3. Treatment of SARS-CoV-2 with Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) originate from the bone marrow, umbilical cord, placenta, and other tissues and possess differentiation and programming potential with strong immunomodulation and endogenous repair mechanisms. In regenerative medicine, adult MSCs are known to be protective against pulmonary, neurological, hepatic, renal, and cardiovascular diseases. MSCs are known to exert immunomodulatory effects that impact macrophages and promote polarization towards a therapeutic or reparative phenotype [24]. It was evident from a previously published study that CCL2 (a cytokine responsible for the recruitment of macrophages) derived from MSCs exhibited therapeutic effects by assisting macrophage repolarization [24]. MSCs act as immune modulators for both the innate and the adaptive immune systems. These cells are also involved in the switching of the phenotypes of inflammatory macrophages (M1) towards a reparative or therapeutic phenotype (M2), and they are related to decreased TNF-α and increased IL-10 production that is mediated by cellular contact [24].

Immune modulation of MSCs relies upon the activation of effector functions in immune cells. In a previously published study, MSCs were found to suppress lung infiltration and the dissolution of pulmonary edema. MSCs are multipotent, regenerative, and self-renewable and possess the ability to suppress the immune response and to differentiate into type II alveolar epithelial cells in vitro [25]. In a phase 2 clinical trial (NCT03608592) examining patients with ALI and ARDS, MSCs were demonstrated to possess anti-inflammatory activity [26]. Based on the outcome of this safety trial, MSCs may alleviate SARS-CoV-2-associated CS and ARDS and may also prove to be a prospective therapy for chronic respiratory dysfunction and lung fibrosis. MSCs are the prime therapeutic approach for treatment of lungs fibrosis, because they are easy to isolate, and have minimum immunogenicity, reparative characteristics along with anti-inflammatory properties. Clinical trials proved that MSCs possess short-term safety and tolerability with some limited studies focusing on adverse pro-inflammatory and myofibroblastic feature. The possible mechanism behind the protective effect of MSCs in lung fibrosis is that, MSCs migrate to the site of action (i.e., site of injury) and release multiple paracrine factors that are largely mediated by their extracellular vesicles that regulate the epithelial and endothelial permeability and cause decrease inflammation along with promotion of tissue repair. MSCs exclusively exhibit three characteristics in SARS-CoV-2-infected patients, including (i) activation of T cells to relieve excessive immune responses, (ii) maintenance of homeostasis in response to specific injuries associated with the lungs while also promoting regeneration, and (iii) inhibition of inflammation caused by the release of cytokines that is mediated by extracellular vesicles (EVs) or exosomes [27,28]. These exosomes deliver mRNA, microRNA, DNA, proteins, and metabolites to trigger reparative, therapeutic, and regenerative functions in the lungs [29].

4. Exosome: A Nanometer-Sized Envelope Possessing Miraculous Properties

Exosomes are nanometer-sized enveloped small extracellular vesicles that are extracted by ultracentrifugation of spent culture media of cells, and they are generated by the processes of the endocytosis pathway [30]. Almost all cell types release exosomes that contain varied biological information in the form of microRNAs (miRNAs), nucleic acids, proteins, growth factors, and EVs. In addition to facilitating cell-to-cell communication, exosomes are also helpful in determining the cell state by acting as biomarkers for diagnosis and prognosis [31]. Exosomes possess a small cup-shaped spherical morphology with sizes ranging from 30 to 100 nm in diameter. Exosomes also contain conserved proteins, including tetraspanins (CD9, CD63, and CD 81), MHC class I and II, and heat shock proteins (HSP 60, 70, and 90) [32]. Primary cells, hematopoietic cells, viral particles, and cancer cells found in biological fluids such as saliva, synovial fluid (SF), serum, plasma, bronchoalveolar lavage (BAL) fluid, urine, amniotic fluid (AF), pleural effusions (PE), semen, were all found in proximity to exosomes.

5. Biogenesis of Exosomes

Exosome biogenesis is a continuous cellular phenomenon that is initiated by inward invagination of the plasma membrane within the cytosol to form early and late endosomes. Late endosomes can further fuse together to form multivesicular bodies (MVBs) that invaginate to form intraluminal vesicles (ILVs). ILVs formed during the episodes of inward invagination are accompanied by several essential proteins, cytoskeletal components, nucleic acids, growth factors, necessary cellular components. After formation, these MVBs containing ILVs fuse together with the plasma membrane of the cells and are released as exosomes by exocytosis into the extracellular spaces. In previous studies, it was speculated that in the MVB biogenesis process, the budding of exosome cargos and their sorting is either endosomal sorting complex required for transport (ESCRT)-dependent or -independent [32]. Figure 1 demonstrates the mechanism of EVs biogenesis and its isolation.

It is believed that the ESCRT-mediated cargo-sorting phenomenon includes the identification and sequestration of ubiquitinated proteins to specific sites of the endosomal membranes [32]. Furthermore, the sequential association between subunits I, II, and III of ESCRT terminates the complex and initiates the budding process. It was revealed that the sorting protein Vps4 is involved in the detachment of the ESCRT III complex from the MVBs membrane that aids in the formation of ILVs from cleaved buds [32]. However, ESCRT-independent processes involve proteins and lipids such as tetraspanins (CD81) and ceramides. These exosomes associate post-release with recipient cells for efficient uptake. The exosome uptake mechanism involves both clathrin-dependent and -independent events, micropinocytosis, phagocytosis, and lipid raft-mediated internalization.

These exosomes are composed of proteins such as heat shock proteins, cell adhesion proteins, tetraspanin membrane proteins, cell signaling proteins, transcription proteins, and trafficking membrane fusion proteins [32]. A variety of lipid molecules, including phosphatidylserine (PS), phosphatidic acid, cholesterol, sphingomyelin (SM), arachidonic acid and other fatty acids, prostaglandins, and leukotrienes are also present in these exosomes [32]. In addition to these proteins and lipids constituents, exosomes are also rich in non-coding RNAs such as micro-RNA, small nuclear RNA, small nucleolar RNA, long non-coding RNA, piwi-interacting RNA, rRNA, and tRNA [32].

6. Mesenchymal Stem Cell-Derived Exosomes

These exosomes play vital autocrine/paracrine intercellular communication roles via the transfer of biological information to recipient cells. Their nature depends on their origin. For example, exosomes derived from MSCs have been shown to promote more therapeutic/regenerative activities compared to those promoted by MSCs alone. Animal model-based studies examining previously recommended that exosomes allow for the identification of better therapeutic candidates and provide a novel alternative to whole MSC cell-based therapy. These benefits may be attributed to the high safety and long shelf lives associated with exosomes that make them suitable and strong candidates for regenerative medicines and therapeutics for a variety of diseases [33]. It has been observed that MSC-derived exosomes inhibit pro-inflammatory mechanisms and are also associated with the alleviation of oxidative stress and pulmonary fibrosis and the remodeling that occurs in inflammatory lung disease [33].

Exosomes function according to their lineages, where their function is dependent upon the phenotype of their parental cell type. It has been reported that MSC-derived exosomes possess the potential to restore and maintain homeostasis based on their capability for protein and RNA transfer [34]. MSC-derived exosomes express several phenotypic markers, including CD29, CD73, CD44, and CD105 [35]. In addition to these phenotypic markers, MSC-derived exosomes also possess surface receptors (PDGFRB, EGFR, and PLAUR), cell adhesion molecules (FN1, EZR, IQGAP1, CD47, integrin, and LGALS1/LGALS3), signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2), and MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276) [36]. It has been reported that MSC-derived exosomes contain more than 850 unique gene products and more than 150 miRNAs [37,38].

7. MSC-Derived Exosomes as a Prospective Treatment for COVID-19

MSC-derived exosomes can serve as an attractive target for treating COVID-19 and its associated complications such as lung injury and ARDS. These exosomes of MSC lineages possess immunomodulatory, tissue repair, and antiviral properties. A recent study suggested that MSC-derived exosomes can easily be substituted for MSCs, as these exosomes exert the same effects in the treatment of COVID-19 as do MSCs [61]. In another study, it was demonstrated that MSC-derived exosomes could inhibit the influenza virus due to their ability to transfer miRNAs and mRNA into lung epithelial cells to aid in the reduction of cellular apoptosis and viral replication [62]. In a study conducted in 2018, it was observed that MSC-derived exosomes induced a reduction in TNF-α, IL-1β, NF-κB, and matrix metalloprotease 9 (MMP-9) levels in the lung and caused decreased expression of IL-6 gene and increased production of IL-10 [63]. In another study, it was found that the transfer of miR-146a via exosomes exerted immunomodulatory effects through IL-1β augmentation [64].

Researchers are now discovering potential therapies against SARS-CoV-2 infection that include COVID-19 treatment based on MSCs or their derived exosomes [65,66,67,68]. As the pathogenesis of SARS-CoV-2 is similar to that of most of the previously encountered viruses that cause ARDS and lung injury, it has been proposed that based upon previous treatment approaches using MSCs or MSC-derived exosomes, the usefulness of exosome therapy should be explored in the context of SARS-CoV-2.MSC-derived exosomes were previously reported to elicit a positive response in ARDS and to inhibit cytokine storms by transferring mRNA and miRNAs to lung tissues [69,70]. In a preclinical model of non-infectious acute lung injuries or bacterial sepsis in the lungs, MSC-derived exosomes have shown protective effects mediated by miRNAs and inhibitory mRNAs [71]. It was suggested that pulmonary fibrosis of the lungs can be prevented in COVID-19-induced pneumonia through the use of MSC-derived exosomes. The rigorous anti-inflammatory activities of MSC exosomes can be used as an infusion in COVID-19 confirmed cases to prevent inflammatory responses.

Exosomes have been evaluated previously in the context of SARS-related coronavirus infection, and it was determined that exosomes containing the SARS-CoV spike protein “S” generate neutralizing antibody titers [72]. Kuate injected xenogenic exosomes without any adjuvant into SARS coronavirus-infected subjects and observed the presence of sufficient neutralizing antibodies [72]. In one recent clinical trial (NCT04276987), MSC-derived exosomes were found to be capable of treating severe subjects with novel coronavirus-associated pneumonia when used as an aerosol upon inhalation [73]. In a recently published prospective non-randomized open-label cohort study, the efficacy of allogenic BM-MSC-derived exosomes in the treatment of severe COVID-19 was tested in 24 SARS-CoV-2-infected subjects with moderate-to-severe acute respiratory distress syndrome [74]. A single injection of 15 mL was administered to the enrolled subjects intravenously, and the effects were monitored from days 1 to 14 post exosome injection [74]. A survival rate of 83% was observed, and out of 24 patients, 17 recovered, 3 remained critically ill (although stable), and 4 died due to unrelated treatment [74]. The authors concluded that exosomes derived from MSC lineages can provide a promising candidate for therapy in SARS-CoV-2 infection [74]. In another Chinese clinical trial, it was found that human MSCs and their derived exosomes could aid in the treatment of lung injury associated with novel coronavirus pneumonia (COVID-19) [75]. In a recently active clinical trial (NCT04389385), COVID-19-specific T-cell-derived exosomes were used via a metered-dose inhaler to treat pneumonia associated with SARS-CoV-2 infection [76]. Table 1 shows the prospective role of mesenchymal stem cell-mediated exosomes in SARS-CoV-2 infection [5,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114].