Pooled evidence from preclinical and clinical studies for stem cell-based therapy in ARDS and COVID-19

COVID-19 has severely devastated many lives across the globe. It has been speculated that stem cell-based therapy for COVID-19 treatment could be able to subsidize the effects. In preclinical and clinical studies, stem cell-based therapy has successfully eliminated inflammatory cytokines in ALI, ARDS, and COVID-19. Clinical trials have produced a variety of promising results for validating stem cell therapy in COVID-19 patients. For instance, exosome-based therapy (ExoFlow) showed an 87% survival status, and MSC-based therapy (Mesoblast) achieved an 83% survival rate in moderate to severe COVID-19 patients. This review debates the advantages of cell-free therapy, i.e., stem cell-derived exosome-based therapies, over stem cell-based therapy. This review aims to question whether the immunomodulatory effect of stem cells differs based on their origin and also tries to find possible answers for the best stem cells for treating SARS-CoV-2 infection. Graphical abstract The role of stem cells and their extracellular vesicles in the upregulation of regulatory immune cells, growth factors (EGF, FGF, VEGF), and anti-inflammatory cytokines (IL-6, INF-α, galectin-1, notch-1, PDL-1) that promote the tissue regeneration at the injured site. The right side of the image depicts the downregulation of inflammation-inducing immune cells, pro-inflammatory cytokines, and chemokines that could also enhance COVID-19 therapy. Supplementary Information The online version contains supplementary material available at 10.1007/s11010-022-04601-2.


Introduction
The novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has spread across the globe and posed a great threat to human beings. The worst-hit countries based on death rates due to COVID-19 are the United States of America, Brazil, Mexico, India, Russia, and the United Kingdom [1]. The pandemic has hindered millions of people's lives and affected economic conditions globally. Scientists and doctors are persistently taking measures to reduce and find a treatment for this pandemic disease. Research has succeeded in establishing various hypotheses and some clinical studies on new vaccines based on the current knowledge of coronavirus and the pathophysiology of the disease. Now, the re-emergence of the pandemic is worse. The most unfortunate situation is that the people from developing countries were also severely devastated economically. Many people lost their jobs and migrated from urban areas to rural areas due to the inability to manage their expenses. According to a survey, about 71% of household members lost their job, and about 61% of people shut down their small businesses [2]. Developing countries and the economic impact of COVID-19 did not spare developed countries. However, economic turbulence was lesser than in developing countries [3]. If this condition persists, then there would be a need to amend the strict lockdown, which would further create economic problems. Hence, there is a need to identify a treatment method that should not only cure coronavirus patients but also heal the detrimental effects that occurred during infection without providing any side effects.

Pathophysiology of COVID-19
The pathophysiology of the disease can be divided into three stages: the Asymptomatic stage (initial stage), the Symptomatic stage (mid-stage), and the Progressive to ARDS stage (terminal stage). The asymptomatic stage marks an entry point for the virus. The virus enters via the nasal route, and the spike protein on the virus interacts with human epithelial cells through an Angiotensinconverting enzyme (ACE-2) receptor [4]. S protein has an S1 subunit and an S2 subunit, which has a receptorbinding domain (RBD). It is observed that the ACE-2 receptor sequence from 331 to 524 residues binds to the S protein of the virus [5]. The S1 subunit forms a homotrimer and binds to the S2 portion [6]. The interaction between the S protein and the ACE-2 receptor is activated by multiple proteases like TMPRSS2 and cathepsin CTSL, which cleaves S1/S2 protein and triggers the viral membrane fusion process [7]. Furthermore, in silico analysis 1 3 confirmed that the region surrounding K353 and N501 amino acids play a crucial role in the interaction. Interestingly, Villa et al. synthesized DNA aptamers sequences using SELEX technology that could bind to the ACE-2 receptor, thereby inhibiting the interaction between ACE-2 and S protein [8]. The two major variants of coronavirus, i.e., delta variant and omicron variant, show mutant sequences in the S protein. This mutation increases the virulence of the variants [9]. The Wuhan-Hu-1 (wild type) variant has 1273 amino acids, while the delta variant and omicron variant have 1271 and 1270 amino acids, respectively. Kumar et al. performed a computational comparative study by analyzing the docking score between spike proteins of these variants and human ACE-2 proteins. The docking score for the wild type was − 500.37, while for the omicron variant, it was − 539.81, and for the delta variant, it was − 529. 62. These results revealed that the Omicron strains are more vulnerable to binding to ACE2 than the Delta strain. Thus, researchers predict that the virus has evolved gradually in immunocompromised patients, which might have played a crucial role in its virulence development [10].
This disease was previously believed to affect only the respiratory system, causing ARDS. However, some studies have shown that it could also affect the digestive, cardiovascular, urinary, and reproductive systems, apart from the respiratory system. One of the major speculations comes from the level of mRNA transcript of the ACE-2 receptor on the different cell surfaces of various tissues [11]. According to this study, the amount of ACE-2 receptors found on lung epithelial cells is comparatively less than those found in the duodenum, heart, gall bladder, small intestine, kidney, and testes [12]. However, it is unable to understand the pathophysiological condition of coronavirus and why it majorly affects the respiratory system rather than other systems. The probable reason might be that the respiratory system is the entry point for the virus and is more easily accessible than other parts of the body during the initial period. Later after the disease progresses, it may affect other body parts leading to multiple organ failures [12]. It is found that SARS-CoV-2 also affects various other cells like endothelial cells, pericytes, as well as astrocytes in the brain [6,13,14]. Scientists predict that SARS-CoV-2 could pass through olfactory mucosa and could reach brain cells [13,15].
The symptomatic phase indicates the replication of the virus in the respiratory tract and the lungs. This leads to tissue damage and destroys the architecture of the lung tissue, which affects its functioning and leads to severe respiratory illnesses like shortness of breath, difficulties in breathing, and pneumonia-like symptoms. However, it is also seen in lesser numbers (1.0-3.8%) that coronavirus causes nausea and vomiting as early symptoms of COVID-19, thus affecting the digestive system [12]. At this stage, the immune system gets activated against the intruder, i.e., coronavirus. Innate and acquired immunity get elicited, which results in leukocytopenia [16]. The innate immune system produces various inflammatory cytokines and chemokines, which attract other immune cells to infiltrate the affected region. This creates immune-mediated lung injury and initiates ARDS.
During the progression phase of the disease, the cytokinemediated injury shows adverse effects, leading to fatal conditions. Adverse effects were observed in the patients due to an increase in inflammatory monocytes and neutrophils, CD14 + CD16 + monocyte-derived macrophages, and proinflammatory cytokines [17,18]. Bilateral diffuse alveolar damage, fibroblastic proliferation in the airways, and the circulation of hyperactivated CD4 + and CD8 + lymphocytes were also observed [16]. Some studies have also shown intra-alveolar hemorrhages and fibrin cluster formation, and abundant intra-alveolar neutrophilic infiltration. Apart from the lungs, the liver also showed mild sinusoidal dilatation [19]. This indicates that the tissues might be injured in this stage due to the viral outbreak from the cells and the inflammatory responses from immune cells. The Berlin definition classifies ARDS based on the PaO 2 /FiO 2 ratio and chest scanned images and time period of respiratory distress [20]. Even though COVID-19 disease obeys the ARDS Berlin definition, there are distinct differences between COVID-19 pneumonia and ARDS [21]. The first pattern observed is multiple ground-glass lesions that evolve due to loss of aeration in the lung tissues. Furthermore, diffused alveolar damage, alveolar flooding in the presence of fibrin and hyaluran, and intense remodeling. These are some of the characteristic symptoms of COVID-19 [22]. This hampers respiratory function and causes organ damage. Inflammatory immunological reactions, i.e., cytokine storms, elevate the severity of the disease. Immune response against the virus is by secreting interferons, which also trigger other immune cells to infiltrate [23]. This is observed in some infectious and non-infectious diseases that lead to hyperinflammation. However, MSC-based stem cell therapies may neutralize this cytokine storm by activating suppressor cells as well as repairing and regenerating the injured sites. Researchers from many parts of the world are targeting and suppressing the inflammatory response.

Stem cell-based therapy
Stem cells are human-originated, undifferentiated cells that have the ability of self-renewal and the potential to differentiate into various tissue types. Stem cells can undergo immunomodulation, provide anti-inflammatory responses, and repair and regenerate injured tissues [24,25]. Stem cells are generally of two major types based on their origin: embryonic stem cells (ESCs) [26] and adult stem cells. Human embryonic stem cells are pluripotent in origin, while adult stem cells are multipotent cells that have the potential to differentiate into particular cell lineages. There are different types of adult stem cells based on their location, i.e., bone marrow-mesenchymal stem cells (BM-MSCs) [27], dental pulp derived mesenchymal stem cells (DPSCs) [28], Warton Jelly mesenchymal stem cells (WJ-MSCs) [29], placental stem cells (P-MSCs) [30], amniotic fluid-derived stem cells [31], hair follicle stem cells [32], urinary stem cells [33], breast milk-derived stem cells [34], adipose tissue MSC [35], olfactory mucosa MSC [36], menstrual blood MSC [37], Umbilical cord Mesenchymal Stem Cells [38], induced pluripotent stem cells (iPSCs) [39]. ESCs are used in exceptional cases since the isolation of human embryonic cells is limited and cannot be extracted in large numbers because of ethical issues. The cytoplasmic contents of stem cells are different from differentiated cells; thus, it possesses regeneration and immunomodulatory characteristics [40]. The stemness of a cell is reduced in this transition from pluripotency to multipotency to unipotency (a differentiated cell). Some examples of cytoplasmic contents of stem cells are immunomodulatory factors, chemokines, secretomes, proteomes, growth factors, cytokines, and small molecules such as prostaglandin E2 (PGE-2), dibutryl cAMP, nitric oxide (NO), concavalin A, indoleamine 2,3-dioxygenase (IDO), and morphogenic factors [40,41]. These secretomes have a paracrine effect on other cells, which might contribute to cell migration, stimulation, angiogenesis, or anti-apoptotic processes. MSCs play a critical role in regulating the inflammatory microenvironment and with immune cells like T-cells, neutrophils, B-cells, macrophages, natural killer cells, and dendritic cells [42]. Thus, changes occur due to the secretory by-products of stem cells that regulate the immunological aspects in the biological niche; this characteristic phenomenon of stem cells is known as Immunomodulation. Table 1 shows a list of secretion products of stem cells and their function on the effector cells. These secretion products provide an insight into the potential of stem cell therapy which might assist in treatment.

Preclinical studies of stem cell therapy in ARDS or ALI
There are several successful cases of stem cell therapy treating lung-related diseases like influenza, Acute Respiratory Distress Syndrome (ARDS), Acute Lung Infection (ALI), and Chronic Obstructive Pulmonary Disease (COPD). ARDS symptoms are similar to the symptoms of COVID-19. Hence, some of the case studies are relatable. Table 2 shows the preclinical data of ARDS or ALI, which was treated with stem cell-based therapies.
Loy H et al. provided an insight into the mechanism of human UC-MSCs cells and their regeneration properties in the lung injured site by conducting experiments on Influenza A(H5N1) virus-associated ALI [53]. UC-MSCs regulated ion transporter protein channels for clearance of the alveolar lumen. Immunomodulation properties were also exhibited by UC-MSCs, which decreased the inflammatory factors. Exosomes of UC-MSCs were also injected into the mice to evaluate whether exosomes also exhibited similar responses, and it was found that exosomes also showed convincing positive results [53]. The exosomes and their release contents might have contributed to treating ALI. Exploring the contents of exosomes and ensuring the contents contribute to treating ALI would provide a greater cause.
Kim et al. demonstrated the usefulness of human umbilical cord blood (UCB)-derived MSCs in treating the Escherichia coli-induced ALI mice model. The intratracheal injection of USB-MSCs downregulated the inflammatory responses (TNF-α, IL-1α, MIP-1α, IL-1β, MIP-1β, IL-6, RANTES, and IP-10) and by enhancing bacterial clearance [54]. The same group also studied the antibacterial effects of UCB-MSCs. After E. coli exposure, there was a significant upregulation in toll-like receptors TLR-4 and TLR-2 and β-defensin 2 (BD2) in MSCs, which was associated with bacterial clearance by regulating the TLR-4 signaling pathway [55]. The immunomodulatory function was also observed in hMSCs like USB-MSCs. hMSCs were used for treating pneumonia in E. coli-induced pneumonia rat models. The survival rate of these rat models was 100%, was visualized by an increase in arterial PO 2 , lung compliance, reduction in BAL protein, BAL neutrophils, alveolar E. coli counts, and reduced in histological injury, reduced alveolar thickening, and increased air-space volume [56]. However, optimization of the dose of hMSCs forms very essential criterion in using stem cell-based therapies. The use of hMSCs is more encouraged than UCB-MSCs since isolating UCB-MSCs from the umbilical cord is restricted due to availability. These provide evidence for using stem cells as therapeutics and the role of stem cells in downregulating inflammatory cytokines.
A combination of hUC-MSCs and FTY720 (a novel immunosuppressive agent) was investigated by Huang et al. in an LPS-induced lung injury mouse model. The clinical efficacy in alleviating lung injuries was higher with the combination treatment of hUC-MSCs and FTY720 than with using hUC-MSCs or FTY720 alone. Transcriptomic analysis by using a gene expression chip identified potential gene targets Nr1h4, Nol3, Cyp17a1, Prkg2, and Rps6ka6 related to ALI/ARDS [57]. Genetic expression analysis enabled the 1 3 highlight of the possible genes involved in the aggravation of the infection. Thus, this study speculates that targeting these genes may provide alleviation to COVID-19 patients. Xiang et al. investigated the Menstrual Blood-Derived Mesenchymal Stem Cells (MenSCs) in treating LPS-induced ALI (Fig. 1). In the LPS-induced mice model, decreased inflammation and a severe reduction in the BALF were observed [58]. MenSCs may have reduced the inflammation which was observed in the histological tissues. Even though therapies might provide promisable results in pre-clinical T-cells i. Inhibition of T-cell proliferation ii. Reduction in IL-17 production iii. Stimulation of regulatory T-cell (T reg ) differentiation [48] Natural killer cells (NK cells) Hypoxia-inducible factor 1 i. NK cell-mediated cell lysis is inhibited [49] ESCs T-cells Fas-L i. Induces apoptosis in activated T-cells Human adiposederived mesenchymal stem cells (AD-MSCs) Neutrophils Not mentioned i. The neutrophil influx was reduced, which decreased the inflammatory response of neutrophils in the injured site ii. Myeloperoxidase (MPO) is one of the most cytotoxic enzymes produced by neutrophils against bacteria, and its concentration was reduced [52] i. The concentration of inflammatory cytokines like IL-6, TNF-α, IL-1β, and MIP-2 was reduced studies, using menstrual blood for stem cell isolation is still dilemmatic. Due to sources of stem cell isolation, there is a requirement to use antibiotics to avoid secondary infections. Thus, there is uncertainty about the interference of these antibiotics in stem cell-based therapies. One of the major limitations of using MenSC is reproducibility, and each woman may have a different menstrual cycle and hormonal changes, which depend on the characteristics of the stem cells. Thus, the potential of the therapy may differ even between doses in the same patient. Silva and their team explored the best source of mesenchymal stem cells to treat pulmonary ARDS. Despite mesenchymal stem cell sources, MSC treatment reduced lung inflammation and lung fibrosis, thereby improving lung function and decreasing apoptosis in the lung, liver, and kidney. Their experiment showed that bone marrow BM-MSCs and adipose tissue AD-MSCs reduced TNF-α, IL-1β, KC, TGF-β, and VEGF levels but increased KGF. Lung-resident MSCs (L-MSCs) showed no significant differences in TNF-α, IL-1β, and KC, TGF-β, VEGF, and KGF levels. Hence, BM-MSCs and AD-MSCs showed greater potential than L-MSCs. However, further studies are required to confirm their definite mechanisms of action [59]. In another independent investigation, it was observed that L-MSCs had a crucial role in regulating Treg cells and Th17 cells in the LPS-induced ALI model. The administration of lung-resident L-MSC reduced lung damage and inflammation detected in the BALF. It was also observed that LRMSC could reduce inflammatory cytokines and increase the expression of KGF-2 surfactant protein C Fig. 1 Physiological changes occurred after stem cell injection. I X-rays images of saline-injected control group having no lesion in the lung tissue, LPS-induced rat depicting lung injury, hemorrhage, and inflammation, which were reduced after MenSC administration (LPS + MenSCs group). II H&E staining showing the pathological differences in the control group, LPS-induced group, and MenSCtreated group. III Lung dry/wet ratios of these models were evaluated. Scale bar: 50 µm Reproduced from [58] and reprinted from Creative Commons Attribution 4.0 licence (CC BY-4.0) (SPC). This study found that the administration of LRMSC upregulated Tregs and downregulated Th17 cells by which LRMSCs improve lung injury [60]. Thus, the nature of MSCs changes based on the source, health conditions of the donor, and patients.
Mokhber Dezfouli et al. emphasized the beneficial effect of intrapulmonary autologous transplantation of bone marrow-derived mesenchymal stromal cells in treating LPS-induced ARDS in rabbits (Fig. 2). They confirmed that MSCs decrease the severity of clinical symptoms and inflammation. Lung CT images showed decreased inflammation and mucous accumulation, which was confirmed by cytokine profiling. (Fig. 2I). After MSC administration, inflammatory cytokines (TNF-α and IL-6) were found to be reduced in the blood and BAL. Necropsy and histopathological staining also further confirmed that no injury was observed in the treatment group (Fig. 2II) [61].
Jackson and his group studied the crosstalk between MSCs and macrophages to understand its impact on the phagocytosis mechanism in the E. coli pneumonia mouse model of ARDS. They demonstrated that MSCs could transfer mitochondrial contents through nanotube (TNT)-like structures, which could enhance the phagocytic ability of macrophages and thus improve the function of macrophages against ARDS [62]. Li et al. demonstrated the beneficial effects of syngeneic MSCs treating H9N2 avian influenza virus (AIV)-induced acute lung injury in mice. After the infusion of MSCs through the caudal vein, a significant reduction of chemokine and proinflammatory cytokine levels was observed. Also, a reduction in the recruitment of inflammatory cells into the lungs was observed. The survival rate in the MSCs-treated group was 100%, whereas, in the control group, it was 80%. The histopathology studies showed a reduction in the alveolar space inflammation and improvement in the lung histopathology in the H9N2 AIV-induced lung injury model following MSC treatment. Arterial blood gas analysis was performed, which evaluates the partial pressure of arterial oxygen (PaO 2 ), a saturation of arterial oxygen (SaO 2 ), the partial pressure of arterial carbon dioxide (PaCO 2 ), and pH values showed that MSCs infusion increased PaO 2 , SaO 2 , and pH values, decreased PaCO 2 , which indicate effective protection of the lungs. These results indicate the potential of MSCs for treating clinical avian influenza [63]. Chan et al. investigated influenza disease interventions in H5N1 Influenza A virus-induced Balb/C mice models [64]. Infection by the Influenza virus showed dysregulated alveolar fluid transport due to the inhibition of epithelial sodium channel activity. However, this condition was treated in BM-MSC-treated mice, and there was a significant increase in lung alveolar fluid permeability. The population of macrophages/monocyte in the bronchoalveolar lavage (BAL) fluid was preponderately alveolar macrophages (M2 phenotype) [64]. M2 phenotype macrophages are alternatively activated macrophages, unlike the classically activated macrophages (M1 phenotype). The polarization of M2-type cells might be due to the secretions of MSCs. The function of M2 phenotype macrophages is of the opposite nature to that of activated macrophages (M1 phenotype). They are regulatory in nature and have anti-inflammatory responses that are required to reduce the inflammation in injured lung tissues [65]. The concentrations of cytokine and chemokine proteins were evaluated, and significant improvement was observed in the MSC-treated group compared to the fibroblast-treated group. Overall, in vivo, MSC therapy reduced acute lung injury and increased the survival rate in aged H5N1-infected mice. These preclinical studies recommend that MSCs-based therapy might be highly beneficial to the patients [64]. Analysis of preclinical studies of ARDS enhances understanding of the potential of stem cells in lung lesion healing, immunomodulation for clearance of toxic substances, and lung tissue regeneration.

Clinical study of stem cell therapy in ARDS or ALI
Stem cell therapy has been successful in treating ARDS and ALI. Although many preclinical studies support the idea of stem cell therapy, clinical trials would provide further insights into the dosage of stem cells required, possible hazardous effects, and side effects. Simonson et al. investigated the in vivo effect of non-HLA-matched allogeneic bone marrow-derived MSCs in two patients with severe refractory ARDS on a compassionate use basis. After an infusion of 2 × 10 6 cells per kilogram, both patients demonstrated improved lung function and were discharged from the ICU and hospital. No adverse changes were detected in both patients, including no changes in hemodynamic parameters or in oxygen saturation levels. Beneficial effects were associated with the reduction of inflammatory biomarkers such as interleukin-6 (IL-6), IL-8, and IFN-γ levels. Also, total K18 (epithelial cell death marker) and ccK18 (epithelial apoptosis marker) levels are decreased after the infusion of MSCs, suggesting lung epithelial cell recovery [66]. The findings clearly suggest the beneficial effect of MSCs in lung-protective roles in ARDS. However, analysis has been done only on a few patients, and there is a need for investigations on more patients to conclude the clinical efficacy of the MSCs.
Matthay et al. conducted clinical trials on 60 patients, of which 40 were treated with MSCs, and the rest were controls. The study showed that there was no adverse effect due to MSC treatment. However, in the treatment group, the oxygenation index was higher in the case of MSC-treated patients. The expression of proinflammatory cytokines and chemokines was severely decreased in the treatment group [67]. Another study by Zheng et al. was conducted using hAD-MSCs for treating ARDS. The results showed a decrease in the levels of SP-D proteins and the inflammatory cytokine IL-6. Patients were followed up for 28 days after the treatment in order to record any adverse effects of the hAD-MSC infusion [68]. Most of the clinical symptoms were ameliorated by combination therapy, and the MSC treatment regime has a death rate of 17. Four patients were followed for more than 5 years and had no adverse effects. A multi-centered study was conducted in UK and USA, which was two doses open-label. All the patients were monitored for any adverse effects or changes in PO 2 /FiO 2 levels [69].
According to several clinical study reports, more than 80% of COVID patients had lymphopenia, and more than 50% of ICU patients had high levels of TNF-α and granulocyte colony-stimulating factors, two of the major causes of a cytokine storm [70]. Another study was conducted by Liu et al., where they investigated the clinical pathology and progression of SARS-CoV-2-infected ARDS patients. There were 109 COVID patients of the following categories: the elderly and those complicated with secondary diseases like diabetes, cerebrovascular disease, and kidney diseases. It was found that in each of these conditions, there are higher chances of acquiring SARS-CoV-2-induced ARDS [71]. Consequently, it is necessary to understand stem cell therapy for ARDS and ALI. A single-centered, open-labeled clinical trial was conducted for H7N9-induced ARDS in 17 patients. The mortality rate in the MSC-transplanted patients was significantly lowered in the experimental group. Before MSC treatment, the patients showed ground-glass opacities and fibrillations in the chest radiography images. After the treatment, radiographic images were followed up at different intervals, i.e., 1 week, 24 weeks, 1 year, and 5 years (Fig. 3). The patients started to improve after 1 week of treatment. Additionally, it was observed that MSC therapy did not have any adverse effects on the patients even after 5 years of follow-up (Fig. 3E) [72]. Hence, it can also be believed that stem cell-based therapies would provide beneficial results. The mode of action of stem cell therapies involves inhibiting the virus-induced cytokine storm by regulating macrophages (M1 phenotype), suppressing T-cells activation, increasing the concentration of T reg cells, suppressing dendritic cells, hampering NK cell-mediated cell lysis, affecting B-cell proliferation and antibody production, decreasing the levels of inflammatory factors, increasing anti-inflammatory factors, and regenerating injured tissues.

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Averyanov et al. conducted the first-in-human high-cumulative-dose stem cell therapy for treating idiopathic pulmonary fibrosis (IPF). A high dose of BM-MSCs (1.6 × 10 9 cells/ml) was administrated to 20 patients [73]. The patients in the test group had increased lung function than the placebo group. The patients were monitored for 52 weeks to examine the adverse effects; however, no such adverse effects were observed [73]. Many in vivo and clinical studies exhibit that MSC-based therapies improve lung-related diseases like COPD, ARDS, and IPF. And the major symptoms of SARS-CoV2 is also inflammation of the lungs, pneumonia, and lung tissue damage. Since the pathophysiology of SARS-CoV2 is similar to ARDS and some other lung-related diseases, stem cell therapy for SARS-CoV2 might also provide similar results to ARDS. It also highlights that these therapies may be safe for COVID patients [74]. Table 3 also depicts evidence for using stem cell-based therapies against SARS-CoV2.

Clinical studies of stem cell therapies in COVID-19
Clinical trials are essential for discovering any drug, therapy, or treatment and for considering the safety of the patients. Clinical trials reduce unwanted or inefficient medical treatment, which causes adverse reactions. The following are ongoing and completed stem cell-based therapies for COVID-19 listed on the U.S. National Library of Medicine (NLM) website, ClinicalTrials.gov. Clinical studies are ongoing worldwide, and researchers are trying to find solutions by discovering new drugs or therapies, or vaccines for COVID-19. In India, Stempeutics has successfully received approval from DCGI (Drugs Controller General of India) for phase 3 clinical trials for COVID treatment using BM-MSCs [75]. Ye et al. assessed the efficacy of human DP-MSCs in severely affected COVID-19 patients. Intravenous injection of DP-MSCs suspension (3.0 × 10 7 cells/ dose) was provided thrice a week, while a placebo or saline was given to the control group. The assessment of the study was performed by counting immune cells, kidney and liver functionality tests, and immunological tests. These are some of the essential tests that provide information on whether stem cell therapies are effective and whether they cause any bystander effect on other surrounding microenvironments [76]. Safari et al. conducted a preclinical study by using a combination of BM-MSC along with nicorandil, which is an ATP-sensitive potassium channel opener. The combination of these two as therapeutic agents improved lung tissue damage and cell death [77]. Thus, this combinatorial approach might counteract COVID-19.    (Fig. 4) [78]. In our analysis, it is found that BM-MSCs (24 cases) and UC-MSCs (20 cases) are extensively used for stem cell-based therapy for COVID-19. However, now the question arises as to which stem cells could provide better results or promise better results and are suitable for the treatment. Or will there be any difference overall in treatment when stem cells are derived from different origins? Or which of these cells provides more concentration of anti-inflammatory by-products? Which cells are better obtained without many ethical issues? These are some of the questions that need to be answered when assessing both cells for treatments. Lee et al. compare the immunomodulatory effect of placenta-derived mesenchymal stem cells (P-MSCs) with BM-MSCs and AD-MSCs, and the study reveals that P-MSCs have much higher immunomodulatory capability than BM-MSCs and AD-MSCs [79]. However, the availability of P-MSCs is very scare due to a lack of awareness of placental stem cell banking is limited [80]. Furthermore, Atluri et al. also predict that hUC-MSCs could be the best source for COVID-19 treatments. UC-MSCs are better than BM-MSCs due to availability, scalability, faster doubling time, and non-invasive extraction [81]. Other than these two major cell types, few cases were also performed by cardiosphere derived cells (CDCs) [82], olfactory mucosal lining derived MSCs (OM-MSCs) [36], Haematopoietic stem cells (HSCs) [83], Stem Cell Educator-Treated Mononuclear Cells (SCE-MCs) [84], CAStem cells [Immunityand matrix-regulatory cells (IMRCs) derived from ESCs] [85], P-MSCs [86], Extracorporeal derived MSC [87], DP-MSCs [88], AD-MSCs [89]. In the cases of CDCs, OM-MSCs, and HSCs, there is always a concern about whether these cells would differentiate into their respective lineages rather than perform their immunomodulation function. Furthermore, SCE-MCs function was quite different from the usual stem cell therapy. Stem Cell Educator (SCE) technology was patented by Tianhe Stem Cell Biotechnologies for treating autoimmune diseases like Type 1 diabetes, Alopecia Areata. SCE uses multipotent cord blood stem cells (CB-SCs). CB-SCs have proven their immunomodulatory properties in some autoimmune disorders. Because the properties of CB-SCs differ from those of other stem cells, researchers are attempting to incorporate these CB-SCs in order to combat COVID-19. However, this study is still in the preliminary stage [84]. Cell-free therapy has also undergone a clinical trial platform, and there were four studies that used exosomes or extracellular vesicles as their drug for intervention [90][91][92][93]. Out of these two studies was for the nasal route is their mode of drug delivery, which might be highly recommendable since coronavirus also targets the lung most [91,92]. If the anti-inflammatory factors and growth factors from exosomes could target more of the lesions of the injured lung tissue, then this method may prove more advantageous than intravenous drug delivery.
Stem cell therapy has gained attention in the research community because of its self-renewal, pluripotency, and regenerative capacity. Moreover, there have been numerous studies on using stem cells for therapeutic usage for about two decades. Hence, many researchers have moved toward stem cell-based therapy for combating COVID-19. Many researchers have performed safety assessment clinical trials on the effect of stem cell secretions on COVIDinfected tissues. Some studies have also shown evidence where pulmonary regeneration has happened similarly in vivo studies in humans. In vivo studies, the alveolar type II (AT2) has the capability to proliferate and differentiate into alveolar type I (AT1) cells mimicking the differentiation ability of stem cells. If stem cells get differentiated into AT2 cells, thereby enhancing pulmonary regeneration could be one of the beneficial improvements in achieving a cure for COVID [94]. Numerous researches have provided optimistic results [95].  (Fig. 5). MSCs (1 × 10 6 cells/kg body weight) were transplanted by intravenous drip with saline, and patients were observed for 14-days. Remarkably, after 2-4 days of MSC transplantation, symptoms like pyrexia and dyspnea disappeared. Also, there was a decline in C-reactive protein level, aspartic aminotransferase, creatine kinase activity, and myoglobin, which suggested an immediate recovery of pulmonary function. Furthermore, mass cytometry (CyTOF) analysis showed a decrease in the number of active CXCR3 + CD4 + T-cells, CXCR3 + NK cells, and CXCR3 + CD8 + T-cells and an increase in CD14 + CD11c + CD11b mid regulatory DC cell population. Similarly, TNF-α was also significantly decreased, while anti-inflammatory cytokine IL-10 level increased along with an increase of chemokines IP-10 and VEGF, which suggests the ability of MSCs in controlling cytokine release syndrome. Importantly, this is the first report that shows the negative gene expression of ACE2 and TMPRSS2 in MSCs, which suggests the safety of MSCs in COVID-19 treatment [96].
Shu et al. conducted a clinical study on COVID patients using intravenous infusion of hUC-MSCs (Fig. 6) [97]. It was a single-center study where they found that the patients in the hUC-MSC treatment group recovered and improved, while three patients from the control group did not survive [97]. A similar study was also conducted by Meng et al., where they used UC-MSCs to treat 18 patients (9 in the treatment group and 9 in the control group). Both groups were provided with standard treatments. It was found that there was no adverse effect due to the administration of UC-MSCs. 2/9 of patients from the UC-MSC treatment group had shown symptoms of transient facial flushing and pyrexia. One of the patients suffered from transient hypoxia after 12 h of UC-MSCs administration [98].
Li et al. used menstrual blood from healthy females to extract mesenchymal stem cells for treating two critical COVID-infected patients [99]. These isolated cells were checked for genetic abnormalities by karyotyping. Menstrual blood-derived mesenchymal stem cells (MenSCs) were transplanted onto two patients, one 37-year-old female, and a 71-year-old male. Both these results showed promising results. This 37-year-old patient suffered from fever and dyspnea for almost 9 days and 4 days, respectively. Initially, drugs like oseltamivir and arbidol hydrochloride did not significantly improve the condition. Later, MB-MSCs infusion was administered, and symptoms gradually improved as levels of saturated O 2 , partial pressure of O 2 , Fig. 5 Examination of infected lungs of COVID-19 patient. On Jan 23, no signs of pneumonia were observed. On Jan 30, ground-glass opacity and pneumonia infiltration was observed on multiple lobes of the lungs. After 2 days of MSC treatment, on Feb 2, pneumonia invaded the whole lung, which had gradually reduced (Feb 9 and Feb 15). Reproduced from [96] and reprinted from Creative Commons Attribution License 1 3 and the fraction of inspired O 2 improved. Along with these improvements, the lymphocyte counts increased, inflammation indicators decreased, initial chest CT scans showed large patchy high-density lesions, and after 6 and 10 days of treatment, absorption of the exudate lesions in the bilateral lungs was observed. MB-MSCs treatment was successful, and the patient was negative for COVID in the nucleic acid test. MB-MSC treatment also showed convincing results in 71-year-old male patients [99].
Another case in China, where COVID-19 pneumonia was treated by human umbilical cord Wharton's jelly-derived MSCs. Before the treatment, the patient showed roundglass opacity and pneumonia filtration in both lungs. Along with this, a crazy-paving pattern was observed due to GGO with inter and intralobular septal thickening. Both these symptoms were reduced drastically after the intravenous infusion of WJ-MSCs. The patient improved significantly with a decrease in the symptoms, and gradual recovery of pulmonary function was observed post 1 week of discharge [100] (Fig. 7).
In a study conducted by Singh et al., instead of the most widely used MSCs, they used Cardiosphere-derived cells (CDCs) [82]. CDCs are progenitor cells from heart tissue, and they have been used in clinical trials for myocardial infarction, heart failure, pulmonary arterial hypertension, Duchenne muscular dystrophy, and hypoplastic left heart syndrome. It is also proven that CDCs have immunomodulatory and anti-inflammatory functions, which are essential for treating the cytokine storm released by coronavirusinduced inflammation. Six patients were treated with CDC-based intravenous infusion. Four patients showed tremendous improvement. One patient had a slow recovery rate compared to the other four patients, and another 1/6 was the control. Levels of IL-1α and IL-1β did not vary much; however, levels of ferritin and C-reactive protein (CRP) decreased as expected. Even though the results are encouraging, however, there are certain concerns about using CDCs for COVID-19 treatment [82]. It has been shown that CDCs have limited ability to differentiate into non-cardiac lineage cells [101]. Hence, there are possibilities that it could differentiate into cardiac cells in other areas of the body, which is not desirable. Furthermore, it is also observed that CDCs have a higher potential for forming exosomes than MSCs, which concludes that CDCs could provide more beneficial results in cardiac-related stem cell therapies than COVID-19 [102].
Furthermore, various clinical cases demonstrated the success of stem cell therapy, and there was gradual commercialization via Pluristem Therapeutics, Inc. The company treated 7 patients suffering from ARDS and COVID-19-associated inflammations by administrating Pluristem's PLX cells in 3 medical centers in Israel [103]. Another Australia-based company named Mesoblast also proves that MSC-based therapy has shown an 83% success rate in ventilator-driven COVID-19 patients [104]. Athersys, a commercial biotechnology-based company, has patented Multistem stem cell therapies for various other diseases like neurological, inflammatory, immune, and cardiovascular disease, along with treating COVID-19 [105,106]. Similarly, there are some other companies that have been permitted by the FDA to conduct clinical trials, e.g., Cynata Therapeutics and Celltex Therapeutics [107,108]. Cynanta is currently working on its Cymerus MSCs for Phase II trials, while Celltex is using AD-MSCs and is proceeding toward Phase II studies [109,110].

Preclinical study of stem cell-derived EV or MV or exosomes-based therapy in ALI or ARDS
Another advancement of stem cell therapy is cell-free therapy using extracellular vesicles (EVs) of stem cells. Cellfree therapy is based on considering stem cells as a source for extracting therapeutic molecules such as EVs/MVs/ exosomes instead of the whole stem cell as a therapeutic agent [111]. One of the advantages of cell-free therapies is these molecules can be formulated such that they can be administered as inhalants [112]. The International Society for Aerosols in Medicine (ISAM) recommends that in COVID-19, the primary route of infection is through the lungs, and inhalation as a route of delivery could lead to an effective treatment strategy [113]. Moreover, this route of delivery is being administered before the COVID-19 pandemic for other lung cancer diseases like asthma, COPD, cystic fibrosis, pneumonia, pulmonary hypertension, and ARDS [114]. Every cell ejects EVs in the form of exosomes (40-150 nm in diameter) or microvesicles (150-1000 nm in diameter) (MVs) [115]. The physiological function of EVs is communication, cell signaling, and defense [44]. MVs are products of exocytosis of the plasma membrane along with cellular components and cytoplasm, whereas exosomes originate from multivesicular bodies that are formed during endosomal maturation. Like MVs, exosomes also contain cellular components [116]. As mentioned earlier, stem cellbased therapy works on paracrine-based signaling. Similarly, EVs are regulated via the paracrine effect. However, one of the major advantages of cell-free therapies is that there is no risk of tumorigenicity and lower immunogenicity [115]. And there are various types of RNAs in the cytoplasm of a cell. Some of these RNAs are also likely to get trapped in the EVs. It was observed that EVs predominantly contain rRNAs, however, quantities of mRNAs, miRNAs, and tRNAs are relatively lesser [117]. The physiological and biological activities of exosomes are conserved and similar to those of their parent stem cells. EVs also possess anti-apoptotic, immunomodulatory, angiogenic, and tissue-regenerating functions [117].  MSCs, and mouse splenocyte interaction with MSCs [120]. The cytokine profile of EVs showed 34 different inflammation-associated cytokines (sICAM-1, IL-5, CCL5, IL-6, IL-10, CXCL12, IL-13, RANTES, IP-10, Serpin E1). Furthermore, it was observed that the concentration of sICAM-1, CXCL12, and CCL5 were increased, and concentrations of IL-10, IL-6, IL-5, and IL-13 were reduced drastically. PBMCs interaction with EVs was performed to assess the uptake of EVs by granulocytes, lymphocytes, and monocytes. The order of uptake decreased in the following order: granulocytes, monocytes, and lymphocytes. Isolated splenocytes were activated using lipopolysaccharide (LPS) or concavalin A (ConA), which secreted IFN-γ and TNF-α when EVs interacted with activated splenocytes within 24 h, EVs reduced the concentration of IFN-γ and TNF-α [120]. The above-mentioned studies were in vitro-based studies; however, it should be considered that reciprocating the in vitro studies in vivo and then for clinical trials is the ultimate success for any experimental research. However, in vivo analysis of EVs might have provided intricate details, such as whether EVs induce tumorigenicity or other adverse effects.
In a study conducted by Khatri et al., they studied the attenuation of the Influenza A-induced lung injury model in pigs by BM-MSCs-derived EVs [121] (Fig. 9). In vitro hemagglutination studies of different types of Influenza virus strains (swine/TX/98; H3N2, human/CA/09; H1N1, gull/MD/1995; H9N5, swine/MN/08; H1N1, chicken/NY/ H7N2) were studied, and it was observed that EVs showed complete inhibition of hemagglutination activity within 1.25 and 5 μg/ml concentrations. MSC-EVs also drastically reduced the apoptosis of influenza-infected lung epithelial cells. In the SwIV-induced acute lung injury pig model, after 12 h of intratracheal administration of MSC-EVs (80 μg/ kg body weight), there was a reduction of influenza virus infection by 100-folds. Anti-inflammatory cytokine IL-10 Fig. 8 Immunopathogenesis of the coronavirus and the therapeutic potential of the MSC and their exosomes. Coronavirus infects ACE2-expressing cells and type II alveolar epithelial cells. The influx of T-cells, neutrophils, macrophages, and B-cells is induced by cytokines and chemokines secretion. The localization of the inflammatory cells at the injured site further leads to the production of proinflammatory cytokines, which is known as a cytokine storm, that acts as a major reason for ARDS in COVID-19. These inflammatory responses may cause lung fibrosis, apoptosis of the alveolar cells, edema, and organ failure. Immunomodulatory properties of stem cells are transferred to the injured site through exosomes that reduce inflammation-induced lung injury. The immunomodulatory properties of MSCs and their exosomes suppress inflammation and reduce inflammation-induced lung injury. Once the infected cells move into the bloodstream, the virus can move to many other organs causing various conditions. This figure was adapted from reference [118,119] expression was increased while associated inflammatory cytokines (TNF-α and CXCL10) were reduced by BM-MSCs derived EVs [121]. EVs show improved results in treating respiratory-related diseases in in vitro and in vivo conditions. These studies show that there are possibilities for using cell-free therapies to treat ARDS for COVID-19, and Table 4 supports the use of cell-free therapies. In the current scenario, limited clinical trials are being performed using exosomes derived from MSCs.

Clinical studies of stem cell-derived EV or MV or exosome-based therapy in COVID-19
It is clear from the studies that exosome-based therapies can be more beneficial than stem cell-based therapies. Scientists have also tried many strategies to improve exosome contents for a better immunomodulatory effect. However, there are certain criteria to be considered when cell-free bodies are used for the treatment. It was found that extracellular vesicles have higher expression of tissue factor CD142 has been correlated to the severity of the infection [122]. CD142 acts as a procoagulant that initiates clotting leading to the systemic inflammatory response [123]. Thus, it is speculated that CD142 could be considered a biomarker for COVID-19 diagnosis. Furthermore, cell-free vesicles need to be thoroughly investigated for the absence of CD142 in the treatment regimens.
Researchers have hypothesized that using recombinant DNA technology increases the anti-inflammatory cytokines, increasing miRNA, which could further enhance the  The results showed an 87% survival rate in the patients, and the rest of 16% of the patients expired due to reasons unrelated to the treatment. Efficacy of the exosomes was evaluated by the increase in the lymphocyte count by 46% and the reduction in the mean neutrophil count (32%), mean C-reactive protein (77%), ferritin (43%), and D-dimer (42%). These results provide positive hope for treating COVID-19 [90]. Table 5 shows the clinical cases of treating COVID-19 with cell-free therapies.

Precautions and criteria for using stem cells
The successful translation of stem cell-based therapies for COVID-19 depends on various factors related to the production, packaging, and delivery of stem cells. Isolation of stem cells, maintaining the stemness of cells, improving the stemness quality, characterization of stem cells, Good Manufacturing Product (GMP) handling, shelf life, the dosage of stem cells, and the half-life of stem cells. Stem cell therapies require a higher quantity and continuous supply for treatment. In this case, due to the compromised immune system of the patients, autologous stem cells are not the best option, hence, there is a requirement for pooled heterologous stem cells handled under the GMP facility [129]. As mentioned in the research, the average concentration of stem cells required for one dosage of intravenous injection of stem cells is one million cells or, in some cases, more than a million [35, 38,130]. Multicentric clinical trials for stem cell therapy or cellfree therapies require billions and trillions of cells.
ESCs may not meet this condition since ESCs are derived from blastocytes' inner cell mass, which is obtained after 5 days of culturing of the zygote [131]. It is unethical to extract an enormous number of stem cells for a particular trial or treatment. However, according to the pluripotency of ESCs, it might be the best candidate for stem cell therapies. Nevertheless, adult stem cells have produced convincing results. In the isolation of MSCs from bone marrow, it is found that repeated aspirations may decrease the yield of MSCs. Thus, initial aspiration is highly crucial for the culture and expansion of cells. MSCs yield also depends on an individual's age and health status [131]. Properties of stem cells include the self-renewal ability to proliferate, differentiate, and regenerate. These properties depend on the stemness of the cell. It is recommended that the MSCs should have 3-5 passage numbers for stem cell therapies. This leads to risk in achieving the required quantity of cells by expansion affecting the stemness. On the other hand, there are studies that demonstrate that antioxidants, expression of the MRPS18-2 (mitochondrial ribosomal protein S18-2), expression of the retinoblastoma-associated protein (RB), ligand-receptor interaction between cell-cell adhesion through N-cadherin, and scrapie responsive gene 1 (SCRG1)/bone marrow stromal cell antigen-1 (BST1) could maintain the stemness of the cell [132][133][134]. These study results are obtained in small-scale studies, and the scale-up of these studies is cumbersome.
The development of clinical-grade stem cells for therapies should be conducted according to the GMPs. There are strict rules and regulations set by various organizations like the United States Food and Drug Administration (USFDA), the European Medicines Agency (EMA), the Japanese Pharmaceuticals and Medical Devices Agency (PMDA), and other regulatory agencies. GMPs ensure to scrutinize the potential risks involved and impose rules for manufacturing, testing, and application of the final product [129]. Stem cells must be evaluated for molecular cytogenetic disorders or aberrations to avoid oncogenic activities arising due to in vitro extensive expansion of cells. TF/CD142 expression must be significantly less in the stem cells as these proteins ameliorate the MSC's hemocompatibility, thromboembolism, and massive intravascular thrombosis. Deficient TF/CD142 expression reduces anticoagulant activity in the in vivo system [135]. Some of the other essential tests include phenotypic pluripotency assays, histone modification, DNA methylation assays, and viral and bacterial contamination tests [136]. The shelf life of stem cells is very short since they cannot be passaged more than 3-5 times. However, stem cells can be cryopreserved and stored. Clinical-grade stem cells are a paramount requirement for these therapies since mishandling stem cells could cause adverse effects or even death in patients. Stem cell therapies are a boon with tons of restrictions. Even though many studies report the successful regeneration and recovery of patients worldwide, the fate of a stem cell inside the human body is unpredictable.

Conclusion
Worldwide prevailing studies on clinical trials are dominantly working on discovering new anti-viral drugs and vaccines, and very little research is being concentrated 1 3 on stem cells or their exosomes as therapeutic candidates. This review article aims to focus light on these aspects for those considering these areas as potential candidates. Stem cell-based therapies have a paracrine effect on the nearby cell, which guides the damaged cells to regenerate and boosts the immune system by producing cytokines and chemokines. However, there is a certain hypothesis for stem cell-based researchers that is also encouraged by researchers nowadays. There are some similarities in the symptoms when compared to ARDS and COVID-19; moreover, stem cell-based therapies and cell-free (exosome) therapies have shown great recovery in these patients. Hence, researchers and clinicians have to invest in the idea of using these therapies for treating COVID-19.
And several clinical studies mention that stem cells and their derivatives are safe and efficient.