Mesenchymal Stem Cells: A New Piece in the Puzzle of COVID-19 Treatment Felipe Saldanha-Araujo1,2, Emãnuella Melgaço Garcez3, Amandda Evelin Silva-Carvalho1 and Juliana Lott Carvalho3,4* 1Hematology and Stem Cells Laboratory, Health Sciences Department, University of Brasília, Brasilia, Brazil 2Molecular Pharmacology Laboratory, Health Sciences Department, University of Brasília, Brasilia, Brazil 3Multidisciplinary Laboratory of Biosciences, Faculty Of Medicine, University of Brasilia, Brasilia, Brazil 4Genomic Sciences and Biotechnology Program, Catholic University of Brasília, Brasilia, Brazil COVID-19 is a disease characterized by a strong inflammatory response in severe cases, which fails to respond to corticosteroid therapy. In the context of the current COVID-19 outbreak and the critical information gaps regarding the disease, several different therapeutic strategies are under investigation, including the use of stem cells. In the present manuscript, we provide an analysis of the rationale underlying the application of stem cells to manage COVID-19, and also a comprehensive compendium of the 69 clinical trials underway worldwide aiming to investigate the application of stem cells to treat COVID-19. Even though data are still scarce, it is already possible to observe the protagonism of China in testing mesenchymal stem cells (MSCs) for COVID-19. Furthermore, it is possible to determine that current efforts focus on the use of multiple infusions of high numbers of stem cells and derived products, as well as to acknowledge the positive results obtained by independent groups who publicized the therapeutic benefits provided by such therapies in 51 COVID-19 patients. In such a rapid-paced field, up-to-date systematic studies and meta-analysis will aid the scientific community to separate hype from hope and offer an unbiased position to the society and governments. 1). Soon after, genome sequencing studies demonstrated that such cases were associated with a new coronavirus, named Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2). The disease caused by such a virus was named coronavirus disease 2019, or simply COVID-19 (2). The COVID-19 pandemic has taken aback governments, health systems, and also scientists. Still, the formidable challenges of understanding the biology of SARS-CoV-2 and the pathophysiology of COVID-19, in order to develop effective treatment and immunization protocols, have led to a fast worldwide mobilization of the scientific community. Countless studies and position articles have been published regarding COVID-19 and are set to expand rapidly. Stem cells constitute important assets for animal-free experiments, providing cheaper, and ethical alternatives to animal studies. In this sense, scientific papers have already described the application of stem cells to generate new data regarding several aspects of SARS-CoV-2 (3), similar to what has been done with other viral diseases in the past (4). In addition to the possible application of stem cells in basic research, such cells are also appealing tools for immunomodulation, as shown by our group and others (5–12), as well as tissue regeneration, with a great deal of accumulated knowledge regarding the underlying mechanisms of action and decades of clinical experience, as shown by our group and others (13–18). It is well-established that Mesenchymal stem cells (MSCs) are able to control the functions of most if not all immune cells, and that such effects occur through a network of mechanisms including direct cell-cell contact, and secretion of soluble factors with immunosuppressive function (19, 20). MSCs may therefore be promising tools for the management of disorders involving immune system dysregulation, which may be the case of COVID-19. In the context of the world-wide COVID-19 pandemic, clinical trials have been initiated, even though much necessary information regarding the disease is still lacking. Over 1,700 clinical studies have been registered worldwide, focusing on investigating COVID-19 and trying to validate novel therapeutic regimens, immunization protocols, and anti-viral strategies (World Health Organization International Clinical Trials Registry Platform). Among them, 69 involve the use of stem cells and stem cell-derived products. In the present manuscript, we provide an up-to-date analysis of the rationale and current studies involving the clinical application of stem cells to manage COVID-19. Even though data are still scarce, it is already possible to determine the state of the art, the main groups dedicating efforts in the theme, and also the short-term perspectives for COVID-19 management using stem cells. 21). They constitute enveloped, positive-sense RNA viruses, capable of infecting various mammals, including humans (21). The SARS-CoV-2 belongs to the Nidovirales order, Coronaviridae family, Coronavirinae subfamily. The Coronavirinae is further divided into the alpha, beta, gamma, and delta coronaviruses. While coronaviruses alpha and beta seem to have originated from mammals (especially bats), the gamma and delta coronaviruses seem to derive from pigs and birds (22). Prior to the present SARS-CoV-2 outbreak, coronaviruses were only thought to cause mild infections in humans. Now it is considered that among the seven coronaviruses subtypes that are capable of infecting humans, the beta coronaviruses–including SARS-CoV-2 -, may cause severe and fatal diseases. SARS-CoV-2 spreads mainly through the respiratory airways through droplets that are shed from the respiratory secretions of infected persons, but also by direct personal contact (23). Recently, the SARS-Cov-2 has been isolated from fecal samples of severe pneumonia patients, suggesting other transmission routes (24). It has been shown that SARS-Cov-2 is capable of infecting angiotensin I converting enzyme 2 (ACE-2) receptor-positive cells. Such a receptor is expressed by a wide variety of cell types, including the lung epithelial and capillary endothelial cells, in which it successfully replicates. Inflammatory cells were shown to be potentially infected by SARS-CoV viruses, but only leading to abortive infection (25). Still, viral entry engenders both innate and adaptive immune response, which initiate in loco, but soon are detectable in the serum of COVID-19 patients (26). The local tissue alterations provoked by the virus infection are characterized by intense inflammation, inflammatory cell migration, and edema (Figure 1). The subsequent tissue destruction and dysfunction are proportional to viral load and ACE-2 expression, which is increased in patients under Angiotensin-converting enzyme inhibitors /angiotensin receptor blockers therapy (27–29). Local tissue damage engendered by the viral infection is detectable by radiological analysis, even before the patient presents pneumonia symptoms. FIGURE 1 Figure 1. Putative mechanisms of action of MSCs against lung inflammation caused by COVID-19. During SARS-CoV-2 infection, lung ACE-2 positive cells, such as epithelial and capillary endothelial cells are infected, leading to cell damage, inflammatory signaling, and the release of cytokines and chemokines. The inflammatory milieu promotes the activation of local macrophages, dendritic, and endothelial cells, which further secrete soluble factors and promote the migration of circulating monocytes, granulocytes, as well as lymphocytes. This leads to a feed-forward process, characterized by inflammation, tissue destruction, and organ dysfunction. MSCs and their secretome can be used to counteract inflammation through contact-dependent and also paracrine processes. MSCs, Mesenchymal Stem Cells; Tregs, Regulatory T-cells. Created with BioRender.com. COVID-19 can also affect the heart, liver, kidneys and alter the immune system (30), presenting a wide range of clinical outcomes that range from mild to common, severe, and critically severe states (26). In the latter scenario, patients require advanced life support, which is of great concern to public health systems (31). The inflammatory response caused by SARS-Cov-2 is both the primary mechanism of viral elimination, but also tissue destruction and dysfunction (Figure 1). The internalization of the virus in tissue and immune cells leads to activation of nuclear factor-kappa B (NF-kB) pathway and secretion of a myriad of inflammatory factors, including IL-1, IL-17, TNF-α, and INF-γ (32–34). Hyperinflammation and cytokine storm, both of which can promote multiple organ failure, have been observed in severe and critically severe patients (35) justifying current efforts to test the therapeutic benefit of anti-inflammatory interventions, including corticosteroids, immunosuppressors, and inhibitors of Janus kinase (36). Mesenchymal Stem Cells (MSCs) can be obtained from different tissues and are characterized by regenerative and immunomodulatory properties, which render them exciting tools for cell therapy. As reviewed by our group and others (10, 37, 38), MSCs present remarkable angiogenic, healing, antiapoptotic, and immunomodulatory potential. Furthermore, due to the low expression of MHC-I, MHC-II, and costimulatory molecules, they can be generally recognized as immune evasive and safe when used in allogeneic settings (39). The immunomodulatory mechanisms of MSCs include cell contact-dependent (40) and paracrine processes, including the secretion of TNF-Stimulated Gene-6 (41), IL-10 (42), indoleamine 2,3-dioxygenase (43), adenosine (9), and also extracellular vesicles (7). Such processes lead to lower immune cell maturation and activation, in addition to promoting the differentiation of T-cells into regulatory T-cells (Tregs) (38). Over 1,000 clinical studies have been performed up to date investigating the therapeutic potential of MSCs for various purposes, including diseases in which the immune system response is exacerbated, such as rheumatoid arthritis (44), Crohn's disease (45), systemic lupus erythematosus (46–49) and graft- vs. -host disease (50). Despite the fact that many of such trials are still in progress, the available data obtained during the last 30 years clearly show that MSCs constitute promising tools in the treatment of inflammatory diseases. Considering inflammatory diseases, most consistent data relate to the use of MSCs in the treatment of the graft-vs. -host disease, highlighting the potential of MSCs to improve clinical signs of inflammation and favoring patient survival (50–53). Similar observations have been made in the context of other inflammatory disorders. For instance, it has been reported that MSCs infusions promote a reduction of the inflammatory status and ameliorates the clinical signs of patients with rheumatoid arthritis (54, 55). In Crohn's disease patients, several published studies revealed that MSCs induce perianal fistula healing (56–59). In recognition of the cited and other studies, FDA and EMA have conceded commercial approval for some MSC-based products targeting specific indications (e.g., Alofisel and Remestemcel-L, which are indicated to treat anal fistulas in Crohn's disease and graft- vs. -host disease, respectively). Frequently, the treatment with MSCs follows intravenous administration of the cells. Interestingly, it has been shown that in this scenario a significant percentage of MSCs are rapidly trapped in the lungs upon intravenous injection, and also that lesioned sites increase MSC migration and retention (60). In the lung capillaries, the same occurs: upon injury, the local production of the pro-inflammatory mediator Angiotensin II (Ang II) is increased, and drives MSC migration in vivo through interactions with the Angiotensin II type 2 receptor (61–63). When trapped in the lungs, MSCs show the same immunomodulatory behavior as described in other body sites, such as the capacity of releasing anti-inflammatory cytokines (64), and antimicrobial peptides (65) (Figure 1). As revised by Khoury et al. (66), at least 6 preclinical studies have been executed in order to investigate the therapeutic effects of MSCs in rodent (5 studies) and porcine (1 study) models of influenza virus infection. Two out of six studies described a lack of efficacy of MSC treatment in such models, but the most recent investigations (4 out of 6) have revealed positive outcomes, such as survival rate and decreased virus-induced inflammation. The capacity of influenza viruses to infect MSCs (which has not been assessed in vivo), host age, the varied MSC sources (umbilical cord and bone marrow), the different cell doses and administration routes involved in the studies may all help explaining the dissonant observations among research groups. Clinically, the anti-inflammatory effects of MSCs have also been investigated in the context of Acute Respiratory Distress Syndrome (ARDS) with different etiologies (67–69). In a more recent study, perhaps as a hint for the application of MSCs for COVID-19 management, Chen et al. have infused MSCs into influenza A (H7N9) patients and obtained a significant reduction in mortality, compared to control group (70). In the cited study, 17 patients with ARDS caused by H7N9 infection were treated with menstrual blood-derived MSCs. While the control group (treated with antiviral agents, oxygen inhalation, etc) presented 54.5% mortality, only 17.6% of MSC-treated patients expired. Interestingly, 4 patients were followed during 5 years, with no harmful effects during this period. Overall, the achieved results of MSC therapy for ARDS are promising, but important information is lacking in the literature, such as details on the management of control subjects, the time between MSC infusions, details regarding registered deaths, as well as other outcomes of interest, such as ventilator-free days and ICU stay. Therefore, considering the acquired experience of the scientific and medical communities regarding the clinical application of MSCs to modulate the immune response, as well as the limited–but existent–pieces of evidence regarding the safety and efficacy of MSC therapy for viral respiratory infections, MSCs have entered clinical investigation for COVID-19 treatment. The current state of the art is reviewed
