Mesenchymal stem cell‐derived extracellular vesicles for the treatment of acute respiratory distress syndrome

Abstract Acute respiratory distress syndrome (ARDS) is a serious and potentially fatal acute inflammatory lung condition which currently has no specific treatments targeting its pathophysiology. However, mesenchymal stem cells have been shown to have very promising therapeutic potential, and recently, it has been established that their effect is largely due to the transfer of extracellular vesicles (EVs). EVs have been shown to transfer a variety of substances such as mRNA, miRNA, and even organelles such as mitochondria in order to ameliorate ARDS in preclinical models. In addition, the fact that they have been proven to have the same effect as their parent cells combined with their numerous advantages over whole cell administration means that they are a promising candidate for clinical application that merits further research.

using latent class modeling in previously conducted ARDS randomized controlled trials. The subphenotypes have been termed hyperinflammatory and hypoinflammatory. 10 The hyperinflammatory subphenotype is present in around 30% of ARDS cases and is indicated by factors such as raised levels of inflammatory biomarkers, higher prevalence of vasopressor usage and lower levels of serum bicarbonate. In addition, the hyperinflammatory subphenotype is marked by higher rates of sepsis as well as a higher mortality rate. 10 After the existence of the subphenotypes was established, several large ARDS RC trials have been retrospectively analyzed, taking into account the presence of subphenotypes, and this has led to differences being observed in the responses to treatment between the phenotypes. For example, the preliminary study which established the subphenotypes found that low positive end-expiratory pressure (PEEP) gave better results for mortality than high PEEP in the hypoinflammatory subphenotype whereas high PEEP gave better results in the hyperinflammatory subphenotype in the ALVEOLI trial. 10 This was despite the original analysis of the trial showing no benefit to mortality. 11 Further post hoc analysis of the FACCT trial 12 has also shown significant differences in responses between phenotypes to liberal and conservative fluid management strategies. 13 In addition, although the HARP-2 study had previously found no significant difference in 28-day survival in ARDS between a placebo and simvastatin, 14 reanalysis incorporating subphenotypes found that simvastatin led to significantly higher 28-day survival in the hyperinflammatory subphenotype. 15 The potential of precision medicine lies in identifying novel therapeutics aimed at the subpopulation within ARDS most likely to respond and new therapies should be developed in the view of these findings.

| Mesenchymal stem cells (MSCs) in ARDS
MSCs-based therapy is considered as a promising approach for ARDS because of their ability to target major aspects of ARDS pathophysiology.
When used in preclinical models of ARDS, MSCs have been shown to greatly reduce inflammation and while the mechanism behind this is still not known precisely, it is known that MSCs reduce the levels of many pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 while increasing the levels of cytokines which reduce inflammation like IL-4, IL-5, and IL-10. 16 In addition, MSCs promote bacterial clearance both directly by secreting antimicrobial peptides and proteins such as LL-37 17 and lipocalin 18 as well as indirectly by activating host monocytes, macrophages and neutrophils which then phagocytose the bacteria. [19][20][21][22][23][24] Their secretion of substances such as keratinocyte growth factor (KGF) has been shown to be essential in alveolar fluid clearance and restoration of epithelial permeability. 19,25 For further information about the properties of MSCs in ARDS, consider consulting the reviews by Johnson et al 16 and Walter et al. 26 Due to these therapeutic effects, MSCs are being actively developed toward clinical application. MSCs have been shown to be safe in early phase clinical trials such as the START trial phase 1 and 2a. 27,28 In addition, a study known as MUST-ARDS conducted by Athersys Inc. with a patented bone marrow derived adult multipotent progenitor cell product named "MultiStem" found a lowering of 28-day mortality and an increase in both ventilator and ICU free days using the treatment. 29 As well as these, the parallel trial, REALIST studying the administration of umbilical cord derived mesenchymal stem cells in ARDS is currently at the recruitment stage for phase 1 (NCT03042143).
MSCs have been proven to have an immunomodulatory effect through multiple mechanisms. 30 Although initially, it was thought that they would promote regeneration of the injured lung tissue through engraftment and trans-differentiation, now it has become apparent that engraftment plays little to no role in their therapeutic action. 31 However, it is known that they modulate host cells through direct cell-to-cell interactions and through the release of paracrine factors including biologically active agents such as KGF, 25 indoleamine 2,3-dihydrogenase, 32 and prostaglandin E2. 33 Accumulating evidence suggests that one of the most important effectors in paracrine mechanisms of MSC effect are extracellular vesicles (EVs) which seem to be able to recapitulate the therapeutic effect of their parent MSCs. 34

| Issues with MSCs: Reasons to investigate MSC EVs
The need to investigate MSC EVs stems from the issues found with whole cell administration. Until recently, the tumor formation risk due to MSCs has been assumed to be fairly low owing to their short life span in vivo with many studies suggesting that they have an inhibitory effect on tumor growth, 35 such as in liver cancer caused by hepatocyte growth factor (HGF). 36  stimulate tumor growth. Although no tumors have ever been detected which have been formed directly due to MSCs in clinical trials involving MSCs, the fact remains that they do possess ability to promote the growth of tumors and this property requires further research before MSCs can be considered safe for use in patients. 37 Furthermore, despite their low immunogenicity, MSCs have been shown by Romieu-Mourez et al 38  Moreover, the storage of MSCs using cryopreservation requires preservatives such as DMSO. DMSO treatment produces fewer MSC colonies when plated and the survival of those colonies is reduced, especially with higher DMSO concentrations. In addition, the expression of some genes such as Bak and Bcl2 were increased when using the DMSO compared to the fresh MSCs. 41 Furthermore, the process of freezing and thawing has been found to lower the viability of MSCs which could have an adverse effect on their therapeutic efficacy in patients. 28 It is due to these concerns that new treatments that do not involve the administration of live cells are increasingly being investigated. It is widely accepted that MSCs provide protective paracrine effects, which are in large mediated by the secretion of EVs and therefore therapeutic potential of EV-based therapy is being actively explored. The use of MSC-derived EVs as a cell-free therapeutic offer several advantages

| Definition and nomenclature of EVs
EVs are small circular structures surrounded by a phospholipid membrane which are released by cells and act as a package for various substances. When they were first discovered and for considerable time afterward, EVs were thought to be pieces of debris. It was assumed that they originate as a result of damage to the cell or due to the process of replacement of the cell membrane. 42 However, it is now known that EVs are vital in intercellular communication as they can transport a variety of substances large distances across the body and modulate functional activities of the target cells.
As of yet, there is no consensus on the classification of EVs. However, EVs can be categorized broadly using three criteria: their size, the method by which they are formed in the parent cell, and the contents which they carry. One of the categories of EVs are "microvesicles (MV)." These are normally fairly heterogeneous, and their diameter ranges from 50 to 1000 nm. 43 They are formed when the cell membrane projects outward from the cell and detaches, budding off the membrane, forming a closed sphere containing cytoplasm. The release of microvesicles can be stimulated by many factors such as oxidative or shear stress, hypoxia, or injury. At a molecular level, the cause of stimulation of microvesicle release depends on the cell type.
In many cells, such as dendritic cells, calcium ions can act as a second messenger to stimulate the release of microvesicles, whereas in others, the phorbol ester activation of protein kinase C can have the same effect. 44,45 Another category of EVs are exosomes. In contrast to microvesicles, the diameter of exosomes is reasonably homogeneous with the diameter range being from 30 to 100 nm. 43 Another way in which exosomes differ from microvesicles is that although microvesicles are formed by budding off from the cell membrane, exosomes have their origins within multivesicular bodies in an endosome, in which multiple exosomes are kept while inside the cell. They are then released out of the cell through exocytosis when the multivesicular bodies fuse with the cell membrane. 45 This process is reliant on regulation of cytoskeletal changes by p53. 46 The third category of EVs is apoptotic bodies. These are EVs which are released by cells as they are undergoing apoptosis and contain material that is about to be phagocytosed such as organelles and sections of DNA. Unlike the other EVs, these are over 1 μm in diameter. 47

| EV isolation, characterization, and purification
Currently, there is no globally accepted standard for the isolation, characterization or purification of EVs and methods used depend on the material from which EVs are extracted, the volume of the sample and the application of the EVs. 48 Although EVs are often extracted from a variety of biofluids such as plasma, serum, or urine, according to a survey carried out by the ISEV (International Society for Extracellular Vesicles), the most common starting material for EV extraction used by their members was conditioned cell culture media. 49 According to the same survey, the most common isolation method was ultracentrifugation, particularly among researchers using conditioned cell culture media. This typically involves two stages with the first stage composed of spins at increasing speeds to sediment structures which have a higher buoyant density than EVs. The second stage involves speeds of over 100 000g in order to sediment EVs. This is followed by washing and microfiltration of the EV suspension in order to purify the EVs. 50,51 Although this method is the one used most often, it is not without problems. While washing increases purity, the number of EVs obtained is lower. 51 Also, factors such as centrifugation speed, type of rotor and centrifugation time have an effect on the purity, yield and sedimentation efficiency and therefore must be optimized according to the experiment, making standardization difficult. 52,53 This, combined with the fact that it cannot be scaled make it unsuitable for large scale EV isolation for therapeutic purposes.
In contrast, density gradient centrifugation, the second most widely used technique gives a greater EV purity and also higher amounts of EV proteins and RNA than ultracentrifugation. 51 Although sucrose is the most commonly used cushion material, recently, it has been demonstrated that iodixanol can better preserve the size of the vesicles. 54 However, this is likely not applicable in a clinical setting either due to its complexity, cost and amount of time consumed.
Ultrafiltration, the next most commonly used method separates EVs by size and is arguably a simpler process, and a viable alternative to ultracentrifugation, especially when combined with size exclusion chromatography. 55 In addition to these techniques, precipitation of EVs through the use of various substances such as PEG (polyethylene glycol) is also used often. This has the benefit of being scalable and when combined with ultracentrifugation, gives sufficient EV purity. 56 Hence, this is a viable method for large scale production of EVs for clinical use and has been used in one clinical study. 57 This scalability is also seen with techniques such as size exclusion chromatography which can also be combined with ultrafiltration or ultracentrifugation to create EVs in an efficient way which has potential for standardization according to the ISEV. 34,[58][59][60] The characterization of EVs can be done by four distinct methods according to guidelines from the ISEV. 60 First, the cell source of the EVs (MSCs in the case of MSC EVs) should be quantified; next, the amount of EVs, derived from specific number of cells, should also be quantified. This can be achieved through techniques measuring EV number such as nanoparticle tracking analysis and flow cytometry and supplemented by quantification of the total levels of protein, lipids, or RNA. 61 According to the MISEV2018 guidelines, the presence of EVs should be demonstrated by the analysis of at least one transmembrane protein associated to the plasma membrane (eg, CD9, CD63, CD81) and one cytosolic protein in EVs (eg, TSG101 and ALIX).
Next, the subtype to which the EVs belong should be characterized by analysis of the protein composition of the EVs using Western blotting and by analysis of nucleic acids using PCR. 61 Single EV analysis may also be carried out using imaging techniques like transmission electron microscopy. 62 In addition, co-isolated components present in the sample should also be characterized as it is questioned whether these could contribute to the effect of EVs. 34

| Mechanisms of action of EVs
EVs interact with their target cells through receptor-mediated binding after which they can either fuse with the cell membrane to release the contents into the cell or be taken into the cell through endocytosis, a process in which they are placed into an endocytosed vesicle. 45 EVs can carry a variety of substances such as lipids, multiple species of RNA, various proteins including enzymes, and transcription factors and even organelles such as mitochondria. In addition to acting as complexes that essentially carry signals between cells, EVs also transfer receptors from one cell to another. This has been shown for example by Barry et al who observed that EVs were able to transfer CD41 originally made in platelets to target endothelial cells. 63 Their ability to deliver proteins means that they can target specific mechanisms within the cell. For example, it has been shown by Sarkar et al that EVs can deliver caspase-1 which acts to induce cell death in smooth muscle cells. 64 Their ability to transfer mRNA and miRNA also means that they can alter the transcriptional landscapes of target cells.

| MSC EVs in early phase clinical trials
Multiple recent studies have presented preclinical data addressing the reparative and regenerative properties of MSC vesicles following injuries to the kidney, heart, liver, brain, lung, hind limb ischemia injury 70 as well as various immune disorders. [71][72][73] The mechanisms have been primarily mediated through the transfer of the content from the vesicles to the recipient cells, changing the function and/or phenotype.
The first evidence of clinical administration of MSC EVs to patient was reported in GvHD in 2014 with promising results. 57 Currently, there is one ongoing phase 1 study investigating the therapeutic effect of MSC EVs (exosomes and microvesicles) in type 1 diabetes (NCT02138331). However, the study has passed its completion date and the status has not been updated yet. The same team conducted a subsequent randomized, placebo-controlled, phase 2/3 clinical study to investigate the safety and therapeutic efficacy of human cord blood-derived EVs in inhibiting the progression of grade III and IV chronic kidney disease. The results of the trial suggest that MSC-EV administration was safe, had a significant effect on the amelioration of overall kidney function as well as the modulation of inflammation. 74 In addition, a phase 1 clinical trial to assess the safety and efficacy of MSCs and MSC EVs for promoting healing of large and refractory macular holes is currently at the recruitment stage (NCT03437759).

| MSC EVs in preclinical models of ARDS
Although the effect of MSCs themselves in preclinical models of ARDS has been studied very well, the study of the therapeutic effect of MSC-derived EVs in ARDS is fairly new and the knowledge base is not currently extensive ( Figure 1 and Table 1).
One of the seminal studies on the therapeutic potential of MSC EVs for the treatment of lung injury was carried out by Zhu et al. 75 In their experiment, they induced ARDS in mice using the intratracheal administration of endotoxin from Escherichia coli. EVs were isolated from bone marrow-derived MSC-conditioned medium using ultracentrifugation, which is the standard method followed by most studies.
The effect that the EVs had on the mice was compared with the effect of MSCs. It was found that MSC EVs reduced lung inflammation and reduced oedema to the same levels as MSCs.
Interestingly, it was demonstrated that KGF mRNA was transferred from the EVs to mouse lung cells and expressed. However, the authors themselves admit that the way in which KGF concentration was determined (using ELISA) may not have been sufficient to come to this conclusion as it is not certain whether it detected only human KGF or whether it also detected mouse KGF.
Our group has found that EVs are the major component of the MSC   showed that EVs reduced inflammation, indicated by a 73% reduction in the influx of neutrophils and macrophages as well as a 49% reduction in the level of MIP-2. They also showed a reduction in oedema and the permeability of endothelial-epithelial barrier to protein. It was also shown that CD44 receptors are essential for the uptake of EVs into cells. Khatri et al conducted an experiment investigating the effect of MSC EVs in influenza virus induced ARDS in a pig model. 79 They found that in pigs, the replication of the viruses was reduced by the administration of EVs. They also found that there was a reduction in the death of alveolar epithelium cells. This effect was shown to be, in part, due to RNA transfer via EVs. However, this was shown by the effect being abrogated by pre-incubation of EVs with RNase, so no specific RNA was found to mediate the effect. As in other studies, a reduction in pro-inflammatory cytokines was also observed.
Gennai et al carried out an experiment in ex vivo perfused human lungs. 80 These were lungs that had been rejected for transplantation and the purpose of the study was to find out if injured lungs could be improved to a potentially transplantable standard using MSC EVs. It had been known already that MSCs could restore fluid clearance in ex vivo lungs, 81  were injured with cytomix (IL-1β, TNF-α, and IFN-γ). 83 It was revealed that there was an increase in the level of angiopoetin-1 (Ang1) mRNA and protein in the injured endothelium which was treated with the EVs, and it was also found that the pretreatment of the EVs with Ang1 siRNA stopped their effect, indicating that the transfer of Ang1 mRNA from EVs plays a crucial role in the mechanism of EVs. In addition, it was also found that the internalization of EVs was required for the MSCs to produce their effect.
Although previously mentioned studies all used bone marrow derived MSCs EVs, Varkouhi et al used EVs extracted from MSCs derived from umbilical cord and found that they attenuated acute lung injury in rats. 84 This carries significance as umbilical cord MSCs are less invasive to obtain, have a higher proliferation capacity, and can be cultured for longer. 85 The same study compared the effect of normal EVs with those primed with interferon-γ. Although both led to a lowering of mortality, the primed EVs were much better than normal EVs in improving multiple parameters of lung injury such as the alveolar protein leak and alveolar-arterial oxygen gradient.
The study also identified an improvement of macrophage phagocytosis, an increase in bacterial killing, and an increased production of endothelial nitric oxide synthase as possible mechanisms. However, more detailed mechanisms of these effects remain to be elucidated. In addition, it was found that the primed EVs were larger on average than the normal EVs. However, the reason for this is unknown and requires further investigation.
The role of EV-mediated transfer of microRNAs is also being increasingly recognized as an important mechanism of their biological effect. In the recent study by Yi et al, 90 it was found that exosomal transfer of miR-30b-3p resulted in inhibition of serum amyloid A3 (SAA3).

| Current challenges in translation of MSC EVs to clinical practice
The main challenge with translating MSC EVs to clinical practice is that differences in EV isolation, purification, and characterization methods mean that there is often a large degree of heterogeneity in EV preparations. This is at odds with the homogeneity required for clinical application. Therefore, criteria for the standardization and optimization of EV production should be established. Proper characterization would allow further study into the differences between the efficacy of the different types of EVs. In a position paper, members of four societies (SOCRATES, ISEV, ISCT, and ISBT) propose harmonization criteria for MSC EVs to facilitate data sharing and comparison, which should help to bring the field closer toward clinical applications. 34 This includes suggestions such as the possibility of immortalizing MSC cell lines to ensure reproducibility although more study is required into the changes that immortalization may cause. Although EV production is not stopped due to immortalization, immortalized MSCs have been found to have features such as lower plastic adherence, 92 so the changes in EV composition needs to be examined.
Furthermore, additional studies are required to find ways to scale up the production of EVs as they are needed in large quantities to be therapeutically effective and development of GMP protocols for EV production is also essential. Moreover, although a few protocols for the biomanufacturing of exosomes have been reported, 93,94 biomanufacturing of larger microvesicles remains largely unexplored.
To use EVs as an off-the-shelf therapy, their stability and storage must also be examined further. In addition, the potency of the isolated EVs must be assessed using standardized disease-specific potency assays, which are currently lacking.
Although multiple mechanisms have been discovered which modulate the function of EVs, more work also needs to be carried out to further understand the contents within EVs, especially the differences between naïve and pretreated EVs. Also, their distribution within the body after administration and the ways in which they move through endothelial barriers need further investigation.
Moreover, the establishment of subphenotypes within ARDS patients represents a major shift in the way ARDS is viewed and future therapies, including MSC EVs should be developed in line with this emerging evidence.
In addition, research so far seems to have focused mostly on bone marrow MSC EVs. Hence, other sources should also be explored.

| CONCLUSIONS
In conclusion, the ability of MSC-derived EVs to ameliorate the causal factors of ARDS in preclinical conditions, both in vivo and ex vivo, is clear. However, although studies have concluded that EVs have similar therapeutic effects to MSCs themselves, further research into the mechanisms of action of EV-based therapeutics and manufacturing methods as well as disease-specific potency assays is essential.
Despite these problems that must be solved, the use of EVs holds promise and merits further research due to their therapeutic potential.