The concept of MSCs originated from the non-hematopoietic stem cells present in bone marrow (BM) (23). Researches on MSCs can be traced to 1960s, when Tavassoli and Crosby clearly established the proof of bone and marrow generation in de novo potential of BM transplanted to extramedullary site (23, 24). Subsequently, a series of seminal studies conducted by Friedenstein and his colleagues demonstrated that only a small subset of BM cells was responsible for the osteogenic potential of BM allograft (25). Along with the development of hematopoietic stem cell research, the groundbreaking work from Tavasolli, Friedenstein, and Owen revealed possible existence of a second type of stem cell in bone marrow (23). The term “mesenchymal stem cells” was first coined by Caplan in 1991 (26). However, it was not until 1999 that the term gained widespread popularity (23, 27).

MSCs are considered as a heterogeneous subpopulation of adult multipotent cells with self-renewing and differentiation potential (28). MSCs show a fibroblast-like morphology under the microscope. According to the minimal criteria to define human MSCs proposed by the International Society for Cellular Therapy in 2006, MSCs must meet the following three criteria: 1) plastic adherence in standard culture conditions; 2) expression of specific surface antigens such as CD105, CD73, and CD90, but lacking expression of CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR; 3) trilineage differentiation into osteocytes, chondrocytes and adipocytes in vitro ( 29). In addition to bone marrow, MSCs can also be isolated from the other multiple tissues, including adipose tissue (30), placenta (31), umbilical cord (32), Wharton’s jelly (33), dental pulp (34), periodontal ligament (35), synovial membrane (36), periosteum (37), skin (38), skeletal muscle (39), tonsil (40), amniotic fluid (41), uterine cervix (42), peripheral blood (43), among which bone marrow-derived mesenchymal stem cells (BM-MSCs) are studied most extensively (44).

MSCs have been shown to be a promising therapy for various diseases such as autoimmune disease (45), graft-versus-host disease (GvHD) (46), inflammatory disease (47), neurodegenerative disorder (48), cardiovascular disease (49), wound healing repair (50) and diabetes mellitus (51) due to their tissue repair and immunomodulatory properties (21, 52). One of the important properties that make MSCs play therapeutic role in diseases mentioned above is their capacity of migration to injured or inflammatory sites when injected systematically, which is called the homing effect (44, 53). Migration of MSCs occurs in response to a large amount of chemotactic factors, including both growth factors (GFs) and chemokines generated in the damaged tissues, such as platelet-derived growth factor-AB (PDGF-AB), insulin-like growth factor 1 (IGF-1), RANTES (regulated upon activation, normal T-cell expressed and secreted), macrophage-derived chemokine (MDC), and stromal-derived factor-1 (SDF-1) (54, 55). Meanwhile, a myriad of relevant receptors expressed on MSCs membranes have been detected, including tyrosine kinase receptors PDGF-receptor (R) α, PDGF-Rβ, and IGF-R and chemokine receptors CCR3, CCR4, CCR5, CXCR4 (55). Later, the surface proteins podocalyxin-like protein (PODXL) and α6-integrin (CD49f) highly expressed in the early cultures of MSCs have been found to enhance their migration to injured tissues after systemic administration (56). Adhesion molecules and integrins such as VCAM-1 and VLA-4 contribute to the migration of MSCs to the inflammatory sites as well (57). In targeted location, MSCs can mediate productive repair by secreting bioactive factors with the capacity of anti-apoptotic, immunomodulatory and angiogenic effects and interacting with injured host cells (12, 52, 58–60). Another reason for the widespread use of MSCs is its robust immunomodulatory property. MSCs exert immunomodulatory effects by suppressing innate and adaptive immune responses, which involve a variety of immune cells. MSCs have been demonstrated to not only interfere with all major stages of the dendritic cells (DCs) life cycle: differentiation, maturation, and activation, resulting in the formation of immature DCs with an inhibitory phenotype but also alter the cytokine secretion profile of DCs, as evidenced by decreased secretion of tumor necrosis factor α (TNF-α) and interleukin-2 (IL-2) and increased IL-10 secretion (17, 61, 62). In addition, MSCs inhibit the cytotoxic effects of natural killer (NK) cells and interferon γ (IFN-γ) secretion. They also modulate immune responses bysuppressing T lymphocyte activation and proliferation. MSCs can induce T-cell anergy and promote the formation of regulatory T cells (Tregs). They enable CD4+ T cells to generate a functional Treg population but suppress the activation, proliferation and differentiation of proinflammatory T cells during the CD4+ T cells induced to differentiate toward Th1 or Th17 cells (63). Furthermore, differentiation, antibody production, and chemotactic behavior of B cells are affected by MSCs as well (17). The exact mechanism underlying this immunosuppressive effect of MSCs is not well understood, but it is believed to involve soluble factors secreted by MSCs, such as prostaglandin E2 (PGE2) (61), tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) (64), nitric oxide (NO) (65) and transforming growth factor-β1 (TGF-β1) (66). Notably, there are complex interactions between antigen-presenting cells (APCs), adaptive immune cells, and MSCs. APCs, especially blood CD14+ monocytes, play a critical role in activating MSCs through the release of IL-1β, which then enables MSCs to inhibit T lymphocytes (66).

The mechanism of MSC-mediated immunosuppression varies among different species. For instance, a study demonstrated that MSCs from human and non-human primates mediated immunosuppression by IDO whereas MSCs from mouse by NO (67). Moreover, the immunosuppressive ability of MSCs may not be inherent, but rather is elicited in the presence of proinflammatory cytokines and is chemokine-dependent (46, 67). When exposed to certain combinations of cytokines, MSCs produce high levels of inducible nitric oxide synthases (iNOS) and chemokines which drive immune cells migration into proximity to MSCs, where immune cell function can be suppressed by the high levels of NO (46). Proinflammatory signals activated MSCs to secret PGE2 and TNFα stimulated gene/protein 6 (TSG-6), both of which are negative feedback loops of inflammation, leading to a shift in macrophages from the M1 phenotype (proinflammatory) to the M2 phenotype (anti-inflammatory) (68). MSCs can also be activated by microenvironment generated by injured tissues to express factors that appear to specifically meet the needs of the tissues (69). Special storage conditions, low cell engraftment and poor survival in the lung, and cell senescence during in vitro expansion are the main obstacles limiting MSCs clinical translation (15, 20, 70, 71). However, several strategies have been developed to optimize MSCs therapy in ARDS, such as genetic modification and pretreatment with a series of preconditioning strategies (20), which will be discussed in detail in the section of functional optimization of MSCs and their derived EVs.

It is now widely accepted that the therapeutic potential of MSCs is largely attributed to their secretome, especially their derived EVs. EVs are nanometer sized particles secreted by almost all cell types. Previously, EVs were generally classified into exosomes, microvesicles, and apoptotic bodies according to their size and intracellular origin (18). The Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines updated the nomenclature of EVs since there has not yet consensus on specific markers of EVs subtypes. Therefore, based on the physical characteristics such as size, EVs can be categorized into small EVs (sEVs) with a diameter <100nm or 200nm, and medium/large EVs (m/lEVs) with a diameter>200nm (72). MSC-derived EVs express some specific surface molecules reflecting the cells of origin, such as CD29, CD73, CD44, and CD105 (18). EVs have been considered to participate in the intercellular communication via delivering their contents into the recipient cells, including proteins, RNA, DNA, amino acids, lipids, metabolites, and even organelles, changing the function and/or phenotype of the recipient cells under the physiological and pathological processes (73, 74). For example, insulin-like growth factor-1 receptor (IGF-1R) mRNA contained within MSC-derived EVs could be transferred to tubular cells, where it was translated in the corresponding protein, resulting in increased cell proliferation (75). EVs originated from MSCs not only exhibit similar or even superior behavior and function as MSCs but also overcome the disadvantages of live cell injection (76). As a promising cell-free therapeutic approach, MSC-derived EVs have many compelling advantages like high stability and biocompatibility, easy storage, no risk of iatrogenic tumor formation, low immunogenicity and easy to cross the biological barrier compared to their parent cells (77, 78). Over 150 miRNAs and 850 proteins have been identified from cargos of MSCs-derived EVs, which contribute to their therapeutic efficacy in a wide range of pathological conditions, including cardiovascular disease, neurological disease, acute kidney injury, liver injury and inflammatory lung diseases (74, 78–83). miRNAs are small non-coding RNAs enriched in EVs that can be internalized into the target cells and subsequently interact with specific binding sites to alter behavior of cells in physiology or pathology conditions. In recent years, with the deepening of researches on contents of MSC-derived EVs, multiple cytokines, growth factors, and prostaglandins found in EVs have been revealed to mediate immuno-modulatory properties (84).

MSC-derived EVs inherit many of the properties of their parental MSCs in the treatment of inflammatory diseases, such as immunomodulation, homing effect and tissue repair functions. After intravenous injection, MSC-derived EVs can also migrate to the injury and inflammatory sites, which may be attributed to the increased vascular permeability at the site of inflammation (76, 83, 85). As with MSCs, MSC-derived EVs display a similar immunomodulatory ability through interacting with various effector cells of the immune system (84). Co-culture of MSC-derived EVs with peripheral blood mononuclear cells (PBMCs) in vitro reduces PBMC proliferation (83, 86). MSC-derived EVs also modulate CD4+ T cell subsets, as evidenced by suppressing T cell induction to Th1/Th17 subtypes, while enhancing Tregs level and function significantly (83, 87–89). The proliferation, activation, and apoptosis of B lymphocytes are also inhibited (90). MSC-derived EVs can induce macrophage polarization towards the anti-inflammatory M2 phenotype (91). Moreover, it was reported that the proliferation, activation, and cytotoxicity of NK cells were inhibited by human fetal liver (FL) MSC-derived EVs (92). The underlying mechanism by which MSC-derived EVs regulate the function of CD4+ T cells and NK cells may involve TGFβ signaling (92, 93). Nevertheless, MSC-derived EVs promote Tregs immunosuppression capacity not by contacting with CD4+ T cells directly, but by triggering the polarization of T cells through interacting with APCs such as monocytes and B cells in PBMCs, which may be the mediator between MSC-derived EVs and Tregs (88). Specific miRNAs contained in MSC-derived EVs, such as miR-191, have been identified to inhibit immune response through interacting with specific proteins, such as death-associated protein kinase 1 (DAPK1) after being transferred to macrophages (94). Phinney et al. demonstrated that MSCs modulated macrophage function by transfer of partially depolarized mitochondria and regulatory microRNAs employing two different types of EVs, which enhanced macrophage bioenergetics and suppressed Toll-like receptor (TLR) signaling, respectively (95). Proinflammatory stimuli can enhance the immunosuppressive functions of MSC-derived EVs, in which multiple anti-inflammatory miRNAs, such as miR-146, miR-34, miR-21 were strongly upregulated under the stimulation of inflammatory cytokines and transferred to macrophages, resulting in M2 polarization (83, 96, 97). In addition to miRNAs, deep RNA sequencing and proteomic analysis of MSC-derived EVs revealed that several mRNAs and proteins with anti-inflammatory properties were highly enriched in cytokine-stimulated EVs compared to non-stimulated EVs (83). Anti-inflammatory mRNAs and proteins of particular importance include IDO mRNA, macrophage inhibitory cytokine 1 (MIC-1), Galectin-1 (Gal-1), and latent-transforming growth factor β-binding protein (LTBP) (83). This may explain its potent anti-inflammatory effects and therapeutic potential in various inflammatory diseases. Furthermore, some chemokines and chemokine receptors such as CCL2, VEGFC, and CCL20 carried in MSC-derived EVs can recruit immune cells to the vicinity of EVs and subsequently induce immunosuppressive effects (98). At the same time, the complex composition of EVs allows them to target different pathogenic pathways and act synergistically to enhance their therapeutic function (83).

It has been reported that MSCs were initially trapped by the lung and gradually accumulated in the liver and other organs after intravenous infusion, which might be due to the cellular diameter and attachment potential, while intravenously administered EVs are predominantly distributed in the spleen followed by the liver, and then the lungs and kidneys (99, 100). Regarding to the homing capability, MSCs are considered to migrate towards damaged or inflammatory sites in response to chemokines produced at the site of injury and chemokine receptors expressed on their own surface (101), while MSC-derived EVs appears to exhibit an inherent tendency towards injured tissues (102). MSC-derived EVs have many advantages over MSCs in treating various inflammatory diseases. EVs are nanoscale particles with smaller diameter, which means virtually no risk of microvascular obstruction, especially pulmonary capillaries. This feature also allows them to reach target locations faster to execute their tissue repair functions (101, 103). Besides, MSCs require special storage conditions (liquid nitrogen), while EVs are more stable and can be stored at -80°C for a long time. Amplifying stem cells in vitro with inappropriate processes and culture conditions may increase their immunogenicity (104). In contrast, EVs are poorly immunogenic, which enables them to prevent therapeutic cargoes from rapid degradation in the body (99). And MSC-derived EVs have increased circulating half-life (105). Moreover, MSC-derived EVs have the ability to cross the blood-tissue barrier (101). Despite these advantages, there are still many technical challenges to overcome in the process of clinical translation of MSC-derived EVs, which will be elaborated in the sixth part.

Current data have suggested that MSCs and their EVs exert strong antimicrobial effects through indirect and direct mechanisms (106, 107). MSCs could inhibit a range of clinically relevant pathogenic microorganisms growth directly in vivo and in vitro, which was mediated in part by the secretion of antimicrobial factors, such as human cathelicidin antimicrobial peptide hCAP-18/LL-37 and hepcidin (108, 109), IDO (110), keratinocyte growth factor (KGF) (111), interleukin-17 (IL-17) (112), and β-defensin 2 (BD2) (113). In a mouse model of Gram-negative pneumonia, murine MSCs have been found to enhance bacterial clearance from the lung due to the upregulation of antibacterial protein lipocalin 2 in response to lipopolysaccharide (LPS) and inflammatory mediators (114). Besides that, the antimicrobial activity of MSCs could be achieved indirectly by enhancing alveolar macrophage (AM) phagocytosis against bacteria and the survival of blood monocytes partly through the secretion of KGF and transferring mitochondria to innate immune cells (111, 115). Macrophages of healthy individuals and those with sepsis that are exposed to MSCs have stronger ability of bacterial phagocytosis and killing (116). It has been revealed that MSC-EVs can enhance bacterial clearance in E. coli pneumonia mice and ex vivo perfused human lung models of E. coli pneumonia as well (117). The mechanisms underlying their antimicrobial activity could ascribe to the increased leukotriene (LT) B4 production in injured alveolus mediated by decreased multidrug resistance-associated protein 1 (MRP1) expression resulting from the transfer of miR-145 by MSC-EVs (118). The anti-microbial effect of MSC-EVs was also partly due to the enhancement of human monocytes and alveolar macrophages phagocytic capacity against bacteria (107, 117).

One of the pathological manifestations presented by ARDS patients is alveolar edema, which is ascribed to increased lung endothelial barrier permeability and reduced alveolar fluid clearance (AFC) (119). AFC is predominately dependent on the function of alveolar epithelial fluid transport. In experiments with perfused human lungs, treatment with human MSCs or MSC conditioned medium (MSC-CM) normalized AFC and restored lung endothelial permeability induced by E. coli endotoxin, thus improving pulmonary edema via secretion of paracrine soluble factor KGF in large part, which might traffick epithelial sodium channels subunit (αENaC) to the apical membrane of alveolar epithelial type II (AT II) cells (120). The researchers also found that human MSC-EVs were as effective as their parent stem cells in reducing lung protein permeability and inflammation in E. coli endotoxin-induced ALI and pneumonia in C57BL/6 mice, which might be due to the increased level of KGF mediated by expression of the mRNA from MSC MVs in the injured alveolus (107, 121). Similar findings were obtained in a study using an ex vivo perfused human lung injured with bacterial pneumonia, which showed increased AFC and reduced lung protein permeability following MSC-EVs administration (117). The presence of hepatocyte growth factor (HGF) in MSC-EVs has been verified to stabilize pulmonary endothelial barrier function, as evidenced by reduced endothelium permeability and apoptosis rate, as well as increased cell proliferation in LPS-induced endothelial cell model (122). Alveolar epithelium forms a tighter monolayer than the endothelium. Epithelial barrier dysfunction in ARDS facilitates accumulation of protein-rich edema fluid and inflammatory cells in alveolar cavity. Previous study noted that allogeneic human MSCs restored epithelial protein permeability across human AT II cells injured by cytomix (a combination of IL-1β, TNF-α, and IFN γ). This beneficial effect on human AT II cells was mediated by angiopoietin-1 (Ang1) release from MSCs to prevent actin stress fiber formation and claudin 18 disorganization following an inflammatory insult in part through suppression of NF-κB activity (123). MSC-EVs may also play a protective role in ARDS through mitochondria transfer. In the ARDS microenvironment, MSC-EVs could restore alveolar-capillary barrier integrity at least partially via mitochondrial transfer to rescue mitochondrial dysfunction (124).

At present, the most widely recognized pathogenesis of ARDS is excessive and uncontrolled inflammatory response in the lung. As a promising cell-based therapy candidates, MSCs and their derived EVs hold great potential for their ability to modulate/suppress innate and adaptive immune systems in the treatment to various inflammatory disease, including ARDS (125). MSCs harvested from different tissues and their EVs could increase survival rate and attenuate systemic and pulmonary inflammation in animals suffering from lung injury induced by various insults, whether administered systemically or intratracheally (13, 54, 121, 126–130). During the early phase of ARDS, neutrophils are recruited into the alveolar space in response to the chemokines released by macrophages and epithelial cells, activated excessively and undergo delayed apoptosis. Nevertheless, neutrophil apoptosis is essential for the resolution of ARDS. The study found that administration of MSC-CM attenuated LPS-induced ALI by inducing neutrophil apoptosis, which might be related to the inhibition of NF-κB signalling pathway (131). Neutrophil extracellular traps (NETs) are structures formed by dying neutrophils as a novel defence mechanism against bacterial infections. However, excessive NETs formation is associated with exacerbated immune responses and causes damage to lung tissue (132). Treatment with MSCs could reduce the oxidative stress and release of NETs, thereby alleviating lung injury and improving survival in LPS-induced ALI of mice (133). Cholinergic anti-inflammatory pathway (CAP) activation has been identified as a critical mechanism underlying the therapeutic effects of MSCs on ARDS, which is mediated by MSC-derived PGE2 (134). MSCs-derived EVs carrier multiple cargoes, in which miRNAs are extensively studied and considered to modulate gene expression and function of target cells under pathological conditions. In a rat model of traumatic ALI, miR-124-3p transferred by MSC-derived EVs could improve oxidative stress injury and inhibit inflammatory response through directly targeting and downregulating purinergic receptor P2X ligand-gated ion channel 7 (P2X7), which was overexpressed in traumatic ALI rats (128). Macrophages are key innate immune cells responsible for ARDS inflammatory response. Recent study revealed that the highly expressed miR-181a in MSC-EVs in the ARDS environment was crucial for the EVs immunomodulatory effect on human macrophages exposed to LPS as well as the mouse model of LPS-induced lung injury via PTEN-pSTAT5-SOCS1 axis (135). A study conducted by Wang et al. demonstrated that MSC-EVs could alleviate ALI via transfer of miR-27a-3p to alveolar macrophages and promoting M2 macrophage polarization (136). Apart from miRNA, mitochondrial components packed in MSC-derived EVs are increasingly attracting researchers interests and widely studied in recent years. Human MSCs could enhance phagocytic capacity and promote anti-inflammatory phenotype in human macrophages stimulated with LPS or ARDS bronchoalveolar lavage fluid (BALF) via EVs mitochondrial transfer (137). Furthermore, EVs from adipose-derived mesenchymal stem cells (AdMSCs) could restore metabolic homeostasis of macrophages and promote a functional shift of LPS-stimulated macrophages from M1 proinflammatory to M2 anti-inflammatory phenotype via transferring mitochondrial components (138). Cytokine IL-27 has been identified as a pro-inflammatory factor in the pathology of sepsis, which is primarily released by dendritic cells and macrophages (139). It was revealed that IL-27 in BALF and serum was elevated in ALI/ARDS patients and associated with disease severity. Blocking IL-27 with neutralizing antibodies could reduce pulmonary inflammation and improve survival rate in a mouse model of cecal ligation and puncture (CLP)-induced lung injury. From this, IL-27 is a critical cytokine in ALI/ARDS and inhibition of IL-27 may represent a promising therapy for ALI/ARDS patients (140). Recent study revealed that EVs from adipose-derived MSCs could alleviate sepsis-induced lung injury in mice by inhibiting the secretion of IL-27 in macrophages (141). Metabolic pathways of macrophages are closely related to their activation state and function. M1 macrophages rely on glycolysis for energy production whereas M2 macrophages rely on oxidative phosphorylation (OXPHOS). Metabolism shift between glycolysis and OXPHOS is involved in M1/M2 macrophage polarization (142). It was reported that EVs derived from BMSCs could attenuate LPS-induced ARDS by modulating alveolar macrophage polarization through inhibiting cellular glycolysis (143). However, it should be noted that not all MSCs exhibit immunosuppression properties. Bone marrow MSCs contain different functional subpopulations, in which IL-17+ MSCs possess antifungal effect but fail to execute MSC-based immunosuppression owing to the downregulation of TGF-β mediated by the NF-κB pathway (112).

It is well-known that extensive apoptosis of the endothelial and alveolar epithelial cells is involved in the pathogenesis of ARDS patients, leading to alveolo-capillary barrier dysfunction and disability of lung edema fluid clearance by alveolar epithelial cells (2, 144–147). MSCs could be activated to reduce apoptosis in UV-irradiated fibroblasts and lung cancer epithelial cells, which may be explained in part by the upregulation and secretion of stanniocalcin-1 (STC-1) (148). Exosomal miR-22-3p from human umbilical cord blood-derived MSCs could suppress inflammatory reaction and pulmonary cell apoptosis rate by reducing frizzled class receptor 6 (FDZ6) in LPS-induced ALI, which further benefited from the upregulated miR-22-3p (149). Ferroptosis is a novel and iron-dependent programmed cell death (PCD) modality (150). Excessive ferroptosis contributed to intestinal ischemia/reperfusion-induced ALI (151). A study revealed that specific delivery of miR-125b-5p by adipose derived stem cells (ADSCs) EVs could alleviate the inflammation induced pulmonary microvascular endothelial cells (PMVECs) ferroptosis in sepsis induced ALI via regulating Keap1/Nrf2/GPX4 expression, thereby improve the acute lung injury (152).

Autophagy is a highly conserved lysosomal degradation pathway that primarily functions as an adaptive role to maintain homeostasis of organisms at both basal and stress-induced increase levels (153). Diverse stimuli including bacterial infection, LPS exposure, sepsis, and mechanical ventilation, etc. have been shown to promote autophagy in multiple lung cell types and animal models of lung injury (154). However, the exact role of autophagy in ALI still remains controversial. Autophagy may play a protective or detrimental role in ALI depending on the underlying causes of lung injury and the stages of disease progression (154). Changes in autophagic flux caused by autophagy dysfunction may underlie acute lung injury during sepsis (155). It was discovered that BMSC-derived EVs could relieve LPS-induced acute lung injury through inhibiting autophagy stress in alveolar macrophages via delivering miR-384-5p to combine with Beclin-1 directly (156).