Mesenchymal Stem Cells Versus Covid-19. Can they Win the Battle?

Bone marrow, adipose tissue, umbilical cord and dental pulp are suggested as convenient sources of MSCs, yet it is not completely elucidated which source of MSCs is optimal (38, 39). BM-MSCs are characterized by easy acquisition, rapid expansion in vitro, minimum immunologic rejection, protracted standing post-transplatationally in the host, preservation of capacity to differentiate subsequently to repetitive passages as well as simplicity of transplantation (40,41). Anyway, the acquisition of BM-MSCs is complicated process and also, size and longevity of BM-MSCs diminishes by ageing (42,43). So, to bypass such obstacles, alternative sources for MSCs isolation are implied (12,13). Umbilical cord blood and adipose tissue have been proposed as an alternate source for isolation and therapeutic employment of MSCs regarding its larger proliferative potential, high cell yields and ease of harvesting (44,45). BM-MSCs, UCB-MSCs and AT-MSCs all have similar morphological as well as operational traits (11). In the context of current pandemic Covid-19, and the entire inflammatory response caused by cytokine storm, as it will be further elaborated in this review, adequate MSCs source should be discussed so that optimal MSCs could be employed in therapy. Since the virus enters the cells through target ACE2 receptor, it is crucial to decide whether MSCs from any of these sources express ACE2 receptor, and therefore is susceptible to Covid-19 infection. Recent study showed that ACE2 is highly expressed in adult bone marrow, adipose tissue or umbilical cord-derived MSC. Distinguishing from placental derived MSCs together with human embryonic stem cell (hESC) or human induced pluripotent stem cells (hiPSC) that express low levels of ACE2 receptor (46). These outcomes should be further taken into consideration when determining on the optimal source of MSC therapy.

In December 2019, non typical infectious respiratory syndrome of idiopathic cause was recognised in Wuhan, China. Having already met with manifestations of (SARS) in 2003, Chinese scientific and medical department could identify a new coronavirus, SARS coronavirus 2 (SARS-CoV-2) as the pathogen (47). SARS-CoV-2 was easily and quickly spread all across the globe, leading to high morbidity and mortality. In order to prevent further spread of the virus, lock-down and other quarantine measures were enforced. Ease of spreading, high morbidity and mortality rate, changed the portrait of this pathogen, so on March 11, 2020, the World Health Organization (WHO) declared COVID-19 a global pandemic. The COVID-19 is clinically heterogeneous, patients may be asymptomatic or have light, moderate, severe or critical clinical traits. Symptoms of COVID-19 may vary, but most common include fever, headache, malaise, cough, bone pain, myalgias, anosmia, impaired taste and respiratory distress. Similar to SARS in 2003, this infectious disease leads to a high probability of ICU admission and mortality (48, 49). The pathogenesis of SARS-CoV-2 has been explained by the interaction and binding of the virus spike glycoprotein (SARS-CoV-2S) to the angiotensin-converting enzyme-related carboxypeptidase (ACE2) on the target cell surface. After binding, spike protein is activated by the cellular transmembrane protease serine 2 (TMPRSS2) which enables the virus to enter the host cell (50), leading to complex over activated inflammation and immune response, known as cytokine storm. The term “cytokine storm” was first established in 1993. to depict a graft versus host disease, and defines as the rapid and sudden efflux of a large number of cytokines that happens as a result of the immune system′ s overactivation by some stimuli (infection, drugs, etc.), begin locally and disseminate systemically, triggering collateral detriment in tissues (51,52). It is elucidated that cytokine storm is linked with the exacerbation of various infectious diseases, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and with the severity of COVID-19 (53). However, it was reported that cytokine storm is one of the main causes of a severe form of the disease in COVID-19 patients and dying, and is associated with thrombosis, massive mononuclear cell infiltration in multiple organs, and high levels of circulating cytokines (54). Notwithstanding that the specific dysregulated molecular causes are still not understood, it is assumed that cytokine storm is caused by disbalance in the regulation of the immune system (i.e., increment in immune cell activation via TLR or another mechanism, decreasing in anti-inflammatory response, etc.) (51). The immune response is being provoked by SARS-CoV-2 entering respiratory epithelial cells and is accompanied by inflammatory cytokine production and weak interferon (IFN) response. Cytokine storm occurs as a result of activation of pro-inflammatory Th1 cells and intermediate CD14+ CD16+ monocytes, macrophages, and neutrophils infiltration into the lung tissue (54). Specifically, pathogenic Th1 cells that produce pro-inflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) can be activated by SARS-CoV-2 very fast. After that, GM-CSF further activates CD14+ CD16+ inflammatory monocytes to secrete immense amounts of IL-6 and tumor necrosis factor-α (TNF-α) (55). Neutrophil extracellular traps (NETs), weak IFN-γ induction, and membrane-bound immune receptors (e.g., Fc and Toll-like receptors), might be some of the causes for massive cytokine release (53,54). One of the possible mechanisms of the cytokine storm is induced by the angiotensin 2 (AngII) pathway, where SARS-CoV-2 triggers NF-κB. ACE2 on the cell surface is occupied by SARS-CoV-2, which results in decreasing in ACE2 expression and an increase in AngII. Also, AngII-angiotensin receptor type 1 (AngII-AT1R) axis can induce TNF-α and the soluble form of IL-6Ra (sIL-6Ra). IL-6 binds to sIL-6R through gp130 to form the IL-6-sIL-6R complex, which can trigger STAT3 in non-immune cells. Both NF-κB and STAT3 are able for activation of the IL-6 amplifier (IL-6 Amp) to induce various pro-inflammatory cytokines and chemokines, such as IL-8, and IL-6, vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1) (56, 57). Some studies have been demonstrated that levels of IL-1β, IL-2, IL-6, IL-7, IL-8, IL-12, inducible protein 10 (IP-10), MCP-1, TNF-α, macrophage inflammatory protein 1 alpha (MIP1A), granulocyte-colony stimulating factor (G-CSF), and IFN-γ were increased, though levels of the Th2 cytokine IL-4 were low in patients with severe COVID-19 (58, 59,60). Other studies showed that the level of CD4+ cells and CD8+ cells was reduced constantly, but neutrophil counts were elevated in patients with a severe form of COVID-19 comparing with mild patients (60). Because of the rapid evolution of the cytokine storm, critically ill COVID-19 patients develop acute respiratory distress syndrome (ARDS) (61). ARDS is described as a multi-factorial syndrome of severe lung injury whose main characteristics are: hypoxemia, pulmonary oedema, diffuse alveolar damage and multiple organ failure. Lungs are not able to provide sufficient oxygen saturation to the alveolar spaces and respiratory support may be required (62). Yet, there are paradoxical outcomes in terms of SARS-CoV-2 positive Chronic Obstructive Pulmonary Disease (COPD) patients. Actually, most of COPD patients did not develop critical symptoms, which is contradictory, due to the fact that pulmonary co morbidity elevates chances of ARDS development and fatal outcome (63, 64). It is assumed that key role in this “protection” plays IL-6. Covid-19-related pneumonia is caused by inflammasomes activation and secretion of IL-1, TNF and IL-6, making IL-6 crucial in ARDS development. IL-6 coordinates the cytokine storm resulting in an immune hyper-reaction and in the inflammatory injury of the pulmonary tissue (65). This is in line with a meta-analysis that emphasized high levels IL6 in the severe forms of Covid-19 (66). Moderately-elevated concentrations of IL-6 found in moderate COPD patients has protective roles against the decay of Covid-19, explaining therefore their better prognosis. Eventually, the drug Tocilizumab, a monoclonal antibody that blocks the IL-6 receptor, is currently used to treat Covid-19 in hospitals (67). These outcomes, lead to conclusion that Tocilizumab must be utilised only in patients with an current cytokine storm, or else, it’s usage may be damaging.

Luckily, MSCs are insusceptible to virus infections due to IFN-stimulated genes (ISG), that can impact every of step of viral cycle (68,69,70). Specifically, PMAIP1, ISG15, IFI6, IFITM, SAT1, p21/CDKN1A, SERPINE1 and CCL2 are ISGs that being expressed in MSCs are shown to halt numerous viral pathogens, but most importantly SARS (69). Team of researches made a list of ISGs constitutively expressed by human MSC and afterwards set up by IFITM1, IFI6, CCL2, ISG15, SAT1 and PMAIP1. In attendance of IFN-γ there was an induction of non-constitutive ISGs, as well as MT1G, CD74, SERPING1, IFNAR2 and MT1X (68). MSCs have this great ability to switch to non-constitutive ISGs up regulation in order to better fight viruses, which can be advantageous in regards to respiratory infections (68). One of the most crucial ISGs is IDO, which not just that have immunomodulatory properties, but also inhibits viral protein bio synthesis (71). These anti viral features, besides familiar MSCs’ immunomodulatory, antiinflammatory, neoangiogenic, regenerative and multipotent potential, definitely make MSCs potent and competent player in a harsh battle against Covid-19.

Since SARS-CoV-2 and COVID-19 first and foremost affect lungs, leading to ARDS, pneumonia and MODS consecutively, plus the fact that MSCs entrap in lungs, traits of MSCs residing in lungs should be thoroughly observed as therapeutic tool for SARS-CoV-2. It is shown that lung residing MSCs promote regeneration and tissue repair (72). Lung residing MSCs are positioned perivasculary and express CD73 which in turn promotes expression of anti-inflammatory genes in MF and vice versa (72). Again, exhibition of this marker is flexible and differ depending on the cell surrounding conditions, and testify how MSCs are sensitive to inflammatory response (73). MSCs respond by production of soluble factors, specifically when in lungsMSCs secrete FGF7 (or KGF) leading to normalization of alveolar clearance (74). Various proteomic and transcriptomic analyses displayed that lung MSCs affect signal pathways in regards to up regulation of Wnt/β-catenin and down-regulation of NF-κB signaling, leading to diminished TNF-α production and eventually to fibrosis reduction (75, 76, 77). MSCs displayed remedial effect in model of ARDS by stimulating surfactant secretion, differentiating into pulmonary endothelial cells and alveolar epithelial cells (78). Hence, most important are exactly these effect in regards to SARS-CoV-2-cytokine storm and ARDS (79, 80), which has already been elaborated in preclinical and clinical studies (81, 82).

Most recent study revised outcomes of MSCs applied in patients with ARDS and Covid-19 related conditions (83). When it comes to ARDS, highlights should be noted; patients with ARDS received BM-MSCs in dose of 10 million cells/kg. It is found no MSCs related adverse effects, except for, three patients who died in next weeks after treatment. Yet, for one who died from multiple spleen, brain and kidney embolic infarctions, it is confirmed by MRI that these changes were present prior to MSC administration (84) suggesting MSCs administration as safe. This study was the first phase of research, that was extended by Matthay et al. (85), and included much larger group of moderate to severe ARDS patients who received intravenously same dosage of human bone-marrow-derived MSCs. This study suggested no infusion-related haemodynamic or respiratory complications, even though higher numerically mortality and severity scores were observed in patients who received MSCs comparing to placebo group, but this difference was not statistically significant. Most important is the finding that showed direct impact of MSCs viability on angiopoetin 2 plasma concentrations, leading to drastic decrease of angiopoetin 2 concentrations, 6h after MSCs infusion which can be beneficial in ARDS. This study is planned to be continued, so more consistent and clear data could enlighten previous conclusions. In spite of insufficient and limited data in regards to clinical outcomes from patients that received MSCs therapy to fight Covid-19 respiratory disease, certain clinical trials emerged and propose MSCs as safe, beneficial and with no detrimental adverse effects (86, 87). Patients in China, on March in 2020, with severe COVID-19 pneumonia, were treated with MSCs, intravenously. In this study for the first time it is showed that MSCs are ACE2- and TMPRSS2- and that MSCs secrete anti-inflammatory factors to prevent the cytokine storm-hence displaying that MSCs have natural immunity to the HCoV-19. This MSCs therapy improved vital condition of patients, reduced level of inflammatory cytokines and chemokines so less mononuclear/macrophages were attracted to injured lung tissue. At the same time bigger number of regulatory DCs were directed to the inflammatory tissue niche. In addition, they noted enhanced levels of IL-10 and VEGF, decreased C-reactive protein and TNF-α levels (80). Among these data most important was the observation of lack of overactivated cytokine-secreting immune cells CXCR3+ CD4+ T cells, CXCR3+ CD8+ T cells and CXCR3+ NK cells (80), which in summary can explain lung regeneration and improvement in critical patients. Later on, in June, a detailed review by Rajarshi stated MSCs therapy in COVID-19 patients as justified and advantageous (88). Other study examined therapeutic potential of Wharton Jelly’s MSCs (hWJCs) delivered intravenously in COVID-19 positive critically ill patient with pneumonia and diabetes. After his vital parameters stabilized, he received hWJCs intravenously. After hWJC adoptive transfer, there were no adverse effects whereas level of serum CRP and inflammatory factors (IL-6 and TNF-α) were drastically decreased, and vital parameters of the patient improved. Plus the level of CD3+, CD4+ and CD8+ T cell were significantly increased after intravenous injection of hWJCs (89). This is the first study of this kind, and though encouraging and promising, should be elaborated and further examined with much bigger group of patients. These preliminary clinical records lead to conclusion that MSCs alone or combined with other therapeutics could enhance the chances of survival and improve overall health condition in Covid-19 patients (80,90). One study examined exosomes (ExoFloTM) derived from allogeneic BM-MSCs as treatment for severe COVID-19 (91). 24 SARS-CoV-2 PCR positive patients received 15 mL dose of ExoFlo intravenously and were observed in regards to safety and effectiveness.

There were no any side effects of ExoFlo treatment 72 hours after treatment. 83% of the patients survived, 71% of patients cured; 13% stayed in critical though stable condition; 16% passed away of causes unconnected to the therapy. On the whole, after treatment, patients’ clinical condition and oxygenation improved. Additionally, there were enhancements in absolute neutrophil number and lymphopenia. Also, there was significant diminishing of C-reactive protein (CRP), Ferritin and D-dimer, confirming therefore beneficial effect of ExoFlo on inflammatory response. Having in mind such data, ExoFlo could be a prommising tool of Covid-19 related pathology, due to it’s safety, ability to halt inflammation, stabilize immune response and oxygenation. In spite of that, further research with bigger group of patients with similar results could guarantee ExoFlo as secure therapeutic approach.

In order to summarize ongoing MSC-based therapies in patients suffering from Covid-19 worldwide ClinicalTrials.gov database was analysed. There are 68 studies that include MSCs treatment in various Covid-19 related conditions-Pneumonia and ARDS. Most of these studies are randomised and open-label studies and in early phase (phase I, I/II, II). Specifically, 29 of the studies are currently recruiting, yet 26 are not recruiting yet. There are 8 completed studies, while there are 3 enrolling and 1 study is withdrawn. Most of the studies utilised BM-MSC, UC-MSC, WJ MSC, AT-MSCs. However, there are fewer studies that utilised allogeneic dental pulp MSCs, placenta-derived MSCs, stromal MSCs, MSC derived exosomes and secretomes. Most of the studies follow basic protocols of disease severity assessment such as inflammatory cytokine profile (TNF-α, IL-6 and IL-10), vital parameters and pulmonary function assessment. Studies groups include both, males and females varying in age. All of these data are summarized in Table 1.

Other clinical trials also accentuate the significance and safety of MSCs usage in the treatment of COVID-19 patients with no reporting of any serious adverse events related to MSCs (92,93,94,95,96,97,98,99,100), although there is a necessity for large randomized multicenter clinical trials in order to determine precise therapeutic potentials of MSC in COVID-19-induced disease (92,93,97). It is also very important to emphasize that MSCs should not be administered in the early period of viral infection because inflammation is very pertinent and advantageous to combat viral infection. “Physiological inflammation” which is very important for control of virus infection and replication, can be abrogated by too much immunosuppression that is induced by inadequate use of MSCs in terms of the time of MSCs administration and their dose (101,102,103,104). However, there is the urge to investigate the safety of using human MSCs in long period follow-up. It is well-known that MSCs exhibit potential risks of adverse events during MSC transplantation (105) in CVDs (cardiovascular disease), neurological disease, GVHD (graft-versus host disease), and orthopedics, such as pro-tumorigenic effect, perturbed differentiation capacity, short surviving after implantation, not so amazing improvements, immune response and infection-related mortality (106). Some animal studies reported that MSCs therapy for CVDs can exhibit some adverse events such as proarrhythmic (107) and tumorigenic ability in heart tissue (108) as well as the ability to differentiate into unwanted tissue type (109). Although many studies suggest the usage of MSCs as a treatment in orthopedics, it is important to note that MSCs are able to take part in ectopically forming bone tissue in non-bone tissues, which is better known as heterotopic ossification (HO) (110). Although MSCs are cells with doubtful-advantage in numerous animal studies that showed MSCs promoted tumor growth and metastasis (111,112), or even suppressed their growth (112), MSCs should be used with caution in clinical studies.