key: cord-0942254-5e0pu0kl
authors: Moradinasab, Susan; Pourbagheri-Sigaroodi, Atieh; Zafari, Parisa; Ghaffari, Seyed H.; Bashash, Davood
title: Mesenchymal stromal/stem cells (MSCs) and MSC-derived extracellular vesicles in COVID-19-induced ARDS: Mechanisms of action, research progress, challenges, and opportunities
date: 2021-04-28
journal: Int Immunopharmacol
DOI: 10.1016/j.intimp.2021.107694
sha: 08c822fd0cbcfb2bd5aa40c68537875b3679eb4e
doc_id: 942254
cord_uid: 5e0pu0kl

In late 2019, a novel coronavirus (SARS-CoV-2) emerged in Wuhan city, Hubei province, China. Rapidly escalated into a worldwide pandemic, it has caused an unprecedented and devastating situation on the global public health and society economy. The severity of recent coronavirus disease, abbreviated to COVID-19, seems to be mostly associated with the patients’ immune response. In this vein, mesenchymal stromal/stem cells (MSCs) have been suggested as a worth-considering option against COVID-19 as their therapeutic properties are mainly displayed in immunomodulation and anti-inflammatory effects. Indeed, administration of MSCs can attenuate cytokine storm and enhance alveolar fluid clearance, endothelial recovery, and anti-fibrotic regeneration. Despite advantages attributed to MSCs application in lung injuries, there are still several issues (__)foremost probability of malignant transformation and incidence of MSCs-related coagulopathy(__) which should be resolved for the successful application of MSC therapy in COVID-19. In the present study, we review the historical evidence of successful use of MSCs and MSC-derived extracellular vesicles (EVs) in the treatment of acute respiratory distress syndrome (ARDS). We also take a look at MSCs mechanisms of action in the treatment of viral infections, and then through studying both the dark and bright sides of this approach, we provide a thorough discussion if MSC therapy might be a promising therapeutic approach in COVID-19 patients.

strategies from all around the world. SARS-CoV-2 is genetically similar to two other beta groups of coronaviruses, named severe acute respiratory coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) with a mortality rate of 10% and 37%, respectively [1, 2] . Despite the lower mortality rate of the novel coronavirus infection than the SARS and MERS epidemics [3, 4] , the longer incubation period made the SARS-CoV-2 more contagious with a much higher total number of deaths [5, 6] . The clinical manifestations of COVID-19 vary from asymptomatic or mild sickness with fever, cough, or lack of breath to severe respiratory failure which requires mechanical ventilation support. Patients with severe COVID-19 are highly at the risk of acute respiratory distress syndrome, secondary infection, coagulopathy, septic shock, and also multiple organ dysfunction (MOD) [5, 7, 8] . Para-clinical findings in patients with COVID-19 demonstrate hyperactivation of the immune system, which in turn results in inflammatory hyper-response, cytokine-storm, and subsequently, damage of the pulmonary tissue, dysfunction of air exchange, and ARDS [9, 10] .

Due to immunomodulatory, anti-inflammatory, and other immune properties, mesenchymal stem cells (MSCs) __ which according to 2019 ISCT suggestion should be expressed as mesenchymal stromal cells [11] __ can play a key role in avoiding or alleviating the cytokine storm. Presently, MSCs are largely used in cell-based therapies and their safety and efficacy, especially for the treatment of immune-mediated inflammatory diseases, have been investigated in numerous clinical trials [12] [13] [14] [15] . A large number of clinical trials introduced MSCs as the promising therapeutic approach toward improving the adverse effects of the SARS-CoV-2 infection via immunomodulatory effects, attenuating the cytokine storm, and exerting anti-inflammatory effects [16] . In this study, we review the historical evidence of successful use of MSCs for lung disease and highlight the therapeutic role of MSCs and their EVs such as immunomodulation, alveolar fluid clearance (AFC), endothelial recovery, and anti-fibrotic regeneration in the treatment of viral infections. Also, through discussing the advantage and disadvantage of this approach we deliberate if MSC therapy might be a promising therapeutic approach in COVID-19 patients.

In 2004, the first inclusive report on MSCs clinical implementation was published, in which allogeneic bone marrow MSCs (BM-MSCs) were injected for pediatric graft-versus-host disease (GVHD), leading to 1year-survival of the patient [15] . Afterward, MSCs have been widely examined as a valuable treatment option for a wide spectrum of tissue/ organ damage especially for immune-mediated inflammatory diseases such as GVHD, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, and ARDS [17, 18] . This is attributed to their ease of isolation and large ex vivo expansion capacity, as well as the ability to interact with other immune cells via secretion of paracrine factors and exerting their immunomodulatory effects [14] . As the lungs are in direct contact with the external environment, they are prone to different infections including viral diseases. Over the past decade, a considerable number of studies shed light on the therapeutic effects of MSCs for lung diseases, such as ARDS [19] [20] [21] [22] [23] , chronic obstructive pulmonary disease (COPD) [24] [25] [26] [27] [28] , Asthma [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] , and idiopathic pulmonary fibrosis (IPF) [43] [44] [45] [46] [47] [48] .

Despite the limited data from clinical studies, numerous reports suggest the therapeutic function of MSCs in preclinical models of respiratory virus-induced lung infections. A report by Chan et al. showed that the MSCs remarkably diminished the impairment of alveolar fluid clearance caused by the avian influenza virus (H5N1) in vitro and reduced A/H5N1-associated acute lung injury (ALI) in vivo [49] . Similarly, another study indicated that umbilical cord MSCs (UC-MSCs) may effectively restore alveolar fluid clearance in A/H5N1-associated ALI [50] . Furthermore, a study in a pig model provided interesting evidence showing that extracellular vesicles from MSCs may attenuate influenza virus-induced ALI [51] . Based on the results of a recent study showing that MSCs could improve the survival rate of H7N9-induced ARDS in both preclinical and clinical studies, the authors suggested that the MSCbased therapy could also be an ideal option for treatment of COVID-19 as they share similar complications such as ARDS and ALI [52] ; however, in the other two in vivo studies in mice infected with A/H1N1, no improvement in inflammation or survival was seen [53, 54] . Besides, MSCs are themselves at the risk of being infected with the influenza virus [55, 56] . Future studies are needed to clarify the safety and efficacy of MSC's therapeutic role in influenza virus infections. The history beyond the therapeutic effects of MSCs in lung injuries caused by viral infections is summarized in Fig. 1 .

The principle mechanism by which MSCs exert their therapeutic functions is interaction with immune cells either via direct cell-cell contact or with the secretion of soluble immune factors (cytokines, chemokines, growth factors, angiogenic factors, and EVs). Indeed, MSCregulated immunomodulation affects both innate and adaptive immunity by regulating the activity of neutrophils, monocytes, macrophages, dendritic cells (DC), T cells, B cells, and natural killer (NK) cells. MSCs modulate the T cell functions including inhibition of T helper (Th17) response, generation of regulatory T cells, shifting from Th1 to Th2 subset, and finally decreasing inflammation. Not only do MSCs restrain the proliferation of CD4 + and CD8 + T cell subsets [57, 58] they can also significantly influence DC development through prostaglandin E2 (PGE2) production, which eventually leads to the production of antiinflammatory Th2 subset via differentiation of DCs to the plasmacytoid subset [59] . Apart from the modulation of the T cell responses, MSCs inhibit the proliferation of B cells and prevent their differentiation into plasma cells resulting in a reduction of immunoglobulin secretion [60, 61] . MSCs can also hamper oxidative burst in neutrophils, diminish the extracellular release of elastase and myeloperoxidase, and eventually weaken neutrophil infiltration and damage [62] . Concerning the immunomodulatory effects of these cells on innate immunity, Dayan et al. reported that MSCs could also decrease M1-type macrophages that possess a significant pro-inflammatory effect, while they increase alternatively activated M2-type macrophages via down-regulation of IL-1β and IL-6 levels [57] . Indeed, the anti-inflammatory properties of MSCs are mainly mediated by the paracrine pathways. They decrease the secretion of the pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-12, and IFN-γ while elevating the level of the antiinflammatory IL-10 mediator [63] . Taken together, all these findings reveal that MSCs could hamper the inflammatory responses, at least partly, through polarization of immune cells to the subsets which possess prominent anti-inflammatory characteristics.

In the case of ARDS, MSCs play a role in clearing alveolar fluid which is disturbed due to cytokine storm in the lungs [64] . It has been reported that human UC-MSCs could improve the AFC impairment by producing a large amount of Angiopoietin-1 (Ang-1) [65] and hepatocyte growth factor (HGF) [50] . Ang-1 serves as a ligand for the Tie2 receptor and plays a key role in vascular stabilization due to its effects on endothelial permeability by inhibiting leukocyte-endothelium interactions and upregulating a tight-junction [66] . HGF, on the other hand, inhibits epithelial apoptosis and also regulates motility, cell growth, and morphogenesis via interaction with the c-Met receptor. Ang-1 and HGF both are considered significant mediators in the restorative properties of UC-MSCs on AFC in A (H5N1)-induced ARDS [50] . Accordingly, it has been reported that HGF could restore lung permeability via inhibition of endothelial apoptosis as well as the protection of tight junctions in a bacterial ALI model [67] .

Given the fact that MSCs can secret a variety of growth factors including HGF, keratinocyte growth factor (KGF), and vascular endothelial growth factor (VEGF), they have significant ability to play essential roles in tissue regeneration in lung injuries via diminishing collagen accumulation and fibrosis [68] . HGF and VEGF can also restore pulmonary capillary permeability and stabilize the pulmonary endothelial barrier [69] . In a study by Aguilar et al., administration of MSCs expressing KGF resulted in the reduction of bleomycin-induced fibrosis, as well as decreasing collagen buildup and maintaining normal alveolar structure [70] . It has been also reported that HGF and KGF from MSCs stabilize B cell lymphoma 2 (Bcl-2), suppress hypoxia-induced factor 1-А (HIF1-α) protein expression, and diminish reactive oxygen species (ROS) production; resulting in an anti-apoptotic effect on the alveolar epithelial cells [71] . To provide a well-conceptualized prospect, we summarized the plausible mechanisms of MSCs in viral infections in Fig. 2 .

Extracellular vesicles (EVs) are membrane-bound vesicles about 40-1000 nm in size, produced by a variety of human cells. Exosome, microvesicles, and apoptotic bodies are three kinds of EVs. Microvesicles are separated directly by budding from the cell membrane, while exosomes are formed within the endosomal network of multi-vesicular bodies (MVBs) and their fusion with the plasma membrane [72] . MSCderived EVs mimic many of the beneficial impacts of MSCs and transport a wide range of bioactive factors such as proteins and lipids, as well as transcription factors, mRNA, microRNA, long non-coding RNA, DNA, and signal transduction molecules [73] . Released EVs are the integral component of the cell-to-cell communication network, and upon transfer to the recipient cells, they can modulate downstream signaling. Compared with their parents, EVs administration is safer because they cannot replicate and form a tumor, they are non-immunogenic, and finally, they do not lead to emboli formation. Also, EVs are more stable and viable at − 80 • C for a longer time [74] .

Termed as cell-free therapy, EVs have attracted special interest for their clinical application. MSC-derived EVs have been investigated in ARDS due to their ameliorating function in ALI by reducing inflammation, improving edema clearance, enhancing macrophages phagocytosis, and attenuating the severity of cytokine storm, and also tissue repair through the direct mitochondrial transfer of host cells [23, [75] [76] [77] [78] . In the study by Zhu et al., intratracheal administration of MSC-derived microvesicles was effective for pulmonary edema in the treatment of endotoxin-induced injury in mice by reducing extravascular lung water, restoring lung protein permeability, and reducing the influx of neutrophils and macrophage inflammatory protein-2 (MIP-2) levels through the expression of KGF mRNA in the injured alveolus [23] . In another study, Intratracheal administration of MSC EVs showed the same results by alleviating lung inflammation and restoring alveolarcapillary permeability in LPS-induced ALI mice. Notably, the effect was partly mediated by Ang1 mRNA, which is considered an important vascular-stabilizing factor [79] . Microvesicles derived from human MSCs could also improve the survival of mice injured with bacterial pneumonia by secreting KGF and decreasing the influx of inflammatory cells, cytokines, protein, and increasing the macrophage phagocytosis [76] . Mitochondrial transfer from BM-MSC carrying microvesicles to pulmonary alveoli ensued protective effects against LPS-induced ALI in mice [78, 80] . Moreover, the transfer of functional mitochondria from EVs was responsible for the elevation of macrophage phagocytosis through the promotion of oxidative phosphorylation [80] . Apart from ARDS, MSC-derived EVs have been also studied as a potential therapy in the management of other lung injuries including idiopathic pulmonary fibrosis, bronchopulmonary dysplasia, pulmonary arterial hypertension, asthma, and silicosis [81] [82] [83] [84] . All in all, MSC-derived EVs due to their advantages over their parents could be considered as an ideal candidate for the clinical application in a wide variety of viral infections, including COVID-19. Fig. 3 provides a schematic representation of the plausible 

SARS-CoV-2 viruses are enveloped with positive-stranded RNA of about 26 to 32 kb in length [85] . After entering the lungs, as the first organ exposed to the virus due to its way of transmission, the virus attaches to its functional receptor angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) through the specific recognition of its surface spike (S) protein. A very similar structure also exists in the SARS virus, however, with a lower affinity binding (10-20 times) compared to the S protein of SARS-CoV-2. Notably, this binding capacity indicated the high transmission power of SARS-CoV-2 [86] . RNA is then released into the host cell and uses the replicate machinery of the infected cells [87] . The ACE2 receptor is abundantly distributed not only in alveolar epithelial cells also in the kidneys, liver, heart, and digestive organs; the fact explains why beyond ARDS, patients might go through organ damage, shock, and death from MOD [9] . Inside the host cell, the virus can trigger a "cytokine storm". Indeed, deleterious unrestrained widespread inflammatory response arising from an excessive secretion of pro-inflammatory cytokines finally leads to deadly clinical manifestations of ARDS. The pathophysiology of SARS-CoV-2 has been largely discussed in several recent studies [88] [89] [90] . Previous studies indicated that MSCs could be at the risk of various viral infections such as influenza virus [56] , hepatitis B virus [91] , and human herpesvirus [92] . On the other hand, not only the lack of ACE2 receptors on MSCs may probably make their recognition by SARS-CoV-2 impossible [63] but the expression of interferon-stimulated genes (ISGs) could also induce resistance in MSCs [93] . Inline, RNAsequence analysis for infused MSCs showed these cells do not express both ACE2 and TMPRSS2, which means MSCs cannot carry risks for cross-contamination of SARS-CoV-2. Infused MSCs also express high levels of type II alveolar epithelial (ATII) specific surfactant protein A (SPA) and surfactant protein C (SPC), indicating the potency of these cells for generating ATII cell [94] . Apart from these interesting characteristics, anti-inflammatory factors such as TGF-β, HGF, leukemia inhibitory factor (LIF), FGF, VEGF, and nerve growth factor (NGF) were highly expressed in MSCs that make these cells applicable candidates in COVID-19 treatment [63] .

To date, there have been several clinical studies publishing data regarding the use of MSCs as the novel treatment for SARS-CoV-2 cases. In the first reported study, a total of ten patients was enrolled. The intravenous (IV) administration of clinical-grade BM-MSCs was done for seven patients, while the remaining three patients served as the control group. They were closely followed for 14 days, no adverse effects were observed after MSC injection in seven cases, and MSC therapy could nearly improve all the patients' symptoms. The results of this interesting study revealed that BM-MSCs inhibit the over-activation of the immune system and prevent cytokine storm. Besides, an increase in the regulatory DC population has been observed [63] .

Liang et al. have shown the safety and efficacy of treatment with UC-MSC in the modulation of the immune response and recovery of the injured tissue of a 65-years-old female severely ill COVID-19 patient. In this reported case study, a patient with a fever, fatigue, cough, and shortness of breath was treated with glucocorticoid and antiviral drugs which were ineffective. After diagnosis as a critically ill-type COVID-19 with ARDS, she was treated with triple IV infusion of 5 × 10 7 UC-MSCs. Following the second infusion, besides clinical improvement, white blood cell (WBC) counts and neutrophils decreased to a normal level, and lymphocytes elevated to normal range [95] . The results published by Shu et al. support that human UC-MSCs can be considered as a priority treatment option for severe COVID-19 cases. In the indicated study, a total of 41 patients were divided into 2 groups: 29 patients assigned to the placebo group and 12 assigned to the UC-MSC group. The patients were followed for 2 weeks after hUC-MSC injection. In the hUC-MSC treatment group no invasive ventilation occurred, no mortality was observed, and the patients' clinical symptoms, including shortness of breath, chest tightness, and fatigue were remarkably improved [96] . Meng et al. also conducted a clinical trial for evaluation of the safety of human UC-MSCs in the treatment of moderate and severe cases of COVID-19 in which 9 patients received 3 cycles of UC-MSCs on days 0, 3, and 6. After treatment, the PaO2/FiO2 ratio was improved, and computerized tomography (CT) scans reported improvement of lung injuries. The lung lesions were controlled for six days and completely disappeared within 2 weeks with no serious UC-MSCs infusion-associated adverse events [97] . In a case series, 8 critically ill patients on mechanical ventilation received PLX-PAD, a placenta-derived mesenchymal-like cell therapy. Despite the little sample size and the uncontrolled study design, the clinical status of patients including respiratory variables, kidney function, and positive end-expiratory pressure were improved [98] ; further supporting the therapeutic efficacy of UC-MSCs in COVID-19 patients especially those with the critically ill status.

Wu et al. reported IV delivery of a cell population derived from human embryonic stem cells (hESCs), termed immunity and matrixregulatory cells (IMRCs), could hamper both fibrosis and pulmonary inflammation in a mouse model of lung tissue damage. With regards to the crisis of SARS-CoV-2 disease, IMRCs were administrated to 2 critically ill COVID-19 patients. Within 14 days following IV injection, both patients significantly recovered from pneumonia and tested negative for SARS-CoV-2. Notably, IMRCs may probably be superior to primary UC-MSCs due to stronger immunomodulatory effects, higher consistency in quality, and anti-fibrotic function [99] . Sanchez-Guijo et al. has tested the safety and efficiency of allogenic AD-MSCs for 13 patients with COVID-19 being under mechanical ventilation and treated with antiinflammatory and anti-viral therapies. Ten out of 13 patients received two doses, 2 patients were treated with a single dose and 1 patient received 3 doses of AD-MSCs (0.98 × 10 6 cell/kg). Clinical improvement was seen in 69.2% of the patients and 7 patients were extubated and discharged. No adverse effect related to cell therapy was observed and the treatment resulted in the reduction of inflammatory factors including CRP, lactate dehydrogenase (LDH), ferritin, IL-6 as well as an increase in B and T lymphocytes [100] . In another clinical study by Chen et al., menstrual blood-derived MSCs were given to 25 patients with the median age of 70 years in a single, double, and triple infusion of 1 × 10 6 cells/kg. After treatment, abnormal signs of CT scan were improved in 16 cases, both PO 2 and SO 2 were increased while the levels of IL-6 and CRP were decreased. While no patients died during hospitalization, three cases underwent treatment-associated side effects, specifically heart failure, liver dysfunction, and allergic rash [101] . To provide a well-conceptualized overview, we summarized the results of previous studies that investigated the therapeutic roles of MSCs in COVID-19 in Table 1 .

In an open-label randomized cohort study conducted by Sengupta et al., the efficiency and safety of allogenic BM-MSC-derived exosomes (ExoFlo TM ) have been investigated for the first time for patients with COVID-19. A single dose of ExoFlo (15 ml via the IV route) was infused into 24 critical patients with COVID-19. They showed that MSC-derived exosomes can downregulate cytokine storm, restore oxygenation, and reconstitute immunity in hospitalized patients with severe conditions. No adverse effects were attributed to the treatment and significant reduction of CRP, ferritin, and D-dimer were seen. Also, the absolute neutrophil/lymphocyte count and oxygenation were improved [102] . Up to now, 8 clinical trials using MSC-derived EVs to treat COVID-19 are registered in clinicaltrials.gov, among which 4 are still in progress ( Table 2 ). Fig. 4 provides a summary of the therapeutic roles of MSCs and MSCs-derived EVs in COVID-19.

Several key points in MSC therapy should be considered to improve the capacity of MSCs in treating COVID-19, including administration route, the period window of administration, optimal dose, source of administration, and last but not least, dose frequency. IV infusion seems to be the most favorable mode of MSC delivery in ARDS and COVID-19 in the majority of clinical trials. It is the least invasive in comparison to intra-arterial or tissue injection. More importantly, due to the trapping property of MSCs, most of the cells injected via the IV route remain in the lungs that is the main organ affected mostly in ARDS and COVID-19. Also, IV administrated MSCs might be beneficial for the recovery of other organs as severe cases might suffer from MOD syndrome. However, the safety issues should be considered during IV delivery of MSCs in COVID-19 patients because of high pro-coagulant tissue factor (TF/ CD142) expression in MSC products. Anticoagulation issues might have less significance in other routes of delivery such as intramuscular leading the cells into the extravascular space directly [112] . The most frequent doses for transfusion of human MSCs range from one-two million cells/kg and never exceed a dose of twelve million cells/ kg. The median dose is 1 × 10 8 cells/patient in the IV route, however, the efficiency dose-response results have shown a narrower minimal effective dose between 100 and 150 million, and less efficacy is attributed to either higher or lower doses [13, 113] . The timing of MSC administration is also significant as the life-threatening cytokine storm, ARDS, and organ failure occur quickly. It was suggested that when symptoms are progressively getting worse, it is the optimum time for MSCs transplantation [63] .

There is a preference for allogeneic transplantation of MSCs over the autologous one in the case of COVID-19 due to the time limitation for treatment, especially in severe cases [114] . On the other hand, MSC capacities are affected by age of the donor. It has been reported that MSCs received from older donors have shown a lower proliferation rate, poor homing, reduced immunomodulatory properties, and a higher percentage of apoptotic cells [115] . This means that autologous alternative is questionable in the elderly due to their liability to suffer severe COVID-19. MSCs from different sources also vary in their biological properties. According to a systematic review study by McIntyre et al., BM-MSCs and UC-MSCs are more effective in the treatment of acute lung disease [116] . However, because of the relatively low count of MSCs from BM, it might not be sufficient for the systematic disease caused by the SARS-CoV-2 virus, and among other available sources, MSCs obtained from umbilical cord __ which has a higher concentration of stem cells and also obtained by a non-invasive method __ seems the most desirable. Moreover, UC-MSCs have a fast doubling time and they could be expanded faster in culture, providing enough cells for multi-organ damage in COVID-19 cases. UC-MSCs have a gene expression profile similar to embryonic MSCs; they have a higher proliferation speed, more plasticity, and unlike ESCs, possess no tumorigenic effects [117] [118] [119] . Taken together, adjusting the safe production of MSCs, in addition to settling an agreement on clinical trials concerning MSCs source and dose, administrative time, and mode of delivery might be pivotal for an effective and safe MSC-based therapy in COVID-19. Notably, pre-clinical animal data recommend the augmented MSCs either by the cytokine pre-activation, genetic engineering, or reprogramming to boost their threats potency [120] , and future investigations should focus to develop modified MSCs that could safely and efficiently exert therapeutic effects.

Several clinical and pre-clinical studies have investigated the role of MSCs and their extracellular vesicles in the treatment of SARS-CoV-19 disease. Although the results of preliminary reports are promising, further trials in a larger cohort of patients are required to validate the definite safety and efficiency of MSCs and MSCs-derived EVs in mollifying symptoms associated with the COVID-19 infection. As per the time of submission of this review -April 2021, there are 74 ongoing studies related to the use of MSCs from various sources in the treatment of COVID-19 that are registered on clinicaltrial.gov. (Table 3) .

Within the past decade, MSC-based therapies have gained interest among researchers because of their promising therapeutic roles in the treatment of inflammatory diseases. However, in regards to COVID-19, most of the clinical trials are yet ongoing and research concerning MSCs and their EVs is in its infancy. There have been only a few published results from clinical trials using MSCs for COVID-19 with none of them enrolled a large group of patients and with a detailed report of adverse effects. Therefore, despite the mentioned advantages attributed to MSCs application in lung injuries including COVID-19, there are still issues requiring to be resolved for the successful application of these cells. One of them involves the clinical-grade production of MSCs in this pandemic situation since a growing number of people are being infected with SARS-CoV-2, many of whom might be eligible for cell-based therapy meaning that we require to have a product ready-to-use on the shelf. Moreover, there are plenty of concerns regarding the stability of MSCs preparation. During ex vivo culture and passaging, MSCs go through cellular and molecular shifts [121] . The changes affect the proliferation, differentiation, homing to the site of injury, secretion of paracrine factors, the exhibition of morphological changes, and even probability of malignant transformation [122] . Previous studies reported that, in a specific vision, SCs may be compared to malignant cells as they can proliferate for a long period, suppress antitumor immunity, and escape apoptosis. Besides, pro-angiogenic factors such as platelet-derived growth factor (PDGF), VEGF, HGF, and Ang-1 released by MSCs can trigger angiogenesis [123] . This means the long-term evaluation of patients who received MSC therapy may be necessary to follow-up their tumorigenic capacity, especially in elder cases with compromised antitumor immunity.

Last but not least, it is reported that some cases of critically ill COVID-19 are highly prone to disseminated intravascular coagulation (DIC) and thromboembolism due to their pro-coagulant state. On the other hand, MSC products from various sources, especially AT-derived MSCs, express high levels of tissue factor (TF). TF is recognized as one of the crucial determinants of cell product hemocompatibility which can trigger instant blood-mediated inflammatory reaction (IBMI) following activation of the host innate immune cascade systems, including complement and coagulation by infused MSCs [124] . Being aware of the risk of lethal thrombotic complication, especially for the reactivated patients, alternative non-IV regimes, standardized hemocompatibility testing for new applied MCs, and administration of anticoagulant before application of high doses of MSCs should be seriously considered [125, 126] . All in all and taking all these concerns into account, we propose that MSCs-based therapeutic strategies should apply for COVID-19 cases only when its significant medical justifications and safety concerns are determined.

Millions of people have been influenced with COVID-19 with no verified treatment available. Currently, supportive therapies, as well as non-specific anti-viral drugs, are mainly used for this purpose; however, MSC therapy seems to be a promising answer to this pandemic. ARDS, the major cause of respiratory failure that is characterized by injury to both the pulmonary endothelium and alveolar epithelium, is the most common complication of COVID-19 cases requiring mechanical ventilation and ICU support. IV-infused MSCs migrate directly to the lungs and secret a wide variety of factors to diminish pro-inflammatory cytokines, reduce pulmonary edema, elevate the rate of AFC, and inhibit cellular apoptosis. MSC-derived EVs has been also shown to downregulate cytokine storm, restore oxygenation, and reconstitute immunity in hospitalized COVID-19 patients with a severe condition. Notably, compared with their parents, exosomes seem to be safer because they cannot replicate and form a tumor, they are non-immunogenic, and they do not lead to emboli formation. Despite the safety and efficacy of MSCs in cell-based therapies have been shown in Phase I and Phase II of numerous clinical trials and several tens of them already made their way to Phase III, this field is in its infancy in COVID-19 and further studies regarding the ideal source and dose, optimum route of administration, and the time window of MSCs administration remains to be done. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The authors would like to express their gratitude to Shahid Beheshti University of Medical Sciences (Tehran, Iran) for supporting this study.

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

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Mesenchymal stem cells: mechanisms of immunomodulation and homing

Human mesenchymal stem cells modulate B-cell functions

Human mesenchymal stem cells modulate allogeneic immune cell responses

Interactions between mesenchymal stem cells and the immune system

Transplantation of ACE2-mesenchymal stem cells improves the outcome of patients with COVID-19 pneumonia

Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome

Allogeneic human mesenchymal stem cells restore epithelial protein permeability in cultured human alveolar type II cells by secretion of angiopoietin-1

Therapeutic targeting of the angiopoietin-TIE pathway

The hepatocyte growth factor-expressing character is required for mesenchymal stem cells to protect the lung injured by lipopolysaccharide in vivo

Mesenchymal stem cells in idiopathic pulmonary fibrosis

Synergism of MSCsecreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway

Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis

Mesenchymal stem cells reduce hypoxiainduced apoptosis in alveolar epithelial cells by modulating HIF and ROS hypoxic signaling

Biological properties of extracellular vesicles and their physiological functions

Therapeutic potential of mesenchymal stem cell-derived microvesicles

Mesenchymal stem cell derived exosomes: a nano platform for therapeutics and drug delivery in combating COVID-19

Therapeutic use of mesenchymal stem cell-derived extracellular vesicles in acute lung injury

Therapeutic effects of human mesenchymal stem cell-derived microvesicles in severe pneumonia in mice

Mesenchymal stem cell microvesicles restore protein permeability across primary cultures of injured human lung microvascular endothelial cells

Mesenchymal stromal cells modulate macrophages in clinically relevant lung injury models by extracellular vesicle mitochondrial transfer

Mesenchymal stem cell microvesicles attenuate acute lung injury in mice partly mediated by Ang-1 mRNA

Mitochondrial transfer from bonemarrow-derived stromal cells to pulmonary alveoli protects against acute lung injury

Therapeutic use of extracellular vesicles for acute and chronic lung disease

Systemic administration of human bone marrow-derived mesenchymal stromal cell extracellular vesicles ameliorates aspergillus hyphal extract-induced allergic airway inflammation in immunocompetent mice

Exosomes mediate the cytoprotective action of mesenchymal stromal cells on hypoxia-induced pulmonary hypertension

Mesenchymal stromal cell exosomes ameliorate experimental bronchopulmonary dysplasia and restore lung function through macrophage immunomodulation

Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology

Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study, The Lancet

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Mesenchymal stromal cells and viral infection

Mesenchymal stem cells are susceptible to human herpesviruses, but viral DNA cannot be detected in the healthy seropositive individual

Intrinsic immunity shapes viral resistance of stem cells

Bone marrow mesenchymal stem cells can differentiate into type II alveolar epithelial cells in vitro

Clinical remission of a critically ill COVID-19 patient treated by human umbilical cord mesenchymal stem cells: A case report

Treatment of severe COVID-19 with human umbilical cord mesenchymal stem cells

Human umbilical cord-derived mesenchymal stem cell therapy in patients with COVID-19: a phase 1 clinical trial

Placenta-Derived Cell Therapy to Treat Patients With Respiratory Failure Due to Coronavirus Disease

Immunity-and-Matrix-Regulatory Cells Derived from Human Embryonic Stem Cells Safely and Effectively Treat Mouse Lung Injury and Fibrosis

Adipose-derived mesenchymal stromal cells for the treatment of patients with severe SARS-CoV-2 pneumonia requiring mechanical ventilation A proof of concept study

Mesenchymal stem cell therapy in severe COVID-19: A retrospective study of short-term treatment efficacy and side effects

Exosomes derived from bone marrow mesenchymal stem cells as treatment for severe COVID-19

Double-blind, randomized, controlled, trial to assess the efficacy of allogenic mesenchymal stromal cells in patients with acute respiratory distress syndrome due to COVID-19 (COVID-AT): A structured summary of a study protocol for a randomised controlled trial

Treatment with allogeneic mesenchymal stromal cells for moderate to severe acute respiratory distress syndrome (START study): a randomised phase 2a safety trial

Mesenchymal stem cell treatment in a critically ill COVID-19 patient: a case report

Umbilical cord mesenchymal stem cells for COVID-19 acute respiratory distress syndrome: A double-blind

Repair of Acute Respiratory Distress Syndrome by Stromal Cell Administration in COVID-19 (REALIST-COVID-19): A structured summary of a study protocol for a randomised

Human umbilical cord mesenchymal stem cells for adjuvant treatment of a critically ill COVID-19 patient: A case report

Mesenchymal stem cells derived from perinatal tissues for treatment of critically ill COVID-19-induced ARDS patients: a case series

Clinical study using mesenchymal stem cells for the treatment of patients with severe COVID-19

Evaluation of the safety and efficacy of using human menstrual blood-derived mesenchymal stromal cells in treating severe and critically ill COVID-19 patients: An exploratory clinical trial

MSC therapies for COVID-19: importance of patient coagulopathy, thromboprophylaxis, cell product quality and mode of delivery for treatment safety and efficacy

Trends in mesenchymal stem cell clinical trials

Current status of mesenchymal stem cell therapy for immune/inflammatory lung disorders: Gleaning insights for possible use in COVID-19

Aging alters tissue resident mesenchymal stem cell properties

Efficacy of mesenchymal stromal cell therapy for acute lung injury in preclinical animal models: a systematic review

Umbilical cord as prospective source for mesenchymal stem cell-based therapy

Stem cells in the umbilical cord

Umbilical cord-derived mesenchymal stem cells: their advantages and potential clinical utility

Safety profile of good manufacturing practice manufactured interferon γ-primed mesenchymal stem/ stromal cells for clinical trials

Aging of mesenchymal stem cell in vitro

Long-term cultures of bone marrow-derived human mesenchymal stem cells frequently undergo spontaneous malignant transformation

Ethical and safety issues of stem cell-based therapy

Mesenchymal stromal cell therapeutic delivery: translational challenges to clinical application

Attention should be paid to venous thromboembolism prophylaxis in the management of COVID-19

Intravascular mesenchymal stromal/stem cell therapy product diversification: time for new clinical guidelines