Potential roles of mesenchymal stem cells and their exosomes in the treatment of COVID-19

1. Abstract 2. Introduction 3. MSCs regulate immune cells in COVID-19 3.1 MSCs interfere with the differentiation, maturation, and function of antigen-presenting cells (APCs) in COVID-19 3.2 MSCs regulate the polarization of macrophages in COVID-19: from M1 to M2 3.3 MSCs regulate lymphocyte subsets and apoptosis in COVID-19 4. MSCs improve ARDS in COVID-19 5. MSCs promote lung regeneration and reverse PF in COVID-19 6. MSC-exosomes for the treatment of COVID-19 6.1 Comparison of MSC-exosomes and MSCs 6.2 Unique advantages of MSC-exosomes over MSCs for COVID-19 treatment 7. Clinical trials of MSCs and MSC-exosome therapy for COVID-19 8. Conclusions 9. Author contributions 10. Ethics approval and consent to participate 11. Acknowledgment 12. Funding 13. Conflict of interest 14. References

The primary pathogenesis is overactivation of the immune system. SARS-CoV-2 continues to mutate and spread rapidly and no effective treatment options are yet available. Mesenchymal stem cells (MSCs) are known to induce anti-inflammatory macrophages, regulatory T cells and dendritic cells. There are a rapidly increasing number of clinical investigations of cell-based therapy approaches for COVID-19.
Objective: To summarize the pathogenic mechanism of SARS-CoV-2, and systematically formulated the immunomodulation of COVID-19 by MSCs and their exosomes, as well as research progress. Method: Searching PubMed, clinicaltrials.gov and Chictr.cn for eligible studies to be published or registered by May 2021. Main keywords and search strategies were as follows: ((Mesenchymal stem cells) OR (MSCs)) AND . Results: MSCs regulate the immune system to prevent cytokine release syndrome (CRS) and to promote endogenous repair by releasing various paracrine factors and exosomes. Conclusions: MSC therapy is thus a promising candidate for COVID-19.

Introduction
According to real-time WHO network data, the worldwide number of confirmed COVID-19 cases to April 22, 2021 was 143,488,236 and 3,055,587 deaths, posing an unprecedented threat to the global economy and to human health [1]. The International Committee on the Taxonomy of Viruses named the COVID-19 pathogen as SARS-CoV-2. This virus gains phagocytic entry into AT2 via interaction with angiotensin-converting enzyme 2 (ACE2) (See Fig. 1). It increases Angiopoietin-2 (Ang-2) levels, lead- ing to long-term and intense activation of pro-inflammatory Ras-related pathways. A high concentration of Ang-2 in the lung interstitium promotes cell apoptosis, releases proinflammatory cytokines and triggers the inflammatory response, thereby causing immune-induced tissue damage and increased vascular permeability [2,3]. In patients with severe disease, the development of CRS is an abnormal systemic inflammatory response that manifests clinically as a rapid and sharp rise in the level of cytokines. These include C-X-C Motif Chemokine Ligand 10 (CXCL10), monocyte chemo-attractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1 (MIP-1), platelet-derived growth factor (PDGF), tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) [4]. This mechanism results in an imbalance between tissue damage and repair, leading to respiratory failure. Patients may eventu- ally die from multiple organ failure. Disruption of matrix metalloproteinases (MMPs) during the inflammatory stage causes complex damage to the alveolar epithelium and to the pulmonary vascular endothelium [5]. Moreover, persistent stimulation of epithelial cells results in senescencerelated phenotypes. Consequently, abnormal interactions between fibroblasts and epithelial cells generate irreversible damage and fibrosis [6].
MSCs can interfere with the antigen-presenting function, differentiation and maturation of DCs by paracrine IFN-γ, indoleamine 2,3-dioxygenase (IDO), transforming growth factor-β (TGF-β), IL-10 and prostaglandin E2 (PGE2) [37]. Consequently, this reduces the activation of DCs and their pro-inflammatory secretions [38,39]. Leng et al. [40] observed a significant increase in the number of CD14+CD11c+CD11 mid low-activity phenotypic DCs on day 6 after MSC transplantation. This prevented the excess proliferation of T cells in COVID-19 patients. The interaction between MSCs and DCs also leads to an indirect conversion of pro-inflammatory Th1 to anti-inflammatory Th2 immunity [41].

MSCs regulate the polarization of macrophages in COVID-19: from M1 to M2
Pro-inflammatory macrophages were reportedly more abundant in the bronchoalveolar lavage fluid from severe compared to mild COVID-19 cases [42]. Zhang et al. [43] proposed that CRS in severe COVID-19 is mainly a virus-triggered macrophage activation syndrome. Viral RNA stimulates macrophages to produce various soluble factors via the activation of TLRs. These factors include IFN-γ, IP10, MCP1, MIP10, granulocyte colony stimulating factor (G-SCF), IL-2, IL-6, IL-7 and TNF-α. In particular, IL-6 and TNF-α cause macrophages to differentiate into M1 (Fig. 1d), thus causing an imbalance in M1/M2. In addition to direct stimulation by viral RNA, ATP is released by the dead cells as DAMPs and binds to the P2X7 receptor (P2X7R). This in turn activates NODlike receptor protein 3 (NLRP3) inflammasomes and increases macrophage-derived IL-1β and IL-18 [37]. The loss of alveolar macrophages is a major underlying cause of refractory respiratory failure in COVID-19 and it has been reported they are almost entirely depleted in severely infected patients [44].

MSCs regulate lymphocyte subsets and apoptosis in COVID-19
Flow cytometry analysis has shown that the number of CD4+ and CD8+T cells in the peripheral blood of COVID-19 patients was significantly reduced, with the degree of reduction being related to the severity of COVID-19 [53]. This phenomenon may be related to the recruitment of T cells from peripheral blood to lung tissue, and to the apoptosis of T cells induced by the virus [54]. COVID-19 patients present with lymphocyte deficiency and over-activation of T cells. These effector T cells are stimulated by pro-inflammatory mediators produced by DCs, macrophages and neutrophils [55,56]. A significant rise in HLA-DR+CD38+ cell levels can manifest in the over-activation of T cells. The proportion of highly proinflammatory CCR4+CCR6+ Th17 cells amongst CD4+ T cells then increases [57]. High expression of IL-17A in Th17 induces the migration of inflammatory white blood cells, leading to inflammatory infiltration and destruction of lung tissue. Additionally, the major histocompatibility complex 1 (MHC-1) of infected cells presents viral antigens, thus activating CTLs to produce high levels of cytotoxic granules such as perforin and granzymes. This implies that over-activation of T cells and the elevated cytotoxicity of CD8+T cells leads to an excessive immune response. T cell-derived cytokines and chemokines such as TNF-α, IFN-γ, IL-2, IL-12, CCL2, IL-18, CCL9, CXCL10, IL-6 and IL-17 are released in large quantities and damage the lung tissue [58]. When the T cell count falls to its lowest level, the concentrations of serum IL10, IL2, IL4, TNF-α and IFN-γ reach their peak on days 4-6. Moreover, the levels of IL-6, IL-7, G-CSF, IP-10, monocyte chemotactic protein-1 (MCP-1) and MIP1a increase significantly, thus causing CRS [59].
Leng et al. [40] reported that on day 4 after MSC transplantation, the absolute lymphocyte count increased to 0.58 × 10 9 /L and lymphocytopenia improved significantly. On days 3 to 6 after transplantation, the level of TNF-α decreased whereas that of IL-10 increased. Similar reversals were reported in another study [60]. T cell counts were also analyzed in a non-randomized, open-label cohort study of COVID-19 patients [18]. This indicated that MSCexosome therapy significantly improved the absolute neutrophil count by a mean of 32% [p value < 0.001] in patients with severe COVID-19. Moreover, the mean CD3+, CD4+ and CD8+ lymphocyte counts increased by 46% (p < 0.05), 45% (p < 0.05) and 46% (p < 0.001), respectively.
The mechanism of action of MSCs in reversing lymphocytopenia and reducing inflammatory mediators in COVID-19 is mainly attributed to the more than 30 soluble paracrine factors such as PEG2, IDO and COX-2 [61]. These have been shown to inhibit the proliferation of CD4+ Th1 and Th17 cells as well as CD8+T cells, and to induce Foxp3+ Treg differentiation (Fig. 1f). They also indirectly inhibit excessive T cell proliferation by interacting with APCs and other immune cells. IL-10 is a critical negative regulator of T cell responses and directly inhibits the ability of T cells to produce pro-inflammatory mediators. IL-10 also reduces the antigen presenting capacities and co-stimulation of macrophages and DCs, thereby decreasing T cell-derived IL-6 and TNF-α, which is one of the essential mechanisms by which MSCs alleviate inflammation [62,63]. This was demonstrated in a recent clinical report by Meng et al. [64] which showed that patients who received MSCs had lower IL-6 levels than those who received placebo [65].
Through their expression of PD-L1 and FasL, MSCs can inhibit abnormally activated Th1 cells, thus inhibiting IL-γ release from Th1. This prevents further macrophage activation in a vicious loop and restores Th1/Th2 balance. Long-term FasL interaction can induce apoptosis of cytotoxic T cells [66].
In COVID-19 patients the average time from symptom onset to dyspnea is 5 days, the average hospital stay is 7 days, and the average time for onset of ARDS is 8 to 9 days [72]. By day 8 to 14 of disease onset, the overexpression of cytokines such as IL-2, IL-6, IL-7, IP10, MCP1, MIP1A and TNFα causes activation of lympho-cytes and macrophages, leading to an excessive inflammatory response [73]. The integrity of alveolar walls and pulmonary capillaries are destroyed, resulting in edema that impairs oxygen exchange and respiration and inevitably develops into ARDS [74,75].
MSCs and MSC-exosomes can effectively alleviate COVID-19-induced ARDS in a dose-dependent manner by increasing alveolar fluid clearance and by improving airway and hemodynamic parameters [76]. MSC-exosomes have been used as intravenous infusion therapy for ALI and pulmonary fibrosis (PF) [23,77,78]. An earlier study showed the exosomes release keratinocyte growth factor (KGF) and Lipoxin A4 which act to prevent long-term lung damage caused by COVID-19 and to promote tissue repair by activating Na+/K+ pumps [79] (Fig. 2d). Importantly, MSCs have been shown to restore epithelial protein permeability, stabilize endothelial fluid leakage, and maintain alveolar-capillary barrier function by secreting Ang-1 [80][81][82]. In addition, MSCs can inhibit cellular signaling pathways mediated by TLRs or PRRs, as well as reducing local immune cell recruitment (Fig. 2c). miRNA-126, VEGF-α, phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) and high mobility group box chromosomal protein 1 (HMGB1) can each restore the vascular endothelial cadherin-catenin (VE-Cadherin) complex and reduce endothelial barrier permeability to relieve ARDS.

MSCs promote lung regeneration and reverse PF in COVID-19
PF is a refractory lung disease that develops due to persistent alveolar injury, repeated destruction, repair, reconstruction, and excessive deposition of extracellular matrix (ECM) [83]. Current studies have determined that only 1% of AT2 cells can regenerate following SARS-CoV-2induced lung injury [84,85].
High expression of IL-17A by Th17 in COVID-19 can induce the migration of inflammatory leukocytes, leading to inflammatory infiltration and the destruction of lung tissue [86]. High levels of TNF-α induce the recruitment of immune cells and reduce antioxidant molecules in parenchymal and endothelial cells, causing lung fibrosis and remodeling (Fig. 1e). MSCs improve angiogenesis mainly through paracrine release of pro-angiogenic/antiapoptotic agents such as Ang, IL-3, MMP-1 and VEGF [87]. They also secrete ECM regulators such as fibroblast growth factor, HGF and MMPs to regenerate damaged tissues [88]. Moreover, MSCs express or secrete ADAM, metallopeptidase with thrombospondin Type 1 Motif 2 (ADAMTS2), basic fibroblast growth factor (bFGF), collagen 15A1 (COL15A1), COL16A2, COL18A1, HGF, high temperature requirement A1 (HTRA1), lipoxygenase (LOX), and tissue inhibitor of metalloproteinases 2 (TIMP2). These regulate the expression of collagen, fibronectin and elastin fibrilaments in lung tissue, thereby alleviating PF [89]. MSCs can also reverse PF by overexpressing MMP-P1 and decreasing collagen-1 (COL-I) production during TGF-β1-induced fibrosis. In conclusion, MSCs can promote angiogenesis and the regeneration of alveolar epithelial cells, prevent the apoptosis of endothelial cells, reduce the levels of TGF-β, TNF-α, COL-I, COL-III, Hydroxyproline and serum ceruloplasmin, inhibit myofibroblast growth, and thereby alleviate or reverse PF (Fig. 2e).
Human embryonic stem cells (hESCs) derived from immune and stromal regulatory cells (IMRCs) have been used to treat lung injury and fibrosis in vivo. IMRCs have superior efficacy to FDA-approved pirfenidone [14] and show excellent efficacy and safety in both mice and monkeys [90].

MSC-exosomes for the treatment of COVID-19
MSC-exosomes are able to transfer cargoes such as mRNA, miRNA, proteins, lipids and even mitochondria to target cells and tissues, resulting in changes to gene expression and in the behavior of target cells. Hence, MSC-exosomes could have a therapeutic role in COVID-19 [91,92]. Preclinical studies have confirmed that MSCexosomes are able to serve as acellular alternatives [78].

Can MSC-exosomes effectively replace MSCs?
A growing number of studies have established that the healing, nutritional, immunoregulation, and antiinflammatory effects of administered MSCs are due to the exosomes they release. These effects of MSCs have been observed in vitro after the addition of MSC-exosomes [37]. MSCs were cleared from the circulation within 24 hours, but MSC-exosomes were detected in lung parenchymal cells and macrophages just 1 hour after injection and remained there for up to 7 days [93]. The efficacy and safety of a single intravenous injection of MSC-exosomes were recently assessed in 24 COVID-19 patients who presented with moderate to severe ARDS. The clinical symptoms, oxygenation, serum markers of acute inflammation, neutrophil and lymphocyte counts all improved in patients who received MSC-exosomes, with no side effects reported [18].
MSC-exosome-transferred miRNAs cause APCs to produce fewer Ag/MHC molecules on their surface, thus resulting in reduced activation of effector T cells. miR-NAs carried by MSC-exosomes also mediate the function of macrophages, NK cells, T cells and B cells to inhibit infection [98].

MSC-exosomes are safer than MSCs for COVID-19 treatment
In the context of COVID-19, MSCs are known to aggregate in the peripheral microvasculature and exacerbate vascular clots, causing central or peripheral vascular dysfunction. This is probably because MSCs express procoagulant tissue factor (TF/CD142) on their surface [99,100].
The small size and low immunogenic effect of MSC-exosomes allows them to pass through small blood capillaries without triggering a blood clot [101]. Because of their strong ability for self-replication and differentiation, the carcinogenicity of MSCs is also another clinical challenge. MSC-exosomes cannot replicate and hence there is no risk of endogenous tumor formation [102].

Advantages of MSC-exosomes for COVID-19: practical considerations
The challenges surrounding the use of MSCs for COVID-19 that still need to be overcome include their immuno-compatibility, stability, heterogeneity, differentiation and migration. The low homing rate of MSCs is also the focus of current research. Although Xiao et al. raised the possibility that CD90 binding to the specific integrins b3 and b5 could to some extent promote MSC homing [103], MSC-exosomes have an important advantage in their homing ability. Due to their nanosized dimension, intravenously injected MSC-exosomes accumulate in COVID-19-damaged organs through blood circulation [104]. MSCexosomes from allogenic sources can also be used immediately after thawing and without washing. In addition, MSCexosomes are easier to use routinely in hospitals compared to MSCs. Finally, the cost of using MSC-exosomes is much lower than that of MSCs.

MSC-exosome as a drug and miRNA delivery system for COVID-19
Designing miRNAs that specifically bind to the SARS-CoV-2 genome could allow disruption of SARS-CoV-2 without any side effects on human gene expression [105]. Thus, MSC-exosomes that carry miRNAs may be a promising new approach to COVID-19 therapy. MSC-exosomes can be loaded with miRNAs either by direct insertion of the nucleic acids, or by collecting the exosomes from genetically-modified MSCs [106]. For example, miR-32, the first miRNA found to target viral RNA, binds to retrovirus PFV-1 transcripts to reduce viral replication [107], while miR-146a has been shown to specifically inhibit COX-2 in lung epithelial cells. miR-375 inhibits the trans-differentiation of myofibroblasts and their synthesis of collagen by blocking P38 [108].
MSC-exosomes are thus a novel intervention tool for COVID-19 treatment that can successfully deliver exogenous miRNAs to exert antiviral function. When combined with antiviral drugs such as Remdesivir, MSCexosomes can therefore serve as an effective drug delivery system [109].

Potential of MSC-exosomes for vaccine development
Spike protein is one of the structural proteins of SARS-CoV-2 that facilitates viral entry into the host cells. Therefore, spike protein is a good target for the development of anti-SARS coronavirus vaccines. Seraphin et al. showed that MSC-exosome-based vaccines containing the SARS-CoV-2 spike protein could induce high levels of neutralizing antibodies [17,[110][111][112].

Clinical trials of MSCs and MSC-exosome therapy for COVID-19
Current treatment trials for COVID-19 include corticosteroids, PD-1/PD-L1 checkpoint inhibitors, cytokine absorption devices, convalescent plasma [113] and anti-malarial and antiviral drugs [114]. Definite clinical benefits from these treatments have yet to be established and their safety and efficacy still need to be validated through Phase II and III clinical trials.
However, clinical trials have shown that MSC therapy and its derivatives are promising candidates for COVID-19 with known safety and efficacy. The United States FDA has approved MSCs for severe COVID-19 patients as compassionate use and progress has been made in this field. A study from Spain involving 13 COVID-19 patients requiring mechanical ventilation reported that no treatment-related adverse events (TRAEs) were observed [115]. After the first intervention with MSCs, clinical improvements were observed in 9 patients (70%) after a median follow-up of 16 days. Seven patients were extubated and discharged, while 4 patients continued intubation (2 with improved ventilation and radiological parameters, and 2 with stable conditions). The research team compared the clinical progress and mortality rates of their study cohort with similar cases in the intensive care unit (ICU). The mortality rate of patients who received MSC therapy dropped from 70-85% to 15% (2/13). Only 2 patients died during the study, one from massive gastrointestinal bleeding unrelated to the MSC treatment, and the other from secondary   Table 2). Preliminary results of NCT04491240 released on 21 October 2020 showed that compared to placebo, the clinical recovery time, C-reaction protein (CRP) and layered double hydroxide (LDH) levels were lower for 10 consecutive days after inhalation of a solution containing 0.5-2 × 10 10 nanoparticles (MSCexosomes) twice daily. These effects may have been me-diated by the contents released from the MSC-exosomes, which included for example miRNA-126, miRNA-290, miRNA-21, miRNA-30b-3p, let-7, miRNA-200, miRNA-145, miRNA-27a-3p, Syndecan-1, HGF and Ang-1 [117][118][119]. Clearly, the application of MSC-exosomes instead of MSC therapy offers significant advantages [120], including more manageable dosing, easier storage, more readily available sources, better stability and lower immunogenicity [121][122][123]. Moreover, its noninvasive administration via inhalation avoids the side effects and pain that are commonly associated with parenteral therapy.
For these reasons, MSC-exosomes are a highly promising, cell-free therapy for COVID-19 [124,125]. The U.S. Food and Drug Administration has in fact allowed the expanded use of MSC-exosome preparations for the treatment of COVID-19 [126]. These include aerosol inhalation of MSC-exosomes, targeted drug delivery based on MSCexosomes, and the development of MSC-exosome-based vaccines [127,128]. However, a phase 3 trial is needed to further evaluate the effects of MSC-exosomes on mortality and long-term lung dysfunction from COVID-19.

Conclusions
COVID-19 treatment is currently very challenging, especially because of its complications and sequelae. Intravenous MSC administration or inhalation of MSCexosomes can improve the overall prognosis for COVID-19 by a variety of mechanisms: (1) through their immune regulation, (2) by promoting tissue repair and regeneration, (3) through their anti-fibrosis effect, and (4) by resuming normal vascular permeability. All these mechanisms can interact to strengthen lung repair and to protect the organs from damage caused by the excessive immune response. Despite the readily available sources, high proliferation rate, minimally invasive or noninvasive administration, and no ethical concerns, several challenges remain to be addressed with MSC and MSC-exosomes therapy. In particular, the dosing and timing of MSC and MSC-exosome therapy require careful consideration, since improper use may aggravate immunosuppression and lead to an unfavorable prognosis for COVID-19.

Author contributions
JM contributed to the conception of the study and led to the submission. XC performed the tables and wrote the manuscript; LL helped perform the figures with constructive discussions; MJ contributed significantly to manuscript preparation; All authors approved the final version.

Ethics approval and consent to participate
Not applicable.

Acknowledgment
Thanks to all the peer reviewers for their opinions and suggestions.

Conflict of interest
The authors declare no conflict of interest.