Stem Cell‐based therapies for COVID‐19‐related acute respiratory distress syndrome

Abstract As the number of confirmed cases and resulting death toll of the COVID‐19 pandemic continue to increase around the globe ‐ especially with the emergence of new mutations of the SARS‐CoV‐2 virus in addition to the known alpha, beta, gamma, delta and omicron variants ‐ tremendous efforts continue to be dedicated to the development of interventive therapeutics to mitigate infective symptoms or post‐viral sequelae in individuals for which vaccines are not accessible, viable or effective in the prevention of illness. Many of these investigations aim to target the associated acute respiratory distress syndrome, or ARDS, which induces damage to lung epithelia and other physiologic systems and is associated with progression in severe cases. Recently, stem cell‐based therapies have demonstrated preliminary efficacy against ARDS based on a number of preclinical and preliminary human safety studies, and based on promising outcomes are now being evaluated in phase II clinical trials for ARDS. A number of candidate stem cell therapies have been found to exhibit low immunogenicity, coupled with inherent tropism to injury sites. In recent studies, these have demonstrated the ability to modulate suppression of pro‐inflammatory cytokine signals such as those characterizing COVID‐19‐associated ARDS. Present translational studies are aiming to optimize the safety, efficacy and delivery to fully validate stem cell‐based strategies targeting COVID‐19 associated ARDS for viable clinical application.

distinct strain of coronavirus related to those that resulted in the severe acute respiratory syndrome (SARS) pandemic in 2003 and the Middle East respiratory syndrome (MERS) pandemic in 2012. 3 Because of its recent emergence, much remains to be elucidated regarding the pathophysiological mechanisms, sequelae and strength and duration of the host immune response in SARS-CoV-2, despite a tremendous amount of research worldwide. SARS-CoV-2 has demonstrated high genetic variability and a rapid mutation rate, and preliminary evidence suggests that immune protection may be limited, providing challenges to the development of vaccines and treatments. 4 The persistent emergence of novel SARS-Cov-2 variants, during COVID-19-related pneumonia, with the subset of patients progressing to ARDS exhibiting a mortality rate of 52.4%. 7 With a median patient age of 51, this case study also underscored the elevated susceptibility of older subpopulation to ARDS and consequently a substantially higher risk of mortality. ARDS is a lifethreatening lung pathology characterized by rapid onset and resulting from a massive and generalized pro-inflammatory immune response in the lungs, circulation and other tissues in  patients; this cytokine storm represents the most life-threatening development in COVID-19 patients. 8 In non-COVID patients, ARDS typically arises as a complication of pneumonia, systemic infection and major trauma, 9 and it is associated with elevated transport of fluid from lung capillaries to alveoli, 10 the air sacs that are the site of gas exchange with the blood, resulting in pulmonary oedema, hypoxemia, and loss of lung compliance secondary to epithelial damage and pulmonary fibrosis.
The cytokine storm characterizing COVID-19 ARDS has also been implicated in tissue damage and embolus formation in multiple organ systems and to play a key role in the pathophysiology of extrapulmonary multiple organ failure secondary to ARDS 11,12 ; indeed, this process is hypothesized to be key in the development of a number of emergent chronic post-COVID-19 pathologies. For instance, emerging evidence investigating the development of 'postviral syndrome' in a subset of post-recovery COVID-19 patients is examining possible corollaries with earlier SARS variants which produced a chronic pathological state resembling chronic fatigue/myalgic encephalomyelitis as a result of viral infiltration to select brain regions. 13,14 Histopathological examination of brain tissue in necropsied patients has also demonstrated neurological complications in a subset of COVID-19 patients implicating non-inflammatory neurovascular damage in clinical manifestations ranging from loss of olfaction/gustation to loss of involuntary control of breathing through medullary centres, with the virus hypothesized to spread to the brain from the upper respiratory tract via the transcribrial route, where angiotensin-converting enzyme 2 (ACE2)-expressing tissues enable viral internalization. 15 Evidence from other coronaviruses in human case reports and in vivo models also suggest the possibility of brain lesioning and fatal encephalitis. 16 ized mortality rates as high as 17% 24 and was noted for mutating to optimize viral-host binding and replication 25 ; the Middle East respiratory syndrome (MERS-Cov) virus, with which SARS-Cov-2 shares 50% sequence homology 23 and which enters host cells via the DPP4 receptor, exhibits a mortality rate of 35%. 26,27 Symptoms of the other human coronavirus strains are generally mild with low rates of mortality and morbidity and these infections are typically associated with the 'common cold', accounting for 10%-30% of all adult upper respiratory infections annually. 22 Coronaviruses are comprised of single-stranded, positive-sense RNA genomes of 28-32 kb in length, the largest genomes of all RNA viruses, and these are translated to non-structural proteins through two open-reading frames. 28 SARS-Cov-2 displays a surface Spike S glycoprotein on its viral envelope that is critical for host receptor binding 29 and invades host cells via interaction with the angiotensin-converting enzyme 2 (ACE2) receptor and is subsequently internalized where it integrates within the host genome and exploits its machinery for viral replication. ACE2 receptors are widely distributed in lung alveolar epithelia as well as nasopharyngeal and oral mucosa cells; extra-pulmonary expression is widely distributed among tissues spanning multiple physiological systems, however, including the liver, kidneys, gut, endothelial and vascular smooth muscle, and brain, providing a mechanism for multi-organ involvement. 30

| Candidatesforcell-basedtherapies
Despite years of efforts by multiple investigative teams aiming to develop viable treatments for ARDS, many candidate therapies have failed to show efficacy. 31 While corticosteroidal anti-inflammatory drugs such as dexamethasone and hydrocortisone [32][33][34] or interleukin receptor antagonists [35][36][37][38][39] have been investigated for ARDS and COVID-associated ARDS with evidence of success for severely ill/ ventilated patient outcomes in a large number of instances, study outcomes to date have also proven inconsistent with these interventions, with steroidal interventions in some cases even elevating mortality in related cases of ARDS with influenza, leading to hesitancy among many clinicians to rely on these-especially given the lack of clarity on risk factors in instances of contrainidication. 33 In the case of broad immuno-suppressant effects with cytokinesuppressant drugs for instance, it has been hypothesized that immunosuppression may worsen infection which in some cases outweighs the beneficial effects of cytokine storm suppression, depending on the stage of disease and pre-existing immune status of the patient.
More recently, certain stem cell-based therapies are beginning to show promising results in mitigating ARDS symptoms. Key examples of these candidate interventions are shown in Table 1. These carry a number of predicted advantages over corticosteroidal or receptor antagonist-based pharmacological interventions, including their intrinsic inflammatory-suppressant properties which combine with a milieu of added supportive therapeutic components which promote for instance cellular repair and normalization of function; their generally non-immunogenic properties as the molecular contents are protected in physiological settings within lipid bilayers, eluding immune recognition; elevated uptake of their secretory components, as carried in extracellular vesicles owing to their recognition as biological carriers, optimizing delivery efficiency compared with non-cellular/non-vesicular molecular therapies; and the sustained secretion of therapeutically relevant factors following administration, which allows for a more protracted delivery within the recipient following each dose while simultaneously removing the need for higher and less well-tolerated single-dose concentrations within a single dose to achieve efficacy.
Mesenchymal stem cells (MSCs) have been given particular attention in recent years as they do not require de-differentiation as is the case with pluripotent sources, yet can still be induced to lineagespecific, directed differentiation. 40,41 MSCs can bypass the technical constraints presented by isolating cells from specific organs, or ethical concerns surrounding use of embryonic cells because, they can be harvested from both autologous and allogeneic sources of relatively accessible tissue sources including umbilical cord, bone marrow, adipose tissue, and placenta. 42

| TherapeuticbenefitsofMSCs
The initiating events for the progression of acute lung injury and

| Anti-inflammatory responses
High levels of pro-inflammatory cytokines, such as TNFα, TGFβ1, IL-1β, and IL-8, have been reported in pulmonary oedema fluid from ARDS patients. 89 TNFα and IL-1β are among a cocktail of cytokines released from macrophages after the immune system is activated. 90 Once released, they act as specific cell-membrane bound receptors to activate a signalling cascade to increase production of pro-inflammatory cytokines, lipid mediators, ROS and cell adhesion molecules. 90,91 The increased production of cytokines, lipid mediators and cell adhesion molecules facilitates migration of the inflammatory cells into tissues and worsens the lung injury as a result. 90 In an in vitro study, the MSCs secreted cytokine IL-1RA, which dramatically lessens the inflammation effect of TNFα and IL-1β expressed by macrophages. 54 IL-1RA acts as a competitive inhibitor to IL-1β by blocking IL-1β's binding site and the production of TNFα by macrophages. 54 MSCs also release F I G U R E 1 Mechanism of action for MSC therapy against ARDS. MSCs restore damaged lung tissue by secreting paracrine factors, transferring mitochondrial DNA, and liberating microvesicles. Secreted paracrine factors restore the alveolar cells by -first-reducing the effect of apoptosis, oxidation, and inflammation, and -second-restoring the fluid buildup and damaged tissues substances such as TSG-6, IGF-1 and LXA4, which induce antiinflammatory responses in murine acute lung injury models by directly acting on the cells inducing inflammation to undergo phenotypic transition. 57,[61][62][63][64]92,93 In addition to abrogating the effect of TNFα, IL-6, and IL-1β, MSCs have also been shown to inhibit recruitment of neutrophils and protein formulation within the inner alveolar space. 94 MSCs can also act to reduce cell attraction and migration as mediators of inflammatory processes, as in lung injury settings. For example, TGFβ1 has an essential role in lung repair and fibroproliferation by promoting collagen synthesis. 95  MSCs showed therapeutic benefit in mice in attenuating acute LPSinduced pulmonary inflammation. 120 In addition, the plasma concentration of sST2 can be used as a diagnostic factor to distinguish ARDS from acute heart failure and serves as a prognostic biomarker to assess the severity of ARDS to determine how supportive treatments and weaning practices should be implemented. [121][122][123] In addition to their anti-inflammatory properties, the therapeutic efficacy of MSCs depends on several other factors such as homing, tissue restoration and protective effects against apoptosis and oxidation. Genetic modification can thus serve as an excellent tool to improve the therapeutic benefits of MSCs.
MSCs have shown tropism to injury sites, but they may lose homing receptors after the large-scale expansion needed to produce enough cells for therapeutic doses. 124 MSCs genetically engineered to overexpress the C-X-C motif chemokine receptor 4 (CXCR4) on their cell surface showed improved homing to sites of tissue injury. CXCR4 interacts with its ligand, stromal cell-derived factor-1 (SDF-1), which has elevated expression at sites of tissue injury. [125][126][127][128] Similarly, MSCs that overexpress the E-prostanoid 2 To further enhance lung repair and restore pulmonary functions, genetic modifications involving anti-apoptosis and anti-oxidation pathways, for example overexpression of heme oxygenase-1, are also being extensively investigated. 75,116,132 These studies of diverse genetic modifications to MSCs have shown that a single modification can yield multiple benefits and suggest promise of this approach. Given the rapid advance of genetic engineering technology to date, we may expect that the huge capacity of MSCs will allow multiple modifications, raising the possibility of synergistic benefits to treat ARDS and increased numbers of genetically modified MSCs translated to clinical studies.  Tables 3 and 4.

| CELL-FREETHER APIE SDERIVED FROMTHES TEMCELLS ECRE TOME: E X TR ACELLUL ARVE S ICLE SANDMIRNA
In light of the key role of secretory factors in conferral of therapeutic benefits from MSC therapy, there is increasing interest in studying the therapeutic benefits of MSC-derived extracellular vesicles (MSC-EVs) as compared to MSCs themselves. [125][126][127] As MSC-EVs are a cell-free treatment, they offer multiple advantages over cell-based treatments. 133 First, as they are non-nucleated and acellular, they cannot proliferate, and thus, there is minimal risk of tumourigenicity. 134 Second, as they do not express HLA antigens, they pose much lower risks of immunogenicity, and graft-versus-host-disease than their source cells, and thus are safer for allogeneic transplantation as they pose a lower risk of host immunorejection. [135][136][137] Third, MSC-EVs are smaller than cells, which allows for better penetration into target tissues. In contrast, because of their larger dimensions cell therapies are restricted from penetrating certain membrane barriers or extravasating from capillaries to key tissues relative to their much smaller secretory vesicles, 137-141 and they carry a higher risk of embolus formation. [142][143][144][145][146][147][148] Furthermore, storage of MSC-EVs does not require cryopreservatives, such as DMSO, which are necessary for long-term storage of MSCs but are detrimental to their viability. In addition, MSC-EVs are less affected by repeated freeze and thaw cycles as compared to MSCs. 136,143,149,150 The secretome of MSCs and other stem cells includes small soluble proteins such as cytokines, chemokines, growth factors, and antiinflammatory factors. 151  incorporate key therapeutic components for delivery. 159,160 There is ample evidence that extracellular vesicles and their factors derived from a variety of cell sources are also able to directly impart It has been demonstrated for instance that murine bone marrow DCs pulsed with diphtheria toxoid (DT) are induced to generate exosomes promoting a DT-specific immunoglobin response. 164 Treatment with exosomes derived from Toxoplasma gondii antigen There is also evidence of secreted exosomes exhibiting directly antiviral properties through their intrinsic contents. Exosomes secreted by macrophages in response to IFN-a have been demonstrated to utilize Hepatitis A receptors to deliver antiviral substances to hepatocytes. 170 Exosomes isolated from human trophoblasts have also been found to exhibit directly antiviral properties in vitro, associated with miRNA cargoes derived from the chromosome 19 cluster as well as a unique peptide and phospholipid repertoire. 84 Intriguingly, Herpes Simplex 1 (HSV-1) viral microRNAs miR-H28 and miR-H29 transmitted via exosomes have also been found to restrict viral cell-cell transmission via IFN-y upregulation, postulated by the authors to represent a mechanism of limiting viral spread to uninfected cells in favour of maximizing transmission to an alternate host. 171 Exosomes containing the spike S protein derived from other variants of SARS-associated coronavirus (SARS-CoV) have also been found to successfully induce the generation of neutralizing antibodies in a murine model 172 The investigation of the therapeutic benefits of MSC-EVs in comparison to MSCs is a rapidly developing field. As of 5 January 2022, there are nine MSC-EV clinical trials, summarized in Table 5.

| CON CLUS ION
The persistent emergence of novel SARS-Cov-2 variants, and with these fears of novel strains capable of effective vaccine escape and/or heightened virulence, combined with continued strug-

DATAAVA I L A B I L I T YS TAT E M E N T
Data sharing not applicable to this article as no data sets were generated or analyzed during this study.