**4. Extracellular vesicles**

Extracellular vesicles (EVs) are currently being studied as potential therapeutic agents for immune related pathologies due to their immunomodulatory and regenerative properties [89]. Interest in EVs has grown due to their ability to have similar therapeutic effects as MSCs as a cell free therapy [89]. What was once viewed as cellular waste products, may now have the potential to treat one of the largest natural disasters in modern history that is the COVID-19 pandemic.

The field of EVs has grown significantly in the recent years leading to the formation of the International Society for Extracellular Vesicles (ISEV) [89]. ISEV defines EVs as particles naturally released from a cell that are delimited by a lipid

bilayer and cannot replicate [90]. EVs are further subclassified as exosomes (40-120 nm), microvesicles (50-1000 nm), and apoptotic bodies (500-2000 nm). Both microvesicles and apoptotic bodies bud-off directly from the cellular membrane and participate in two distinct cellular pathways: apoptotic bodies are products of cell mediated death whereas microvesicles are involved in paracrine communication [89, 91]. Exosome biogenesis, however, differs greatly in that it involves cell membrane invagination and formation of an intraluminal vesicles that undergoes modification in what is called a multivesicular body (MVB) [92]. Once modifications are performed, the MVB fuses with the cell membrane and the ILV's are secreted into the extracellular space as exosomes [92].

Once secreted, EV's carry a variety of nucleic acids, proteins, and lipids that can regulate or alter a plethora of biological processes through effects on cell receptors, adhesion molecules, cytokines, and other cell signaling molecules [89, 93–96]. They have attracted significant attention for their ability to inhibit tumorigeneses, suppress immune responses, promote tissues repair, and have therapeutic effects on neurological disease [96]. A recent study by Schultz et al. performed bioinformatic analysis of mRNA and miRNA cargo of EV's using Gene Expression Omnibus (GEO) database and miRWalk 3.0 servers. The study found that 266 miRNA's within exosomes have the ability to attenuate cell death by inhibiting TNF-a, IFN-y, JAK2, and JAK1 among others. Similarly, 148 miRNA's were identified with 1 or 2 targets of molecules involved in the intrinsic and extrinsic coagulations cascade pathways [97]. Continually, EV's also have the capability of replenishing glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglucomutase (PGM), enolase (ENO), and pyruvate kinase m2 isoform (PKm2), and phosphorylated PFKFB3, all of which are involved in the production of glycolytic ATP. It was proposed that secretions of these enzymes can reduce levels of reactive oxygen species and consequently halt cellular death [96]. In addition, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), extracellular and matrix metalloproteinase inducer (EMMPRIN) have also been found within exosomes further postulating their regenerative effects through angiogenesis stimulation and tissue repair [96].

In preclinical trials, EV's have already demonstrated their immunomodulatory capabilities. In a study by Monsel et al. [98] on pneumonia induced mice, EV's reduced neutrophils and macrophages by 73% and 49% respectively, while decreasing edema and permeability of the endothelial-epithelial barrier to protein [99]. In fact, a recent study demonstrated that EV's reduce levels of inflammatory interleukins: IL-8, IL-6, IL-17 and TNF-α, when transferring anti-apoptotic miR-21-5p to target cells which resulted in reduced edema and lung dysfunction [100]. Additionally, EV's have also demonstrated their efficacy against acute lung injury (ALI) through downregulation of TLR/NF-κB signaling in rat models [101]. A recent study assessed the safety and efficacy of EVs on patients with severe COVID-19 infections. 24 patients were recruited under the specified trial criteria and followed for 14 days [102]. In addition to not having any notable adverse effects to the 15 mL IV dose of exosomes, the experimental group exhibited lower neutrophil count, c-reactive protein, ferritin, and D-dimer indicating an immunomodulatory effect [102]. Additionally, the overall survival rates were 83% with 17/24 patients fully recovered and 3/24 in stable conditions [102]. The study actively demonstrated EVs ability to safely attenuate the cytokine storm associated with severe COVID-19 infections. To fully appreciate the impact of EVs on COVID-19, further studies should be developed. As of February 18, 2021, applying the search word "exosomes" or "extracellular vesicles"

and "COVID-19" on clinicaltrials.gov, results in 9 and 5 listed clinical trials, respectively. One of these trials, (NCT04491240) evaluated the safety and efficacy of exosome inhalation in SARS-CoV-1 pneumonia. Although results are published in c linicaltrails.gov, publication of the article is pending. The same experiment, however, has been approved for phase 2 and is currently enrolling participants (NCT04602442).

The field of EV's continues to show increasing promise as a therapeutic in the battle against COVID-19 based on their ability to carry a variety of cellular and nuclear components in a stable and hypoimmunogenic bilayer [6, 19].

## **5. MSCs and COVID-19**

Due to the mechanisms of action of MSCs as well as their success as a therapy in ALI and ARDS, MSCs have attracted the attention now for their possible use in COVID-19. Leng et al. conducted one of the first studies exploring the case for MSCs in COVID-19 [103]. Ten adult patients with a positive real-time reverse transcription polymerase chain reaction assay and that meet the clinical classification for COVID-19 by the National Health Commission of China were enrolled in the study. Of the ten patients seven patients were in the treatment group, of those seven one was categorized as critically severe type, four were severe, and two were common types. MSCs were administered via IV infusion with 1 x 106 cells per kilogram and patients were assessed for a 14 day period. Two-four days after infusion all patients with symptoms of a high fever, weakness, shortness of breath and low oxygen saturation resolved. None of the patients experienced any infusionrelated nor allergic reactions with no delayed hypersensitivity reactions or infections. Three of the patients that subsequently recovered were discharged 10 days after treatment with one of them being characterized as a severe subtype. In regard to the patient having a critically severe type of COVID-19, their C-reactive protein (CRP) decreased from 19.0 g/L to 10.1 g/L, and their oxygen saturation (SaO2) increased from 89–98% without supplemental O2. The critically severe patient also had significant improvements in lymphopenia, as well as in indicators of liver, myocardial and kidney damage/disease (aspartic aminotransferase, creatine kinase and myoglobin). Chest CT imaging with the characteristic ground-glass opacity and pneumonia infiltration were also reduced by the 9th day after MSC infusion. Overall levels of pro-inflammatory CD4+/CD8+ T cells, TNF-α and conventional DCs all decreased while IL-10, VEGF, HGF and TGFβ increased, promoting a tissue regeneration state. It was also concluded that MSCs were ACE2R and TMPRSS2 negative, theoretically making them immune from possible SARS-COV-2 infection [103]. Additionally, evidence by Sanches-Guijo et al. indicated similar results [104]. Adipose-derived MSCs were used as a treatment for 13 COVID-19 patients. There were no adverse events in the MSC treatment group with no worsening of respiratory or hemodynamic parameters. Clinical improvement was seen in 70% of the patients, seven of them extubated and discharged, and two showing signs of improvement in their ventilatory and radiological parameters, two resulting in fatalities and the rest of the patients in stable condition. Overall levels of CRP, IL-6, ferritin, and D-dimer were decreased [104].These positive effects of MSCs in COVID-19 were further elucidated by Tang et al., the study included two patients with COVID-19 which received three separate IV infusions of menstrual blood derived MSCs [105]. The first patient (Patient 1) was a 37 year old woman with a past medical history of hypertension. Patient 1's levels of CRP, TNF-α, and IL-6 decreased while their SaO2 dramatically increased from 98% on 100% fraction of inspired O2 (FiO2) to 97% SaO2 on 55% FiO2. Initial CXR findings revealed large, patches of high density lesions in bilateral lungs that resolved with treatment along with viral RNA testing. Patient 2 was a 71 year old male that similar improvements in inflammatory markers, SaO2 and CXR findings [105].

Recently, a study conducted by Shi et al. used UC-derived MSCs as a therapeutic in 101 patients diagnosed with severe COVID-19 [106]. The study was a doubleblind, placebo-controlled phase 2 trial with 101 patients randomized in a 2:1 ratio with sixty six patients, with one patient withdrawing, in the treatment group and 35 in the placebo group. Overall chest CTs, age, sex, BMI, and onset of symptoms matched between the groups. The occurrence of adverse events during the study was similar between the treatment (55.38%) and the placebo group (60%) with none directly related to the MSCs. Three IV infusions of UC-derived MSCs with 4 x 107 cells per infusion were administered. High resolution chest CT images were assessed using both radiologist and artificial intelligence software to estimate the total lesion proportion (TLP) via the Hodges-Lehmann estimator of the entire lung. The median change in the TLP was 19.40% in the treatment group 7.30% in the placebo group with the overall difference of 13.31%. Solid lesions were found to decrease by 57.70% in the treatment group with an overall decrease in the groundglass lesions. A 6-minute walk test (6-MWT) was used to assess the restoration of lung function and reserve capability in both groups. The median 6-MWT was 420 meters in the MSC treatment group in comparison with 403 meters in the placebo group [106]. In a similar study using UC-MSCs for COVID-19, Lanzoni et al. conducted a double-blind, phase 1/2a, randomized controlled trial [107]. Twentyfour patients hospitalized for COVID-19 were randomized 1:1 into either the treatment or control group. Two infusions of UC-derived MSCs with 100 20 x 106 MSCs in each were administered. There were two serious adverse events (SAEs) observed in the treatment group while the control group had 16 SAEs, the intervention was deemed safe as it did not lead to an increase in specified infusion related AEs. Overall, the survival rate in the treatment group was far greater than in the control group with 91% of subjects in the treatment group surviving 31 days post first infusion in comparison with 42% in the control group. The time of recovery was also shorter for the MSC group, with a hazard ratio for recovery in the control group vs. the MSC group of 0.29 indicating a lower rate of recovery in the control group. Concentrations of GM-CSF, IFN- y, IL-5, IL-6, IL-7, TNF-α, TNF-β, were also statistically decreased in the MSC treatment group in comparison with control [107].

With the current supporting data surrounding the use of MSCs in COVID-19 as well as their historical efficacy in lung injury models the case for their use on a compassionate basis can be made. In the future more randomized, controlled, multi-centered clinical trials are needed in order to increase the knowledge of the use of MSCs in COVID-19.

#### **6. Ongoing clinical trials**

Clinical trials that utilize MSCs and EVs and that are registered on ClinicalTrials.gov can be seen in **Tables 1** and **2** respectively. The data from current studies are promising and promotes the use of MSCs and EVs as a possible treatment for COVID-19. However, more multi-center, controlled, randomized clinical trias are needed to further solidify the use of MSCs and EVs in COVID-19.










 *Clinical trials registered on ClinicalTrials.gov till January 5, 2021 utilizing sem cells for the treatment of COVID-19.*


#### **Table 2.**

*Clinical trials registered on ClinicalTrials.gov till January 5, 2021 utilizing extracellular vesicles and/or exosomes for the treatment of COVID-19.*
