Investigational Use of Mesenchymal Stem/Stromal Cells and Their Secretome as Add-On Therapy in Severe Respiratory Virus Infections: Challenges and Perspectives

Serious manifestations of respiratory virus infections such as influenza and coronavirus disease 2019 (COVID-19) are associated with a dysregulated immune response and systemic inflammation. Treating the immunological/inflammatory dysfunction with glucocorticoids, Janus kinase inhibitors, and monoclonal antibodies against the interleukin-6 receptor has significantly reduced the risk of respiratory failure and death in hospitalized patients with severe COVID-19, but the proportion of those requiring invasive mechanical ventilation (IMV) and dying because of respiratory failure remains elevated. Treatment of severe influenza-associated pneumonia and acute respiratory distress syndrome (ARDS) with available immunomodulators and anti-inflammatory compounds is still not recommended. New therapies are therefore needed to reduce the use of IMV and the risk of death in hospitalized patients with rapidly increasing oxygen demand and systemic inflammation who do not respond to the current standard of care. This paper provides a critical assessment of the published clinical trials that have tested the investigational use of intravenously administered allogeneic mesenchymal stem/stromal cells (MSCs) and MSC-derived secretome with putative immunomodulatory/antiinflammatory/regenerative properties as add-on therapy to improve the outcome of these patients. Increased survival rates are reported in 5 of 12 placebo-controlled or open-label comparative trials involving patients with severe and critical COVID-19 and in the only study concerning patients with influenza-associated ARDS. Results are encouraging but inconclusive for the following reasons: small number of patients tested in each trial; differences in concomitant treatments and respiratory support; imbalances between study arms; differences in MSC source, MSC-derived product, dosing and starting time of the investigational therapy; insufficient/inappropriate reporting of clinical data. Solutions are proposed for improving the clinical development plan, with the aim of facilitating regulatory approval of the MSC-based investigational therapy for life-threatening respiratory virus infections in the future. Major issues are the absence of a biomarker predicting responsiveness to MSCs and MSC-derived secretome and the lack of pharmacoeconomic evaluations.

immunomodulators and anti-inflammatory compounds is still not recommended. New therapies are therefore needed to reduce the use of IMV and the risk of death in hospitalized patients with rapidly increasing oxygen demand and systemic inflammation who do not respond to the current standard of care. This paper provides a critical assessment of the published clinical trials that have tested the investigational use of intravenously administered allogeneic mesenchymal stem/stromal cells (MSCs) and MSC-derived secretome with putative immunomodulatory/antiinflammatory/ regenerative properties as add-on therapy to improve the outcome of these patients. Increased survival rates are reported in 5 of 12 placebo-controlled or open-label comparative trials involving patients with severe and critical COVID-19 and in the only study concerning patients with influenza-associated ARDS. Results are encouraging but inconclusive for the following reasons: small number of patients tested in each trial; differences in concomitant treatments and respiratory support; imbalances between study arms; differences in MSC source, MSC-derived product, dosing and starting time of the investigational therapy; insufficient/ inappropriate reporting of clinical data. Solutions are proposed for improving the clinical development plan, with the aim of facilitating regulatory approval of the MSC-based investigational therapy for life-threatening respiratory virus infections in the future. Major issues are

INTRODUCTION
Acute respiratory tract infections are among the commonest infectious diseases [1,2]. Until December 2019, the most serious and prolonged outbreaks of these diseases had been observed with infections caused by strains of the influenza viruses type A and type B and by coronaviruses such as the severe acute respiratory syndrome coronavirus and the Middle East respiratory syndrome coronavirus [1][2][3]. Nonetheless, respiratory mortality associated with seasonal influenza has remained elevated worldwide even outside periods of major outbreaks caused by new strains, with global influenza-associated respiratory deaths ranging between 291,243 and 654,832 annually (4.0-8.8 deaths per 100,000 individuals), according to the latest estimate published in March 2018 [4].
The first outbreak of a pneumonia associated with a new coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was reported in China in December 2019 and was followed by a rapid spreading of the infection worldwide [1,5]. At the time of this writing (22 September 2022) the illness caused by SARS-CoV-2 and termed coronavirus disease 2019 (COVID-19) has already killed more than 6.5 million individuals and the pandemic is not over yet [6]. The devastating impacts on healthcare systems, economies, and education and social relationships have resulted in a global crisis with no precedent since the Second World War [7][8][9]. Highly efficacious vaccines have been developed in less than 1 year from SARS-CoV-2 identification [10] and the speed at which many countries have rolled out the vaccination program is unparalleled [9][10][11]. High levels of immunity induced by the mass vaccination efforts and by natural infections have greatly changed the course of the COVID-19 pandemic, but the continued generation of highly transmissible and virulent SARS-CoV-2 genetic variants capable of evading the existing level of immunity and still causing multiple waves of infections is hampering transition of COVID-19 from the pandemic to an endemic phase [12].
Like influenza viruses, SARS-CoV-2 can cause severe pneumonitis and acute respiratory distress syndrome (ARDS) with high frequency in the elderly, in immunocompromised patients, and in those with comorbidities such as obesity, diabetes, chronic cardiovascular disorders, and kidney and liver diseases [1,2,4,[13][14][15]. Invasive mechanical ventilation (IMV) is commonly needed in critically ill patients admitted to intensive care units (ICUs) for seasonal influenza and COVID-19, but patients with COVID-19 require longer duration of IMV and are at greater risk of mortality during the hospitalization than patients with influenza, irrespective of age, sex, and comorbidities [15]. The survivors may not recover completely and may suffer from disabling symptoms for the rest of their lives.
Because the influenza viruses and SARS-CoV-2 can cause serious pneumonitis and ARDS in the same groups of individuals, even minor outbreaks of COVID-19 occurring with a simultaneous influenza wave in the Northern Hemisphere in late autumn and winter could lead to another surge in admissions to ICUs and deaths. In addition, the prevalence of coinfections, which are associated with increased odds of ICU admission and death in those individuals [16], may also escalate because of the easing of non-pharmaceutical measures that greatly reduced the circulation of SARS-CoV-2 as well as the circulation of influenza viruses in 2020-2021, the current absence of a systematic virologic surveillance [17][18][19], and the limited effectiveness of influenza vaccines in high-risk subjects [19].

RATIONALE FOR THE POTENTIAL USE OF MSCS AND MSC-DERIVED SECRETOME AS ADD-ON THERAPY
The severe life-threatening manifestations of influenza and COVID-19 are associated with a dysregulated immune response and hyperproduction of proinflammatory cytokines and chemokines such as interleukin (IL)-1b, IL-6, tumor necrosis factor (TNF)-a, interferon (IFN)-c inducible protein-10, monocyte chemoattractant protein-1, and IL-8 [20][21][22][23][24][25]. The unchecked immunological/inflammatory alterations lead to further tissue damage [20][21][22][23][24][25], in addition to that caused by virus replication alone, and to increased risk of thrombosis [26] not responding to anticoagulation alone [27]. Targeting the immunological/inflammatory dysfunction with glucocorticoids, Janus kinase inhibitors, and humanized monoclonal antibodies against the IL-6 receptor (IL-6R) [28][29][30], in addition to providing maximal supportive therapy [31], significantly reduces the risk of respiratory failure and death in hospitalized patients with severe COVID-19-associated pneumonia, hypoxia, and evidence of systemic inflammation, but the residual numbers of individuals requiring IMV and dying because of respiratory failure remain elevated in the clinical trial setting [28][29][30] and in clinical practice [32]. The evaluation of the efficacy of immunomodulators and antiinflammatory compounds in severe influenza-associated pneumonia and ARDS has generated conflicting results and there is evidence of detrimental effects of glucocorticoids in influenza-related ARDS [31,33,34]. Effective therapeutic options are therefore needed to reduce the use of IMV and the risk of death in hospitalized patients with rapidly increasing oxygen demand and systemic inflammation who do not respond to the evidence-based therapeutic regimen currently recommended by international guidelines (Table 1) [19,35].
Because of this unmet need, the therapeutic potential of mesenchymal stem/stromal cells (MSCs) and MSC-derived products is under evaluation in a huge number of clinical trials [36][37][38][39][40] on the basis of the results of preclinical studies that have demonstrated the ability of intravenously injected MSCs to transiently accumulate in the pulmonary circulation and to exert multiple beneficial effects, including the modulation of immunological responses, the prevention of bacterial superinfections, the promotion of the repair of damaged alveolocapillary barriers, and the alleviation of fibrosis in the injured lungs [40][41][42][43][44][45][46], mainly through paracrine signaling [42][43][44][45]. The MSCs under evaluation are a heterogeneous population of self-renewable multipotent cells that are most commonly harvested from the perinatal tissues (umbilical cord tissue, umbilical cord blood, or placenta), the menstrual blood, adult bone marrow or adult adipose tissue of one or more healthy unrelated donor(s) and are expanded in culture to large quantities for treating many patients [39,42,47,48]. The investigational therapy is either the allogeneic population of MSCs expanded in culture or its secretome, which is composed of soluble factors and extracellular vesicles such as exosomes and microvesicles [38,45]. It is widely recognized that the allogeneic MSCs under evaluation only acquire immunomodulatory properties in inflammatory conditions [49]. The induction of the expression of a predominant immunosuppressive phenotype is known as MSC licensing and has been reported to be elicited in the circulation and at the tissue sites by IFN-c [50,51], particularly in the concomitant presence of one of the proinflammatory cytokines TNF-a, IL-1a, and IL-1b [52]. The importance of the licensing activity of IFN-c is supported by the results of studies in an animal model of graft versus host disease (GVHD), where the recipients of IFN-c -/-T lymphocytes did not respond to treatment with bone-marrow-derived MSCs and died [53].
Licensed MSCs acquire the ability to generate powerful immunoregulatory effects by modulating the proliferation and function of diverse cells involved in the innate and adaptive immunity through the release of biologically active soluble molecules and extracellular vesicles, and the transfer of mitochondria via intercellular communication [49,54] (Fig. 1). Soluble factors with immunomodulatory properties include indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), transforming growth factor (TGF)-b, and IL-1R antagonist (IL-RA). Through the release of IDO and PGE2, MSCs can reduce the proliferation, cytotoxic activity, and cytokine production of effector T lymphocytes, and the proliferation of B lymphocytes. Importantly, MSCs can favor the differentiation and expansion of functional regulatory T lymphocytes (Treg) through IDO, PGE2, cyclooxygenase (COX)-2, and TGF-b [49,54] (Fig. 1). Furthermore, they can promote the generation of IL-10-producing regulatory B cells (Breg) that inhibit the differentiation of effector T cells into T helper-17 (Th-17) lymphocytes [49,54] (Fig. 1). MSCs can also block the activation of effector immune cells via cellto-cell interaction through the association of the programmed death (PD)-1 and its ligand PD-L1 [49]. In presence of macrophage colonystimulating factor (M-CSF), MSCs promote the differentiation of monocytes and type 1 macrophages with proinflammatory activity into M2 type macrophages with antiinflammatory and regenerative properties, which produce IL-10 and TGF-b. MSCs also inhibit the  (Fig. 1). In addition to the process of MSC licensing described above, another mechanism has been recently proposed to explain the immunomodulatory function of intravenously injected allogeneic MSCs [49,55]. The infused cells would undergo apoptosis by interaction with the granules released by cytotoxic CD8 lymphocytes and natural killer (NK) cells of the host, and the apoptotic cells would be taken up by the circulating mononuclear phagocytes. This efferocytosis would induce a sort of reprogramming of the phagocytic cells of the MSC recipient, which would produce PGE2 and IDO themselves and in this manner mediate the immunosuppressive effects of MSCs. The two https://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors(s) and the copyright owner(s) are credited, the original publication is cited, the link to the license is given, and it is indicated if changes were made. CD cluster of differentiation, CTL cytotoxic T lymphocytes, EVs extracellular vesicles, FasL Fas ligand, HGF hepatocyte growth factor, IDO indoleamine 2,3-dioxygenase, IFN interferon, IL interleukin, IL1-RA interleukin 1 receptor antagonist, MHC major histocompatibility complex, M-CSF macrophage colony-stimulating factor, miRNA microRNA, OX40L OX40 ligand, PD-L1 programmed death-ligand 1, PGE2 prostaglandin 2, Th T helper lymphocyte, TGF transforming growth factor, TNF tumor necrosis factor, Treg T regulatory lymphocyte, TSG tumor necrosis factorstimulated gene mechanisms may coexist because there is evidence that viable MSCs cannot be replaced with apoptotic or dead MSCs from a therapeutic perspective [56], and more studies are required to clarify this issue. In addition, it would be important to understand if the reprogramming of phagocytic cells depends at least in part on the biological activity of the extracellular vesicles of the apoptotic MSCs and whether it can also occur with the infusion of MSC-derived extracellular vesicles, which are in large part taken up by the phagocytes of the reticuloendothelial system because of their size.
MSC tracking experiments in an animal model of infectious pneumonitis [57], using real-time, intravital imaging of the kinetics of MSCs in lung vessels, demonstrated the immediate influx of MSCs following their intravascular injection and their persistence in the alveolar capillaries for more than 24 h. Moreover, MSC administration was associated with improvements in the gas-exchange function of the alveolar-capillary barrier, resulting in increased arterial oxygen levels [57]. Figure 2 schematically shows the possible mechanisms through which intravenously injected allogeneic MSCs, or MSC-derived extracellular vesicles, can restore the impaired gas exchange and counteract the effects of the dysregulated immune response and persisting inflammation in severe and critical COVID-19 and influenza. The indicated effects of MSCs and MSC-derived extracellular vesicles are based on the immunomodulatory properties of MSCs discussed above and the results of preclinical studies in animal models of lung injury associated with influenza virus infection [58][59][60][61] or in experimentally-induced ARDS [62]. The figure also shows a possible mechanism through which MSCs become or remain not permissive to viral growth in the inflamed alveoli. This is related to their ability to express INF-stimulated genes in response to INFs such as the type I IFNs (IFN-a/b) produced by infected alveolar epithelial cells and the IFN-c present in the inflammatory infiltrate [63].
The currently approved pharmacological treatment for severe and critical COVID-19 includes the glucocorticoid dexamethasone as standard of care, the humanized monoclonal antibody of the IgG1 class against the IL-6R tocilizumab, and the Janus kinase inhibitor of the JAK/1/JAK2 subtype baricitinib as adjunctive therapies (Table 1) [19,35]. Dexamethasone is known to have a broad antiinflammatory activity, and the transcriptomic data on pulmonary and circulating immune cells from patients with severe COVID-19 has suggested that the therapeutic effect of this glucocorticoid in this disease may be specifically related to TNF-a, IL-1a, IL-1b, IFN-a, and IFN-c signaling but does not involve the IL-6 pathway [64]. Tocilizumab targets IL-6-mediated signal transduction by binding to both the transmembrane and the soluble receptors, and in so doing it irreversibly blocks the proinflammatory and prothrombotic effects of IL-6 for 2-3 weeks, as well as its still desirable effects on the development of an acute-phase response against infections and on the enhancement of bacterial phagocytosis [65]. Baricitinib predominantly blocks IL-6 and INF-c signaling and IL-10 and IFN-a signaling to a lesser extent [66], and similarly to dexamethasone, has a short halflife. Both dexamethasone and baricitinib inhibit the function of type I IFNs involved in viral clearance and must be administered in combination with antivirals in immunocompromised patients. Baricitinib also reduces, albeit to a lesser extent than other Janus kinase inhibitors [66], the desirable regulatory activity of IL-10, which is related to its ability to promote the emergence of Tregs while suppressing the development of Th-17 lymphocytes. None of these therapeutic agents has the direct effects mediated by whole MSCs and by their extracellular vesicles on the viability of alveolar epithelial cells, the repair of the alveolar-capillary barrier, and the prevention of the development of secondary bacterial infections (Fig. 2). In severe and critical influenza, the potential biological effects of whole MSCs or of their extracellular vesicles are unrivaled, because a combination of antivirals and antiinflammatory or immunoregulators is not allowed (Table 1) [19,35].
A mechanistic rationale therefore emerges for the use of MSC-based therapy as an adjunctive therapy in patients with severe and critical COVID-19 who do not respond to Fig. 2 Mechanistic rationale for investigating the clinical use of mesenchymal stem/stromal cells and their products as adjunctive therapy for the management of severe and critical coronavirus disease 2019 and influenza. The pathological mechanisms leading to alveolar damage, hypoxemia, and systemic inflammation are highlighted in red, and the counteracting effects of intravenously injected allogeneic mesenchymal stem/stromal cells that have been induced to express an antiinflammatory/immunosuppressive phenotype systemically and in the inflamed alveoli are highlighted in green. In severe and critical coronavirus disease 2019, virus replication, the proliferation of effector T lymphocytes, the release of proinflammatory cytokines, and the recruitment of leukocytes from the peripheral blood are inhibited by the recommended treatment with antivirals and the glucocorticoid dexamethasone in combination with a Janus kinase inhibitor of the JAK1/JAK2 subtype, such as baricitinib, or with the humanized antibody against the interleukin-6 receptor tocilizumab. This combination also reduces the systemic effects of viral replication and excessive inflammation, but in abolishing the acute-phase response and the IL-6 mediated enhancement of bacterial phagocytosis, the combination of antiinflammatory agents concurs to render the host more vulnerable to pulmonary and systemic infections. Key adjunctive effects of mesenchymal stem/stromal cells are the following: reestablishment of the regulatory function of subpopulations of T and B lymphocytes (Treg and Breg cells) that normally suppress excessive and deleterious immunological/inflammatory responses; activation of the mechanisms involved in the repair of the alveolar-capillary barrier via the release of soluble factors (Ang-1, HGF, and KGF) and extracellular vesicles delivering microRNAs; enhancement of the viability of alveolar epithelial cells through the transfer of healthy mitochondria by intercellular communication; prevention of the development of secondary bacterial infections by producing antimicrobial peptides and by enhancing the phagocytic activity of neutrophils and macrophages through the release of prostaglandin E2. In severe and critical influenza, where a combination of antivirals and antiinflammatory or immunoregulators is not allowed, most of the biological effects of mesenchymal stem/stromal cells highlighted in this figure would be desirable. Generated using in part Science-Slides graphics from VisiScience Corp., licensed use. AEC alveolar epithelial cell, AMPs antimicrobial peptides, Ang angiopoietin, Breg B regulatory lymphocytes, CRP C-reactive protein, CoV coronavirus, COX cyclooxygenase, CTL cytotoxic T lymphocytes, EVs extracellular vesicles, FGF fibroblast growth factor, GCs glucocorticoids, HGF hepatocyte growth factor, IAV influenza virus, IDO indoleamine 2,3-dioxygenase, IFN interferon, IL interleukin, IL1-RA interleukin 1 receptor antagonist, IL-6R interleukin-6 receptor, ISGs interferonstimulated genes, JAKis Janus kinase inhibitors, KGF keratinocyte growth factor, miRNA microRNA, NK natural killer, PGE2 prostaglandin 2, RLR retinoic acid-inducible gene-1-like receptor, TCR T cell receptor, TGF transforming growth factor, Th T helper lymphocyte, TLR toll-like receptor, TNF tumor necrosis factor, Treg T regulatory lymphocyte, TSG tumor necrosis factor-stimulated gene dexamethasone, and in patients with severe and critical influenza who show increasing oxygen demand and systemic inflammation on treatment with antivirals alone. The combination of an MSC-based therapy and glucocorticoids has been already used for the treatment of GVHD and excellent results have been reported in terms of safety and efficacy [67]. Moreover, glucocorticoids at high doses are used as standard of care in studies testing the potential additional benefits of MSC-based therapies in GVHD [68]. The potential future integration of investigational MSC-based therapy into the currently recommended therapeutic management of hospitalized adult patients with severe and critical COVID-19 or influenza is illustrated in Fig. 3.

AIMS AND METHODOLOGICAL APPROACH OF THIS REVIEW
The focus of this review was on the status of the clinical investigations testing the potential use of MSCs and MSC-derived secretome to improve the outcome of patients with severe and critical diseases already managed according to the evidence-based therapeutic approach outlined in  Table 1. The main objectives were the following: to identify progresses in the assessment of the potential added value of the investigational therapy in clinical trials, to highlight unresolved issues, and to discuss how to address them. An extensive literature search was conducted to retrieve all articles reporting on the clinical use of MSCs and MSC-derived products as investigational therapy for lung conditions related to COVID-19 and influenza as described in Table 2. To assess if investigational therapy significantly accelerated the recovery and decreased the mortality of patients with severe or critical diseases in comparison with the recommended/standard therapeutic regimen, controlled prospective clinical trials on the use of the investigational therapy as add-on therapy and single-arm uncontrolled clinical trials on the use of the investigational therapy when the recommended/standard treatments have failed were taken into consideration. Clinical studies in patients in stable conditions on the recommended/standard treatment, clinical studies where the recommended/standard treatment was not described, clinical studies where the recommended/standard treatment did not include antivirals or glucocorticoids or immunomodulators, case series, and case reports were excluded and are reviewed elsewhere [40], together with registered but still unpublished studies.
This review was based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors.

OVERVIEW AND CRITICAL ASSESSMENT OF PUBLISHED CLINICAL TRIAL RESULTS
As of 31 July 2022, 21 published reports were found , including 6 randomized, doubleblind, placebo-controlled trials [71,74,75,81,82,84], 3 randomized, openlabel parallel-group studies [76,77,85], 6 nonrandomized prospective studies with control groups [69,70,72,78,86,89], and 6 prospective, uncontrolled single-arm studies [73,79,80,83,87,88] conducted in diverse countries worldwide (Table 3). A total of 20 reports [69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88] concerned treatment of patients with severe and or critical illness caused by laboratory-confirmed SARS-CoV-2 infection, but 6 of the reported studies also included patients with mild [85] or moderate [69,70,78,79,86] disease, and 1 study [87] was conducted in patients with a condition defined as moderate pneumonia by the investigators, although the reported clinical and laboratory data at baseline (Table 3) reflected a more severe stage according to international guidelines [35,90]. The total number of patients treated with MSCs or MSC-derived products in these studies was 343 (Table 3). One report [89] concerned treatment of patients with ARDS caused by laboratory-confirmed H7N9 influenza virus infection: 17 out of 61 individuals received the investigational therapy, in addition to standard care including antivirals and glucocorticoids, while the others served as control ( Table 3). The characteristics of all studies and their main findings are summarized in Tables 3, 4, 5, and 6. The most frequent source of MSCs was the umbilical cord tissue [70-76, 80-83, 85-87], followed by the menstrual blood [78,84,89] and bone marrow [83,88] (Table 4). In one study the MSC source was adipose tissue [79] ( Table 4). In the report of another study [69] the MSC origin was not mentioned (Table 4). Seven reports [77,79,80,83,85,87,89] lacked the required description of the surface markers and multilineage differentiation ability of MSCs [91], and the only report of a study using extracellular vesicles of the exosome subtype as investigational therapy [83] did not contain the required minimal information about the isolation, analysis, and quantification of the MSCderived product [92]. The report of the study using the secretome of menstrual-blood-derived MSCs as investigational treatment [84] lacked information about the composition of the MSCconditioned medium, which likely contained a mix of extracellular vesicles and soluble factors [93]. The putative immunomodulatory /immunosuppressive properties of the administered MSCs were confirmed by mixed lymphocyte reaction assay only in two studies [81,88], employing umbilical-cord-and bone-marrow- Nonrandomized open-label parallel-group clinical trial on the use of investigational therapy in addition to recommended/standard therapy (add-on therapy) Prospective single-arm study on the use of investigational therapy when recommended/standard therapy has failed

Excluded publications
Any prospective study on the use of investigational therapy in stable patients on recommended/standard therapy Any study on the use of investigational therapy as add-on therapy if recommended/standard therapy was not described Any study on the use of investigational therapy as add-on therapy, or when recommended/standard therapy has failed, if recommended/standard therapy did not include antivirals, glucocorticoids, or immunomodulators, singly or in combination  Table 4). The susceptibility to virus infection of the MSCs to be injected into the circulation of an infected host was tested in a minority of the studies, although infected MSCs may have reduced survival and function as a virus reservoir in the body of the treated patients. The expression of the main cell entry receptor for SARS-CoV-2, the angiotensinconverting enzyme 2 (ACE2), by the infused MSCs was only tested in 2 out of 20 studies concerning COVID-19 [69,78], but the results were reassuring because cells from menstrual blood [78] and those from an unreported origin [69] were marginally ACE2-positive [69] or ACE2-negative [78] ( Table 4). The MSCs from an unreported origin [69] also did not express a protease involved in viral cell entry, the transmembrane serine protease type 2 (TMPRSS2). These results would be in keeping with the in vitro observations that human MSCs from fetal and adult tissues are indeed ACE2-and TMPRSS2-negative and may not be permissive to SARS-CoV-2 [94], but diverse studies have uncovered new mechanisms of viral entry into human host cells [95][96][97][98][99], and these data should be taken into account in clinical trials testing the therapeutic potential of MSCs in COVID-19.
The previously demonstrated susceptibility of human MSCs of bone marrow and cord blood origin to infection with avian influenza A H5N1 virus [100] was not excluded in the only study evaluating the potential therapeutic effectiveness of MSCs in H7N9-influenza-induced ARDS [89]. Thus, the possibility that the various MSC populations infused in the studies reviewed here were infected by the hosted viruses, once injected, cannot be excluded. Nonetheless, the viral load over time was not significantly affected by MSC treatment in the randomized, double-blind, placebo-controlled trials that properly evaluated this outcome in COVID-19associated ARDS [74,82].
The studies testing MSCs in COVID-19 differed greatly in terms of patient selection, MSC dosage, and infusion schedule (Tables 3 and 4). The outcome measures and follow-up periods also varied greatly (Tables 3, 6), precluding the possibility to perform meaningful meta-analyses, as previously recognized by the authors of recent systematic reviews not specifically focusing on the assessment of MSCs and MSCderived products as add-on therapy to the currently recommended standard of care [101][102][103][104]. Considering the safety outcomes, most reports did not contain complete and                        Ctrl group: 1 death due to bacterial septic shock at D 38 after the first infusion   convincing information about the incidence of serious and non-serious adverse events and their relation to treatment (Table 5). For example, in the prospective single-arm study on severe ARDS not responding to standard treatment [73] and in the prospective single-arm study on moderate pneumonitis [87], there was a transient increase in the circulating levels of the fibrin degradation product D-dimer after MSC infusions (Table 6), which was not reported as a treatment-related adverse event although it may reflect the effects of the procoagulant activity of tissue factor (TF)/CD142expressing MSCs from umbilical cord and placenta [105] in patients who already have an increased risk of thrombosis because of the infection. In one of these studies [87], the observed transient increase in the levels of the proinflammatory chemokine IL-6 following MSC infusion (Table 6) was also not reported as an adverse event. It is, however, reassuring to know that in the controlled clinical trial with an open-label follow-up period of up to 1 year [71,72], patients with less severe disease who had received three doses of viable 4 9 10 7 umbilical-cord-derived MSCs/kg of body weight (Table 4) did not show significant reductions in the inhibition rate of neutralizing antibodies against SARS-CoV-2, changes in pulmonary function, alterations of laboratory parameters, or evidence of tumor developments in comparison with the controls (Table 6). In terms of concomitant treatment, some investigators included drugs that were empirically used during the initial phases of the COVID-19 pandemic and that are no longer recommended by current guidelines [35,90], such as hydroxychloroquine and various antivirals used for other infections (Table 3). Nonetheless, in most studies patients received glucocorticoids (Table 3), the current standard of care in severe and critical COVID-19 [35,90], albeit with substantial differences in terms of glucocorticoid type, dosage, and duration of treatment. Three of these studies were randomized, double-blind, placebo-controlled trials of umbilical-cord-derived MSCs in ARDS [74,81,82], and only one [74] demonstrated significantly higher survival rate by day 28 and shorter time to recovery in the group of patients who received MSCs as investigational add-on therapy than in the control group (Table 6). Possible explanations for the discrepancy were MSC dosing ( Table 4) and imbalance of the patients' condition at baseline (Table 3). In the study by Lanzoni and colleagues [74], the mean number of infused cells at each infusion and in total was much higher than in the other two trials, and the cell viability was comparable to that detected in the negative study by Rebellato and colleagues [82], despite the use of thawed cells from frozen cell samples (Table 4). In the same study by Lanzoni and colleagues [74], only 4 of 12 patients (33.33%) in the MSC-treated group versus 7 of 12 patients (58.33%) in the placebo group received IMV, and the baseline levels of IL-6 were significantly higher in the control group (Table 3), suggesting that control patients were at higher risk of death than MSCtreated patients. In the study by Rebelatto and colleagues [82], in which all MSC-treated and placebo-treated patients required IMV in the ICU and received glucocorticoids and anticoagulants as concomitant therapy, the mortality rate was even higher in the group of MSC-treated patients (45.45%) than in the group of patients who received placebo (16.66%), and the MSC-treated patients also showed increased levels of the cardiac troponin I and of creatinine, suggesting further cardiac damage and renal insufficiency between day 4 and day 14 post-treatment (three infusions of 5 9 10 5 cells/ kg of body weight every other day, starting 10.7 days, on average, after symptom onset). In the study by Monsel and colleagues [81], low level of alloimmunization developed in 3 of 21 patients (14.3%) on day 14 post-infusion but an additional 6 of the 21 MSC recipients (28.6%) already had pre-formed antibodies against the human leukocyte antigen of the infused cells before treatment. In this study, antibody-mediated loss of functional cells may contribute to explain the lack of significant effects of the investigational therapy on most efficacy outcome measures, except for a significant decrease of plasma inflammatory markers at day 14 postinfusion (Table 6), but the relative impact cannot be estimated because none of the other studies reviewed here reported data concerning possible alloimmunization.     more rapid and more marked decrease in the levels of CRP in the IT group than in the placebo group, with statistically significant differences at D 3 and D 5; significantly higher reduction of the levels of IL-27, IL-5, IL-17E/ IL-25, IL-18, and growthregulated protein alpha from baseline to D 28 in the IT group than in the placebo group, but no significant difference for the levels of IL-6, IL-1 alpha, IFN-gamma, TNF-alpha, and IL-12 Circulating markers indicative of immune thrombosis, NET, significantly reduced in the IT group Plasma levels of the antibodies against SARS-CoV-2 significantly higher in the IT group than in the ctrl group on D 28 Significantly higher degree of clearance of pulmonary opacities in the severe and critically ill pts in the IT group than in those in the placebo group at D 7 and D 21 by chest CT scan Table 6 continued Author Mortality in  Meng F, et al. [70] 0/9 (0%) 0/9 (0%) IMV in 1/9 pts in the IT group and in 4/9 pts in the ctrl group High-flow oxygen support in 3/9 pts in the IT group and in 5/9 pts in the ctrl group Low-flow oxygen support in 7/9 pts in both groups In pts with most severe disease, PaO2/FiO2 ratio stably improved only in the IT group Time to discharge: 20 D in the IT group and 23 D in the ctrl group No significant differences in duration of symptoms between groups IL-6 decrease within 3 D after the first IT infusion in 2 pts with moderate disease and 2 pts with severe disease, all having high levels of IL-6 at baseline. Similar changes not observed in treated pts with lower levels of IL-6 at baseline and in the ctrl group Chest CT scan results shown for 2 pts with severe disease: better outcome in the treated pts than in the ctrl pts, with improvement detectable on D 6   In another randomized, double-blind, placebo-controlled trial of umbilical-cord-derived MSCs in critical COVID-19 [75], there was a significantly higher survival rate in patients treated with MSCs than in the controls (2.5 times higher overall and 4.5 times higher in patients with more than two comorbidities known to worsen disease outcome), which was associated with a significant decrease in the circulating levels of IL-6 ( Table 6), but the concomitant treatment only included oseltamivir and azithromycin (Table 3), drugs not considered as effective in severe or critical COVID-19 [35,90], and there were no significant differences between groups in terms of length of stay in the ICU and ventilator usage (Table 6). Thus, MSC treatment significantly improved survival in critical COVID-19, but this effect is known to be achievable in similar patients with the very less expensive and easier to use glucocorticoids [35,90].
Taking into account the putative immunomodulatory/immunosuppressive properties of MSCs, it is worth noting that in most placebo-controlled studies involving severely and critically ill patients with COVID-19, investigational therapy was found to significantly reduce the levels of inflammatory markers in comparison with placebo (Table 6). This was not the case for studies involving patients with less severe COVID-19, such as the controlled clinical trial with 1-year follow-up mentioned above [71,72], in which the administration of MSCs as add-on therapy resulted in significant radiological improvements not accompanied by significant functional or laboratory changes by day 28 (Table 6).
Considering the other comparative trials testing MSCs as investigational add-on therapy in COVID-19 [69, 70, 76-78, 85, 86, 88], significantly higher survival rates in the treated groups than in the control groups were reported in three of the eight studies [77,78,88], and the increase in survival rates was more pronounced in critically ill patients than in patients with severe disease (Table 6). In one of these studies [77], which was a randomized open-label parallel-group trial, the length of stay in ICU was also significantly lower in the MSC-treated group than in the control group of critically ill patients. In another randomized, single-blind, placebo-controlled study involving patients with mild, severe, and critical disease [85], the duration of hospital stays (primary endpoint) was significantly shorter in the MSC-treated group than in the placebo group (Table 6). For the patients with severe and critical COVID-19 enrolled in the comparative trials cited above, MSC treatment was associated with significant decreases in the levels of circulating inflammatory markers and/or coagulation markers, and with significantly shorter time to normalization of the total lymphocyte counts in MSC-treated patients than in the controls, although these improvements in laboratory parameters did not consistently translate into reduced mortality and reduced use of IMV (Table 6), possibly because of the low number of enrolled patients. Promising preliminary results were provided by Grégoire and colleagues [88], with the initial analysis of data from the only still ongoing study testing bone-marrow-derived MSCs as add-on investigational therapy in severe ARDS (Tables 3, 4). The concomitant treatment with glucocorticoids and anticoagulants was in keeping with current guidelines [35,90] and efficacy outcome measures were in accordance with those proposed by the World Health Organization (WHO) for COVID-19 clinical research, including the use of the WHO Clinical Progression Scale to evaluate patient trajectory over the course of disease [106]. MSC-treated patients (n = 8) required high-flow oxygen therapy (n = 7) or IMV (n = 1) within 24 h of ICU admission and received three infusions of 1.5-3 9 10 6 clinical-grade cells/kg body weight at an average interval of 3 days starting within 2 days of ICU admission. Matched controls (n = 24) were only retrospectively selected among the patients admitted to the ICU in the same hospital, the major limitation of this study. Although a progression of disease severity was initially observed in two of the seven patients after MSC infusion, survival rate was significantly higher in the MSC-treated group than in the matched control group at day 28 (100% versus 79.2%) and at day 60 (100% versus 70.8%) ( Table 6). The risk of thrombosis was also significantly reduced by the investigational treatment, as indicated by the levels of the fibrin degradation product D-dimer, which were much lower in this group than in the matched control group by day 7 (Table 6).
The two studies testing MSC-derived products in severe COVID-19 [83,84] differed greatly in terms of study design, source of MSCs, type of MSC-derived products, and concomitant treatments. In the randomized, double-blind placebo-controlled trial testing the conditioned medium from menstrual-blood-derived MSCs as investigational therapy in severe disease [84], concomitant treatment agreed with current guidelines [35,90] and the investigational therapy was an effective add-on as it significantly reduced mortality at day 28 and the need for intubation in the treated group in comparison with the control group (Table 6). Treatment was not apparently associated with the occurrence of adverse events, but the adverse event reporting was incomplete, and one patient discontinued after first dose for unknown causes ( Table 5). The second study [83] was a prospective uncontrolled cohort study testing a single intravenous infusion of exosomes from bone-marrow-derived MSCs in patients with mild, moderate, or severe COVID-19-associated ARDS, who showed clinical deterioration for more than 72 h on treatment with the institutional standard treatment, consisting in oxygen support and administration of hydroxychloroquine and azithromycin, two drugs not recommended for the management of similar patients by current guidelines [35,90]. During the follow-up period of 14 days, the overall survival rate was 83%. The recovery rate in the cohort of 20 patients not requiring IMV at baseline was 75%, but the other patients worsened to the point of requiring IMV. In the cohort of patients with severe ARDS at baseline, all three patients remained critically ill, still requiring IMV (Table 6). Overall, a single exosome infusion was associated with a significant improvement of laboratory data, including the markers of inflammation and markers of thrombosis, such as D-dimer (Table 6). The occurrence of treatment-related adverse events was specifically evaluated over a period of 72 h after exosome administration and no adverse events were reported. The cases of pulmonary embolism, acute renal failure, worsening of hypoxic respiratory failure requiring intubation, and four deaths, all occurring at posttreatment days 4-13, were not reported as related to the exosome infusion (Table 5). Thus, investigational therapy was apparently safe and effective at improving the outcome of patients with mild or moderate COVID-19-associated ARDS in comparison with the outcome reported in literature for similar patients [107], even though a large fraction of intravenously injected exosomes is immediately taken up by the mononuclear phagocytes of the reticuloendothelial system in the liver and spleen [108]. However, better outcomes have been reported in clinical trials, for all levels of ARDS severity, with the use of glucocorticoids [107], the current standard of care in severe and critical COVID-19 [35,90], and the addition of Janus kinase inhibitors and monoclonal antibodies against IL-6R, if required [35,90].
Concerning the nonrandomized, open-label, parallel-group study that tested the therapeutic potential of MSCs from menstrual blood when added to standard care in H7N9-influenza-virusinduced ARDS [89] (Table 3), investigational therapy consisted in the intravenous administration of 1 9 10 6 90-95% viable MSCs 3-4 times (Table 4), depending on patients' consent. The group of MSC-treated patients had a significant increase in survival rate and more marked improvement of the inflammatory parameters and D-dimer at discharge than the control group that received standard care alone (Table 6). In addition to the supportive therapy for critically ill patients with multiorgan dysfunction, the concomitant treatment in the MSC-treated group and in the control group included the antivirals recommended by international guidelines (oseltamivir or peramivir) [19] in all patients. Over 50% of them in both study arms also received glucocorticoids, which are not recommended for the treatment of severe influenza because of possible detrimental effects on the outcome [19]. However, the mortality rate in the control group (54.5%) was only slightly higher than that reported in literature [107] for similar patients with severe ARDS [107]. The more than threefold lower mortality rate (17.6%) in the MSC-treated group is impressive but may be explained at least in part by the lower proportion of critically ill patients with severe renal injury at study entry in this group in comparison with the control group (Tables 3 and 6). There was no report of adverse events related to the MSC infusions (Table 5). A 5-year follow-up period was limited to four survivors in the MSC-treated arm and no harmful effects of the MSC transplantation were observed in these subjects (Table 6).

CONCLUSIONS
The results of published clinical studies on the therapeutic potential of MSCs and MSC-derived products in COVID-19 and influenza suggest that MSCs and MSC-derived products may significantly increase the survival of hospitalized patients with severe and critical disease and that this beneficial effect may be related to the putative immunomodulatory/immunosuppressive properties of MSCs and their secretome. However, in COVID-19-associated ARDS, similar or better outcomes have been reported in clinical trials with glucocorticoids alone [107], the current standard of care in severe and critical COVID-19 [35,90], with further improvement attainable with the addition of Janus kinase inhibitors and monoclonal antibodies against IL-6R if required to block disease progression according to current guidelines [90,107]. The studies reviewed here have not consistently demonstrated that adding MSCs or MSC-derived products to this currently recommended therapeutic regimen can reduce the use of IMV and the risk of death in patients still showing rapidly increasing oxygen demand and systemic inflammation despite appropriate therapeutic management. Overall, increased survival rates were observed in 5 of 12 prospective comparative trials that tested MSCs or MSC-derived secretome as add-on therapy in severe and critical COVID-19. Four of these trials were randomized, double-blind, and placebo controlled. The standard of care included glucocorticoids and anticoagulants in all these trials and remdesivir, glucocorticoids, monoclonal antibodies against IL-6R, and anticoagulants in some recent studies, but the proportion of patients receiving these therapeutics in each study varied greatly across studies, and most reports do not contain information about dosing and duration of treatment. The results of the only available study about the use of MSCs as add-on therapy for influenza-associated ARDS are promising, particularly because no immunomodulator/antiinflammatory treatment is currently recommended for severe and critical disease, but the study is a small openlabel trial with imbalances between the arms, which may contribute to explaining the superior efficacy outcome attributed to the MSC infusion. The positive results reported in this study and in 5 of the 12 comparative studies that evaluated the therapeutic added values of MSCs and MSC-derived products in severe and critical COVID-19 must be confirmed in controlled clinical trials conducted in compliance with the current Good Manufacturing Practice and Good Clinical Practice guidelines. Compliance with these guidelines is a condition for the generation of data that can be submitted to the regulatory authorities when seeking the mandatory authorization for the use of a therapeutic candidate outside the setting of a clinical investigation [109].

IMPLICATIONS FOR FUTURE RESEARCH
To comply with the current international guidelines for the clinical development of cell and cell-based therapies, several issues need to be addressed in the design and conduction of future clinical trials and in the reporting of the clinical data. Possible solutions for improving the clinical development plan are proposed in tabular form ( Table 7). The key issue is how to choose the starting time of the MSC-based addon therapy. This challenge is difficult to overcome because the studies reviewed here suggest that blood biomarkers of ongoing inflammation recognized thus far are not consistently predictive of a response to MSCs and MSC-derived secretome when the recommended therapeutic regimen is insufficient to block disease progression. It may be useful to evaluate one of the most recently identified specific biomarkers of prolonged inflammation and dysregulated Table 7 Proposed solutions for the improvement of a development plan aiming for regulatory approval

Challenge Solution
MSC and MSC-derived secretome MSC obtained and characterized according to the criteria of the International Society of Cell & Gene Therapy [91]. Tissue factor and hemocompatibility assessment [105] to be included for the evaluation of the product suitability for intravenous use MSC-derived extracellular vesicles isolated according to the guidelines of the International Society for Extracellular Vesicles [92] MSC-derived conditioned medium characterized for the presence of extracellular vesicles and for the contents of soluble biologically active factors [92,93]. Optimization of formulation and manufacturing [120] Scalable production according to the current Good Clinical Manufacturing guidelines Evaluation of the potency of the product (e.g., immunomodulatory/ immunosuppressive properties assessed by mixed lymphocyte reaction) Use of the Hyperinflammation Syndrome score at enrollment [113,114] Recording and monitoring of C-reactive protein, D-dimers, interleukin-6, serum ferritin concentrations, soluble urokinase plasminogen activator receptor, and leukocyte counts [106,[110][111][112] Main outcome measures All-cause mortality at day 28 and at hospital discharge Clinical progression assessed daily by using the WHO Clinical Progression Scale [106] immune activation, the elevated level of plasma soluble urokinase plasminogen activator receptor, which has been found to predict disease progression in hospitalized patients with COVID-19 and other viral pneumonia more accurately than other inflammatory parameters [109][110][111]. Alternatively, or concomitantly, a composite score such as the COVID-19-associated Hyperinflammation Syndrome score [112,113] may be introduced to evaluate the risk of further disease progression in critically ill patients already receiving the recommended immunomodulatory/antiinflammatory regimen (Table 7), although this score system still needs to be fully validated.
Finally, it should be considered that the high costs of MSC-based therapy would still represent an obstacle to its clinical acceptance [114], even if its effectiveness at reducing the healthcare expenditures associated with the prolonged hospitalizations of critically ill patients were conclusively demonstrated. The costs of obtaining clinical-grade allogeneic MSCs varies depending on the MSC source [114], and the use of cell-free MSC-derived products, such as exosomes and other extracellular vesicles, can greatly increase these costs. The use of properly isolated MSC-derived exosomes would render the costs of the MSC-based therapy particularly high because of the expensive isolation procedure and the necessity to produce a huge number of clinical-grade vesicles to overcome problems with the biodistribution of these nanoparticles after intravenous infusion [108,115]. One of the possible solutions may be to avoid their systemic administration [116], but the first published small single-arm study on the investigational use of repeated doses of aerosolized MSC-derived extracellular vesicles in severe COVID-19 [117] has not provided favorable results in terms of efficacy outcome, and the total dosage of extracellular vesicles (2 9 10 8 nanoparticles per day for 5 consecutive days) is higher than that used for intravenous administration (6 9 10 8 ) [83,118].
Another possible solution would be the systemic administration of exosomes that have been engineered to escape phagocytosis and/or to specifically target the lungs [119], but these manipulations would likely affect some of their desired biological activities against viral respiratory tract infections and would require a lot of preclinical research work before first investigational use in humans, without reducing or even increasing the costs of the final product for the indication discussed here. Thus, the decision to go ahead with a rigorous clinical development of an MSC-based therapy as add-on therapy for the treatment of severe and critical COVID-19, influenza, and other severe viral respiratory infections should also be based on pharmacoeconomic considerations, and studies with an adequate evaluation of the cost-effectiveness of these investigational therapies are highly demanded (Table 7).

ACKNOWLEDGEMENTS
Funding. No external funding or sponsorship was received for this review or publication of this article. The journal's Rapid Service fee was funded by the authors.
Authorship. All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given approval for this version to be published. Disclosures. Sabrina Mattoli is consultant to the European Commission and to the Eureka Association and is shareholder in Novartis Pharma AG and Pfizer Inc. Matthias Schmidt is named as co-inventor on a submitted patent application concerning the generation of engineered exosomes for targeted delivery of biologics to the lungs.
Compliance with Ethics Guidelines. This review was based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors.
Data Availability. All data generated or analyzed during this study are included in this published article.