translation
Most of the EVs in the included studies, whether administered to patients, experimental animals, or in vitro, were reportedly smaller than 200 nm in diameter and derived mostly from MSCs.In at least partial agreement with the ISEV guidelines regarding EV characterization [105], forty-one studies in this review characterized EV preparation using three different protocols, [82].It is worth mentioning that, currently, there are approximately 22 registered clinical trials of phases I, II, and III to evaluate the safety and efficiency of stem cell EVs against COVID-19.Only one systematic review has been published about in vivo animal studies of stem cell EVs against COVID-19, but it did not include any subjects infected by SARS-CoV-2 [106].
However, a specific effect of EVs has not necessarily been proven in these studies, which in general do not strictly establish EV preparation purity, integrity, efficacy, and specificity.This may be due to the various stem cells that were used as sources, separation through different procedures, non-standardized assessment and reporting of EV purity and integrity, and limited support for specific therapeutic effects of MSC-EVs versus non-MSC-EVs and/or co-separating non-EV factors.
Forty studies separated EVs from MSCs; however, these MSCs were derived from various sources, such as bone marrow, adipose tissue, and umbilical cord, possibly with an unclear safety profile.Forty-five out of 48 studies used either UC or precipitation reagents in EV separation.Both procedures are highly non-specific for EVs and were found to give significant variation in reported EV yield and size profile.In the clinical studies, although 4 studies reported safety and promising EV applications in severe COVID-19 cases, the sample sizes were small and in at least one case, commentators remarked on a lack of clarity about EV source and purity [107].Similarly, in preclinical studies, the use of animals with different ages and unspecified breeding conditions may influence relevance of future human trials [108].EVs might exert different actions in vitro and in vivo, and paracrine action of non-EV components of the MSC relesate might contribute to results [22].Despite the recommendations of MISEV2018 [105], none of the studies established a biogenetic origin (e.g., of reported "exosomes" or "microvesicles" as opposed to a mixed EV population) or reported the presence of non-EV components or potential contaminants such as albumin, cytokines, or lipoprotiens from culture media or plasma/serum.Nevertheless, the unreported integrity and half-life of EVs before administration should be considered as an extra source of imposed variation.
EVs might also be engineered to have advantages over native EVs [81,109,110].For example, cells might be engineered to produce EVs that modulate infection-related signaling pathways in recipient cells [81], or present targets for the SARS-CoV-2 spike protein and thus "sponge" the virus [109,110].These bioengineered EVs might enhance the overall yield, bioactivity, and half-life and improve the targeting effect for clinical applications; however, the extended half-life may induce adverse effects such as fibrosis.Thus, more future clinical studies should be carried out to test the validity, safety, and efficacy of these synthetic EVs.
EVs might also be engineered to have advantages over native EVs [81,109,110].For example, cells might be engineered to produce EVs that modulate infection-related signaling pathways in recipient cells [81] or present targets for the SARS-CoV-2 Spike protein and thus "sponge" the virus [109,110].These bioengineered EVs might enhance the overall yield, bioactivity, and half-life, and improve the targeting effect for clinical applications, however, for instance, the extended half-life may induce adverse effects like fibrosis.Thus, more future clinical studies should be carried out to test the validity, safety, and efficacy of these synthetic EVs.
ISEV and the International Society for Cell and Gene Therapy (ISCT) urge that EV studies should consider multiple key points since EV research is relatively new and no adequate quality control and manufacturing obligations are yet in place [111].Among these considerations are the source of stem cell EVs, optimal isolation technique, storage, dosing, and administration route [111].Variations in these key points may affect the reproducibility of MSC-EVs in clinical research against COVID-19.

Cell Source
The cell sources of EVs included human, animal, and in vitro studies were mostly either bone marrow or adipose MSCs.Although the mechanism by which EVs exert their antiviral or anti-inflammatory actions may differ depending on the source, all have shown promising efficacy against lung injuries, as illustrated earlier.However, it is worth mentioning that adipose-derived MSC-EVs were recently reported to increase the thrombosis risk more than bone marrow-derived MSC-EVs, potentially heightening the risk of microvascular injury syndrome in severe COVID-19 patients [112,113].Additionally, Huang et al. confirmed that EVs of different origins might have heterogeneous effects [61].He showed that EVs derived from young MSCs had preferable effects in alleviating acute lung injury and macrophage polarization over EVs derived from aging MSCs in experimental animals [61].

EV Separation Procedure
The procedure for EV separation or concentration could have a significant impact on the therapeutic outcome.Although different techniques, such as ultracentrifugation, size exclusion chromatography, precipitation, and immunoaffinity, were utilized in the included studies, ultracentrifugation was the most commonly reported.Furthermore, the ultracentrifugation sizing procedure and instrument varied from one study to another, which also led to different-sized EV populations.Each ultracentrifugation isolation procedure may hold advantages, such as high isolation efficiency, purity, and concentration, but they may also hold some disadvantages, such as isolating malfunctioning EVs.Harsh and rigorous purification procedures could even result in removal or damage of EV-intrinsic effectors or extrinsic factors that act with EVs to exert their functions [108].Moreover, these different separation procedures may result in EVs of different sizes, concentrations, purity levels, and ultimately function.These differences could challenge reproducibility and complicate rapid EV clinical translation.

EV Dosage and Administration
Major variation among studies may arise due to the absence of predefined patient enrollment criteria.Hence, patients involved in any study should be selected carefully by checking different clinical biomarkers, disease severity, age, and several other considerations.Another main contributor to variation is the dosage of stem cell EVs since it was equivalent to the dosage reported in stem cell-based therapy, among most studies, because the minimal amount of EVs required to induce therapeutic effects without being toxic is not yet defined.EV doses ranged from 1 up to 5 doses of varying concentrations diluted in either saline or sodium chloride.For intratracheal administration, the doses ranged from 1 up to 3 EV doses, while for inhalation and intravenous, they ranged from 1 up to 5 EV doses.Finally, the mode of EV administration is dependent on the disease's severity, where IV is usually selected for direct and faster drug delivery into the bloodstream in severe COVID-19 cases.In contrast, oral route is often used in early COVID-19 patients when there is a need for effective and economical treatment that can be taken at home [114,115].However, the route of administration is also dependent on the drug properties, and the patient's individual circumstances.Intravenous (IV) and inhalation routes were the most commonly used in humans [34][35][36][37][38][39]116], while direct intratracheal application was common in preclinical studies.In COVID-19 patients, intravenous administration holds the potential to target not only injured lung cells, but also multiple organ failures induced by SARS-CoV-2, such as myocardial infarction and microvascular dysfunction [117,118].However, it is still debated to what extent IV administered EVs will reach injured lungs or other organs [119,120].EV inhalation, compared to IV, is simpler, less invasive, offers direct drug delivery to the lung, and can achieve higher drug concentrations at a lower overall dose.The intratracheal route delivers the treatment more directly.

Evidence Profile
The included studies are methodologically sound, as evaluated by the SYRCLE risk of bias tool, which provided a very high rating for assessment of confidence in the domains most related to COVID-19, including inflammatory response and recovery of lung injury in the alveolar epithelium.This suggests the high likelihood of practical application of the findings from the selected animal studies to clinical settings.However, other domains of the SYRCLE risk of bias tool reported a lower level of confidence.This was potentially a result of unclear documentation of various parameters and methodological limitations such as blinding for assessors, group randomization, and concealment that ultimately impact the internal validity of the primary studies.Hence, there is a clear need for better documentation and rigorous methodological discipline in future studies in an effort to improve the reliability and credibility of the resultant publications.

Limitations and Potential Solutions
The strength of our study is limited by the modest number of included COVID-19 patients and the high variation among the EV populations within the studies, which, in turn, limited the data available for a meta-analysis.However, available data on EV clinical trials support their application for effective COVID-19 therapy despite several challenges: (1) difficult and standardized EV separation procedures are lacking, with unclear implications for EV purity; (2) assessment of stem cell EVs in a clinical setting, such as their biodistribution, metabolism, excretion, etc., still cannot be performed; (3) there are no guidelines for large-scale EV production and quality control; and (4) optimal EV dose and dosing regimens must be determined.Further research is necessary to optimize EV production protocols, standardize dosage and administration routes, and conduct large-scale randomized controlled trials to definitively establish the efficacy and safety of EVs in a clinical setting.Additionally, exploring EVs derived from specific stem cell types tailored to target different stages and pathologies of COVID-19 holds promising potential for personalized medicine approaches.

Conclusion
In conclusion, our systematic review of preclinical and clinical evidence demonstrates the immense potential therapeutic role of stem cell EVs in combatting COVID-19, particularly in mitigating the devastating complications of ARDS, ALI, and pneumonia.EVs derived from various stem cell sources hold a favorable safety profile and exhibit potential efficacy in combating COVID-19.Stem cell EVs induce anti-inflammatory properties in COVID-19 patients, evident in the suppression of the proinflammatory mediators, cytokine storm, and neutrophil infiltration, offer a critical approach to managing the detrimental inflammatory response associated with severe COVID-19.Beyond combating the acute effects of COVID-19, EVs display promise in facilitating patient recovery by promoting endothelial cell junction formation and reducing fibrin production, thereby mitigating pulmonary edema and improving lung function.

Fig. 2
Fig. 2 An overview of studies characteristics.A Ratios of pulmonary diseases including ARDS, COVID-19, ALI, and pneumonia.B Stem cells sources to separate EVs with number of studies using each source.C Percentages of different EV separation methods.D Percentages of characterization procedures used for EV identification with number of studies.E EV administration routes reflected by the

Fig. 3 Fig. 4
Fig. 3 Stem cell EVs as therapeutics for COVID-19.A EVs derived from stem cells of various origins were investigated in the form of whole EVs or specific EV-associated cargos.B EVs are administered via different routes, in both clinical and preclinical studies

Fig. 5
Fig. 5 Risk of bias (RoB) assessment graph.A using the ROBINS-I RoB tool for cohort clinical studies, B using the SYRCLE's RoB tool for preclinical studies

Table 1
Characteristics of stem cell EVs employed in included studies

Table 2
Characteristics of studies using stem cell EV-based therapeutics as an anti-COVID19 intervention

Table 3
Characteristics of studies into the role of stem cell EVs in interventions (no specific molecular mechanisms)

Table 5
Characteristics of studies into the role of stem cell EV mRNAs and/or proteins in interventions