The rationale of using mesenchymal stem cells in patients with COVID‐19‐related acute respiratory distress syndrome: What to expect

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)‐caused coronavirus disease 2019 (COVID‐19) pandemic has become a global health crisis with an extremely rapid progress resulting in thousands of patients who may develop acute respiratory distress syndrome (ARDS) requiring intensive care unit (ICU) treatment. So far, no specific antiviral therapeutic agent has been demonstrated to be effective for COVID‐19; therefore, the clinical management is largely supportive and depends on the patients' immune response leading to a cytokine storm followed by lung edema, dysfunction of air exchange, and ARDS, which could lead to multiorgan failure and death. Given that human mesenchymal stem cells (MSCs) from various tissue sources have revealed successful clinical outcomes in many immunocompromised disorders by inhibiting the overactivation of the immune system and promoting endogenous repair by improving the microenvironment, there is a growing demand for MSC infusions in patients with COVID‐19‐related ARDS in the ICU. In this review, we have documented the rationale and possible outcomes of compassionate use of MSCs, particularly in patients with SARS‐CoV‐2 infections, toward proving or disproving the efficacy of this approach in the near future. Many centers have registered and approved, and some already started, single‐case or phase I/II trials primarily aiming to rescue their critical patients when no other therapeutic approach responds. On the other hand, it is also very important to mention that there is a good deal of concern about clinics offering unproven stem cell treatments for COVID‐19. The reviewers and oversight bodies will be looking for a balanced but critical appraisal of current trials.


| INTRODUCTION
The coronavirus disease 2019  pandemic, caused by a novel beta coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), possessing a single-strand, positivesense RNA genome (26-32 kilobases in length), 1 is the definition of the greatest health challenge of our times. Since its emergence in Guangdong, Southern China, late last year, the virus has spread to 213 countries in every continent except Antarctica as of late June 2020. 2,3 Cases are still rising, especially in Russia, Brazil, the United States, and the United Kingdom, even though a declining trend was noted in deaths in recent weeks. Countries are struggling with slowing down the spread of the virus by testing and treating patients, quarantining infected citizens, limiting or banning leisure travel, prohibiting large social gatherings, and closing schools. As of 23 June 2020, more than 9.2 million cases have been reported worldwide with approximately 475 000 deaths. 3 The case fatality rate (CFR) varies from 2.3% to 14.8% depending on the demographics of the nation or region, age, severity of the disease, and comorbidities. Overall, the CFR was reported to be approximately 11% worldwide among COVID-19-positive cases, and the ratio of serious or critical cases is approximately 2%. Older adults between the ages of 70 and 80 years have a CFR of 8.0%, and those aged more than 80 years have a CFR of 14.8%. 4 The CFR was reported to 49.0% among critical cases and was significantly high among those with preexisting comorbid conditions-10.5% for cardiovascular disease, 7.3% for diabetes, 6.3% for chronic respiratory disease, 6.0% for hypertension, and 5.6% for cancer. 4 In contrast, more than 50% of children (younger than 18 years) experienced mild symptoms or were asymptomatic, with fewer than 6% of children developing severe symptoms. 5 COVID-19 has an incubation period of 2 to 14 days; the mean incubation period is 5.2 days. 6,7 Although approximately 30% of infected people are asymptomatic, the onset of illness is characterized by a series of clinical symptoms from mild to severe, including fever (98% of patients), cough, shortness of breath and/or chest pain (76%), and myalgia or fatigue (44%). 8 Less common symptoms are sputum production, sore throat, loss of taste and smell, headache, hemoptysis, and diarrhea. Therefore, disease stage is classified according to the patient's clinical symptoms and laboratory findings as (a) mild type: mild clinical symptoms without pneumonia; (b) common type: fever, respiratory tract and other symptoms with pneumonia; (c) severe type: respiratory distress (respiratory rate is higher than 30 times per minute; in resting state, oxygen saturation is lower than 93%; partial pressure of oxygen [PaO 2 ] to fraction of inspired oxygen [FiO 2 ] ratio lower than 300 mmHg); (d) critical type: respiratory failure requiring mechanical ventilation, shock, and other organ failure requiring intensive care unit (ICU) monitoring and treatment. 9 In addition, patients with acute cardiac injury present with tachycardia or bradycardia. Critically ill individuals may also have acidosis and increased lactate. 6 No specific antiviral therapeutic agents or vaccines for  have been proven to be effective so far. Several therapies, such as ribavirin, remdesivir, favipiravir, and oseltamivir, are under investigation, 4,10 but the antiviral efficacy of these drugs is not yet known. The supportive approach includes corticosteroids, antibiotics, anticoagulants, and oxygen therapy. Recently, convalescent plasma therapy for COVID-19 has been under consideration to prove its safety and efficacy. Promising results (32 participants) and 48 ongoing trials were accumulated in a Cochrane analysis. 11 As defined below, cytokine storm (high serum levels of granulo-

| DIAGNOSIS AND INFLAMMATORY PROFILE OF PATIENTS WITH COVID-19
The diagnosis of COVID-19 is performed by the detection of the virus in respiratory secretions (throat, nasopharynx, sputum, and endotracheal aspirates and bronchoalveolar lavage) by sensitive antigen tests such as polymerase chain reaction assay, even though the testing efficiency is around 60% to 70% in sputum and nasal swab samples. 12 Besides common radiographic tests such as x-ray and computed tomography (CT) imaging, more sophisticated serologic and genomic tests are also available including the full genome analysis by

Significance statement
Although mesenchymal stem/stromal cell (MSC) administration is an unproven stem cell therapy approach for COVID-19 patients, there is a growing demand for new therapies among patients and healthcare workers. Due to preclinical findings and few current clinical data set, MSCs possess remarkable immunomodulatory features, and thus have the potential to recover the pulmonary microenvironment, intercept pulmonary fibrosis, and cure lung dysfunction in COVID-19 pneumonia and respiratory distress syndrome.
However, it is also noteworthy to point out that there is a good deal of concern about clinics offering unproven stem cell treatments for COVID-19, and reviewers will be looking for a balanced but critical appraisal of current trials.
next-generation sequencing. 13 As summarized in Table 1, blood test findings include normal/low leukocyte counts with high C-reactive protein (CRP). There may be lymphopenia; fewer than 1000 lymphocytes has been associated with severe disease. The platelet count is usually normal or slightly low. The CRP and erythrocyte sedimentation rate are generally elevated; however, procalcitonin levels are usually normal. A high procalcitonin level may imply a bacterial coinfection.
The aspartate transaminase (AST) and alanine transaminase (ALT), prothrombin time, creatinine, D-dimer, creatinine phosphokinase, and lactate dehydrogenase may be elevated, and high levels are associated with severe disease. 14,15 CT chest scans are usually abnormal even in those with no symptoms or mild disease. However, starting from the common cases, CT imaging shows infiltrates, ground-glass opacities, and subsegmental consolidation. It may also have abnormal results in asymptomatic patients or patients with no clinical evidence of lower respiratory tract involvement. In fact, abnormal CT scans have been used to diagnose COVID-19 in suspected virus-negative cases; many of these patients become positive on repeat testing. 16 According to the laboratory tests published so far, in a subset of patients with COVID-19, clinical progress is associated with an activation of an inflammatory cascade, called "cytokine storm syndrome," which is mainly due to activated T-helper 1 (Th1) and T-helper 17 (Th17) cell responses. 17,18 It is a diverse set of conditions unified by a clinical phenotype of systemic inflammation, multiorgan failure, and hyperferritinemia 19 and is associated with a wide variety of virus infections, such as severe acute respiratory coronaviruses (including SARS-CoV-2), influenza virus, and dengue virus, and noninfectious diseases. 20 Some patients with COVID-19 progress to this hyperinflammatory condition, often with pulmonary involvement resembling the secondary hemophagocytic syndrome, which is commonly triggered by viral infections. 21 This systemic hyperinflammation results in inflammatory lymphocytic and monocytic infiltration of the lung and heart, causing ARDS and cardiac failure. Huang et al 8 noted that patients with COVID-19 both in the ICU and not requiring the ICU demonstrated higher cytokine profiles compared with healthy adults, characterized by increased IL-1β, IL-2, IL-7, IL-8, IL-9, IL-10, IL-17, GCSF, IP-10, MCP-1, MIP-1α, TNF-α, and some growth factors such as platelet-derived growth factor and vascular endothelial growth factor (VEGF) 8 (Table 1). Huang et al further reported that plasma concentrations of IL-2, IL-7, IL-10, GCSF, IP-10, MCP-1, MIP-1α, and TNF-α were higher in patients in the ICU than in patients not in the ICU. On the other hand, plasma levels of IL-5, IL-12p70, IL-15, eotaxin, and RANTES were found to be comparable between healthy adults and infected patients. Predictors of mortality in a recent retrospective study of 150 confirmed COVID-19 cases in Wuhan, China, included elevated IL-6. 22 Taken together, cytokine profiles of patients seem to lead to mortality that is directly related to virus-driven hyperinflammation. 23 As demonstrated in patients with severe acute respiratory syndrome (SARS) more than 15 years ago, elevated levels of T-helper cell-originated proinflammatory cytokines, that is, interferon (IFN)-γ, IL-1B, IL-6, IL-12, IP-10, neutrophil-originated chemokine IL-8, and MCP-1, were associated with pulmonary inflammation and extensive lung damage. 24  Besides the cytokine storm, COVID-19-related clinical manifestations reflect that patients develop lymphocytic endotheliitis as evidenced by the impairment of many organs, including kidney, heart, small intestine, and lungs. 26 Therefore, COVID-19 pathogenesis in the endothelial cells causes a vascular disease state during which 89% of T A B L E 1 The immunological, serological, and histopathological profile of patients with COVID-19
hospitalized patients showed subsegmental vascular enlargement on their initial CT scans. 27 Studies have also reported evidence of a COVID-19-associated coagulopathy. It has been shown that 90% of patients with pneumonia had increased coagulation activity, marked by elevated D-dimer, a fibrin degradation thromboembolic marker 28 predicting a poor prognosis in COVID-19.

| ARDS IN COVID-19
ARDS, by definition, is a clinical state characterized by an acute presentation of severe and refractory hypoxemia, decreased airway compliance, microscopic evidence of diffuse alveolar damage, and bilateral pulmonary infiltrates excluding cardiac-related edema. 29 Respiratory failure from ARDS has been reported as the leading cause of mortality in COVID-19 cases. 22 Around 10% to 20% of patients with severe COVID-19 may develop shortness of breath, frequently in the second week of the disease, which might be accompanied by or progress to hypoxemia. 8 The mean period from onset of symptoms to dyspnea was reported as 5 days, hospitalization 7 days, and the onset of ARDS 8 to 9 days. 14 Respiratory damage will inevitably progress into ARDS, defined as PaO 2 /FiO 2 lower than 300 mmHg, during days 8 to 14 of the illness, as well as resulting in noncardiogenic pulmonary edema and mechanical ventilation. 8,30 ARDS constitutes an acute hypoxemic respiratory failure that originates mainly from an increase in lung endothelial and epithelial permeability, which result in outflow of fluid into alveoli, leading to noncardiogenic pulmonary edema and decreased arterial oxygenation.
Damage to the lung parenchyma results from multiple mechanisms, including direct injury by the inflaming agent (eg, bacteria and their products, viral invasion, and acid injury after aspiration), by harm in resulting from hyperactivation of the immune system, and by mechanical stretch-induced damage caused by mechanical ventilation. 31 The pathologic features of ARDS in alveoli and their microenvironment in COVID-19 greatly resemble those seen in SARS and MERS infections, 32,33 including uni/bilateral diffuse alveolar parenchyma damage with cellular fibromyxoid exudates, desquamation of pneumocytes, and hyaline membrane formation 34 (Table 1)

| MSC SOURCES
MSCs can be isolated from various tissue sources. The selection of the source is based on their logistical, practical, and in vitro characteristics. Currently, the main sources of MSCs are bone marrow, umbilical cord stroma, adipose tissue, and dental pulp.

| Bone marrow
Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that are able to differentiate into mesodermal lineage. 46 They are isolated from bone marrow aspirates and subsequently expand in vitro. They have been shown to express a variety of cell surface markers, including CD44, CD73, CD105, and CD146, but they lack hemopoietic markers CD11b, CD14, CD34, and CD45. 47

| Umbilical cord
Human umbilical cord stroma, which is known as Wharton's jelly, is a rich source of primitive mesenchymal stromal cells (UC-MSCs or WJ-MSCs). UC-MSCs are multipotent cells that can be differentiated into mesodermal as well as to neuronal lineages. 49

| Adipose tissue
Several studies revealed that adipose tissues contain MSCs, termed adipose tissue-derived stem cells (ADSCs). They are preferentially located in the stromal vascular fraction of isolated adipose explants. In conditional cultures, ADSCs exhibit a multipotent differentiation capacity into cell types originated from three germ layers. 59 ADSCs express CD44, CD73, CD90, and CD105 and are negative for CD31 and CD45. 60 ADSCs can induce proliferation of IL-10-producing regulatory B cells that regulate the immune system by anti-inflammatory potential. 61 Therefore, ADSCs can be used in the treatment of diverse immune-related disorders, including graft-vs-host disease. 62 As the isolation of autologous ADSCs is relatively simple and less invasive compared with BM-MSCs, they have been the first choice in many trials. However, ADSCs still exhibited only moderate benefits in clinical trials.

| Dental pulp
Dental pulp MSCs (DP-MSCs), primarily isolated from the dental pulp, 63 can also be isolated from deciduous teeth, apical papilla, periodontal ligament, dental follicles, and gingiva. 64  They have the capacity to differentiate into mesodermal lineage under certain conditions in vitro. 36 However, DP-MSCs also express stemness-related markers similar to embryonic stem cells, such as Oct-3/4, Nanog, and Sox-2. 65 Like MSCs from other tissues, DP-MSCs also have a strong immunomodulatory ability with a higher suppression rate of T-lymphocyte growth capacities. 66 Moreover, DP-MSCs can suppress lymphocyte proliferation and increase the number of regulatory T cells and IL-10 while decreasing IL-4 and IFN-γ levels. 67 Therefore, dental pulp is considered a novel MSC source to use for the treatment of autoimmune and inflammatory diseases. and tissue inhibitor of matrix metalloproteinases (TIMPs) TIMP1 and TIMP3 decreased after MSC treatment of pressure overload hypertrophy. 74 Moreover, administration of MSCs prevented irradiationinduced lung fibrosis by reducing production of inflammatory cytokines, proliferation of fibroblasts, and accumulation of collagen. 75 They also limit the fibrotic response by reducing myofibroblast differentiation from epithelial cells and fibroblasts. 76 Thus, MSCs are considered as critical elements in ECM remodeling after tissue damage; therefore, they represent a therapeutic tool in recovering normal tissue functions in fibrosis.

| MSC-DERIVED EVS
Recent studies suggest that the signals responsible for the therapeutic effects of MSCs are at least partially linked to the production of EVs.
Simply, EVs are small, round packages Type-I alveolar cell of secretory products enclosed by a phospholipid bilayer membrane, which carry several bioactive components. 77 Although confusion on the nomenclature of EVs exists in the literature, EVs are currently classified into exosomes, microvesicles, and apoptotic bodies according to their cellular origin. 78 EVs encapsulate cargo molecules, including proteins, microRNA, and mRNAs, from their cell of origin. 79   when it is added to cells in which p38 kinase has been activated. 101 However, the augmentation effect of PGE 2 on IL-6 levels is independent of p38 kinase activity, and p38 kinase inhibitors are able to inhibit IL-6 production in activated macrophages by inhibiting PGE 2 synthesis. Collectively, MSC-derived PGE 2 has a central role in controlling the COVID-19-induced IL-6-and IL-10-mediated cytokine storm.
MSCs also have the potential of releasing antibacterial factors and therefore stimulating monocyte/macrophage phagocytosis. 96 The  this study were the small sample size and short-term follow-up as well as the lack of statistical power and randomization. 117 However, this mini trial also showed that infused MSCs remained negative for angiotensin-converting enzyme 2, a cell surface receptor that is required for SARS-CoV-2 virus to attach to the alveolar epithelial cells, 118 meaning that transplanted MSCs did not differentiate and remained free of virus. 119 In an extremely short time interval, as of 5 June 2020, 36 MSC trials for COVID-19 were registered to ClinicalTrials.gov, primarily aiming to rescue patients with severe or critical COVID-19 (Table 2).
Apparently, the number of trials will rise, because we are aware that in the corresponding author's country of residence, two phase II/III tri-