Despite the fact that the indicated classification of EVs is still commonly used in the majority of papers, there has been a growing issue related to the collective definition of different vesicular entities that have been reported so far. EVs encompass rapidly developing, but still relatively new field of scientific interest, with constantly evolving knowledge on their biology, accompanied by emerging experimental approaches and newly developed methodologies. Thus, in 2014 International Society for Extracellular Vesicles (ISEV) in its first position paper has initially provided criteria of EV definition, as well as minimal set of methodological standards and appropriate experimental controls that should be taken into the consideration in EV-related studies, to provide accurate data that reliably supports the stated conclusions (11). Later on, following the progress in the field and further verification of previously established guidelines, ISEV released updated position paper in 2018, pointing out the need for further standardization of experimental approaches (4). Nevertheless, growing evidence demonstrates the lack of consensus and equivocal data on unique markers and subcellular origin of particular EV subsets, with several indications on morphological and phenotype characteristics to overlap between different vesicular fractions (12). Additionally, several new EV subtypes were recently reported, including exomeres, exophers, or migrasomes (13), which demonstrates the complexity of cellular secreting machinery. Moreover, ISEV points out growing overuse of term “exosomes” without clear experimental evidence on their identity, which leads to misunderstanding and misinterpretation of inaccurate data (14). It is also challenging to exclusively isolate homogenous fraction of exosomes without other EV subtypes (15). Furthermore, depending on the type and source of the starting material, as well as an isolation method, there may be a significant variation in the composition of obtained EV pools, additionally impacted by the heterogeneity of the reported protocols (16).

Thus, taking into account recent advances in the understanding of EV biology and the development of methodological approaches, recently established new ISEV guidelines recommend to avoid direct categorisation on “exosome” or “microvesicle” terms and to use general term “EVs” instead. Eventually, some operational terms for EV subtypes, that relate to their biophysical properties that have been well characterized experimentally in particular study, such as “small/large EVs”, “CD81+ EVs” etc. may also be applied (4). Thus, in current review we will use general term of “EVs” that collectively combines several types of vesicular particles reported in the cited literature.

MSCs are multipotent SCs of mesodermal origin, that are able to differentiate into chondrogenic, osteogenic and adipogenic lineages. They may be isolated from various sources, including bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and postnatal tissues such as umbilical cord (UC-MSCs) (96). MSCs are known for their high secretory activity, which includes release of extracellular matrix (ECM) proteins, cytokines, chemokines, growth factors, but also bioactive EVs that may play a role in mediating crosstalk to local and distant tissues (97). This paracrine activity of MSCs makes them also crucial players in immunomodulation, which may trigger mostly anti-inflammatory signaling and suppress excessive activation of immune system components (98). Importantly, MSCs-EVs were demonstrated to share biological activity with their parental cells, that are known to possess immunomodulatory properties. Several studies have demonstrated an impact of tissue origin on potential differences in the functional activity of MSCs, which may be also reflected in the distinct biological activity of their EVs (99).

MSCs possess limited stemness and differentiation potential and thus reduced risk of teratoma formation when compared to pluripotent SCs. On the other hand, they also have relatively high proliferative capacity in vitro and do not require advanced and expensive culture reagents, which allows researchers to effectively reach the level of MSC expansion sufficient for the isolation of EV batches dedicated for the therapeutic applications (100). However, there are still several challenges of the effective use of MSCs as a source of EVs for the therapeutic purposes, including donor variability and need to optimize expansion methods in order to avoid cell senescence. Nevertheless, considering the lack of ethical concerns, ease of isolation from several sources potentially available in both autologous and allogeneic systems, biological safety and low immunogenicity, MSCs have become primary cells of choice for the purpose of the tissue regeneration. A natural consequence of this fact is that researchers are particularly interested in the application of EVs secreted by these cells (101). Thus, numerous studies show that MSC-EVs have significant cytoprotective, regenerative and immunomodulatory potential in several disease models.

ESCs are pluripotent population of cells with unlimited proliferative capacity, capable to give rise into any type of cells within three germ layers. Consequently, ESCs were initially envisioned as potentially ideal type of SCs for the medical purposes (102). However, due to the ethical concerns regarding their sourcing, as well as the risk of teratoma formation, clinical application of ESCs has been highly limited (103). Nevertheless, due to the acellular nature, the use of EVs secreted by already available ESC lines (ESCs-EVs) still remains promising strategy for the regenerative therapies (104). Due to the potentially unlimited quantities of cells, ESCs are often used as starting cells that are differentiated toward more specified progenitors serving as a source of EVs for therapeutic approaches (105). Another interesting approach is to use ESC-EVs to boost the therapeutic efficacy of other SC populations. As an example, ESCs-EVs were demonstrated to reduce senescence and enhance pro-regenerative effects of MSCs in a mouse cutaneous wound model, by activating the PI3K/AKT pathway (106).

iPSCs were initially obtained by the Prof. Yamanaka’s group by genetic reprogramming of somatic cells through the forced expression of key transcription factors such as Oct3/4, Sox2, Klf4, and c-Myc (107). This achievement was awarded by Nobel Prize in 2012 and has opened new chapter not only in the field of stem cell biology, but also in the area of tissue regeneration. iPSCs display pluripotent properties similar to those of ESCs, allowing relatively easy accessibility to pluripotent cells without ethical problems related to the cells of embryonic origin. Consequently, due to their potentially unlimited proliferative and differentiation potential, iPSCs have been widely used for disease modelling, drug discovery and cell-based therapies, resulting in the substantial progress in the field (108).

Along with their paracrine activity, iPSCs have been also recognized as important donors of EVs for the basic research as well as the therapeutic applications. Similarly to ESCs, iPSCs are also differentiated into other cell types that are sources of EVs for the regenerative purposes (109). Interestingly, iPSCs were shown to be able to secrete EVs more abundantly and with higher capability to enter target cells, when compared to the MSCs (110), which may make these cells advantageous in the context of the donor cells for EV-based therapeutic approaches.

Despite the special focus on the pluripotent and mesenchymal SC as main sources of EVs for the tissue regeneration, the pro-regenerative potential of EVs secreted by the several other SCs and progenitor cell was also investigated. As an example, therapeutic efficacy of EPCs- derived EVs was shown in different experimental setups (111). Similarly, protective effect of EVs from neural (112) and cardiac progenitors (113) was also demonstrated.

Cardiovascular diseases (CVDs) are one of the most common causes of death, with limited efficacy of currently available therapeutic strategies. According to the data provided by the world health organization (WHO), CVDs are responsible for about one-third of all death cases worldwide, which corresponds to almost 18 million of human beings every year (114). Cardiac tissue has a limited regenerative capacity and endogenous systems are typically insufficient for the cardiac repair. Once injured, mammalian heart lacks the ability to replace damaged cardiomyocytes, which leads to the progressing loss of its function. Thus, the development of novel therapeutic approaches and identifying intrinsic and external factors together with new potential targets to improve cardiac performance are of special focus (115). CVDs encompass broad spectrum of disorders, but the two major representations of ischemic CVDs are acute myocardial infarction (AMI) and chronic myocardial disease (CMD), which differ in terms of their mechanisms of cause and clinical manifestation, with indispensable role of inflammatory response. Both conditions are life-threatening and lead to the subsequent cardiac remodelling and scar formation rather than regeneration, which can adversely affect function of the cardiovascular system (116).

AMI is a rapid event caused by the coronary artery occlusion by the ruptured plaque that blocks the blood flow, followed by the oxygen deprivation in the myocardium and death of cardiomyocytes. Consequently, due to the insufficient ability of heart to compensate the massive loss of cardiac cells following infarction, injured tissue becomes fibrotic and non-contractile, leading to the heart disfunction such as dilatation, reduced ejection fraction, left ventricle stiffness and its remodelling (117). Current AMI therapeutic strategies include e.g. urgent reperfusion therapy, pharmacotherapy and surgical intervention, including heart transplantation (114). Despite advancement in the treatment, AMI still carries a high mortality rate, with increasing morbidity caused by the several risk factors that are a common part of contemporary, unhealthy lifestyle, such as smoking, obesity, hypertension, lack of physical activity and high exposure to the stress (118). Additionally, patients who survived AMI have a higher risk of recurrent AMI or other CVD-related complications (119). In a consequence, there is a great need for new, more effective therapeutic strategies, including those that would effectively support the natural reparatory mechanisms of the heart muscle and would reduce inflammatory response, minimizing subsequent cardiac tissue deterioration and adverse remodelling.

First attempts in this matter were focused on a cell-based therapies that relied on the administration of several types of stem and progenitors cells, including e.g. BM-MSCs, different populations of cardiac progenitor cells (CPCs) or cardiosphere-derived cells (120–122). However, despite indication on safety and some beneficial effects, the efficacy of cell-based therapies varied depending on a type of cells and route of administration, facing several limitations including low retention in the site of the delivery or a potential immunogenicity (123). Importantly, throughout the recent years there has been an accumulating evidence that the pro-regenerative effect of SCs in the AMI treatment is caused by their paracrine activity that triggers endogenous repair mechanisms and provides immunomodulatory signaling, rather than by their direct differentiation and proliferation in the site of administration (124). Indeed, recent years of studies have brought mounting evidence on protective and pro-regenerative capability of SC-derived EVs in the treatment of AMI and other CVD-related conditions. Thus, due to the unsatisfactory results of cell-based approaches, there has been an increased focus on the alternative solutions, including those related to the utilization of EVs that not only mimic several functional properties of cells of their origin, but also are non-tumorigenic, easy to be stored and may penetrate biological barriers more effectively than the whole cells (125).

It has been widely postulated that EVs from different cell sources may potentially modulate the local microenvironment in a heart tissue toward a regeneration, exhibiting beneficial potential in CVDs treatment ( Table 1 ) (132). The mechanism of EV activity is related to their transfer of bioactive cargo, mainly miRNAs, that are known to be involved in the regulation of cellular processes within a cardiac tissue (133). As an example, pro-regenerative capacity of EVs secreted by iPSC-derived cardiomyocytes was demonstrated to be mediated by the miRNA, indicating the role of miR-106a-363 cluster that represses Notch3 signaling (134). EVs isolated from human iPSCs were also shown to be enriched in several different mRNA and miRNA that may be transferred into human heart-derived cells in vitro, improving their cardiac and endothelial differentiation potential, as well as exhibiting cytoprotective effects (135). Similarly, murine iPSCs-EVs were shown to exhibit anti-apoptotic effect in the murine ischemia/reperfusion (I/R) model via the delivery of their miR-21 and miR-210 (136). Important role of miR-210 was also reported for MSCs-EVs, that were able to enhance angiogenesis in vitro, as well as in vivo in murine AMI model. The mechanism of their action was related to the inhibition of Efna3 gene expression, that is known target of miR-210, acting as an angiogenic suppressor (57). As inflammatory process is indispensably related to the cardiac failure, immunomodulatory properties of MSCs-EVs that alleviate immunological response in the site of injury are of particular focus. The role of the miRNA transfer in the immunomodulatory activity of MSC-EVs was demonstrated, pointing a role of miR-182 that inhibited toll-like receptor 4 signaling and thus promoted macrophage polarization from pro-inflammatory to anti-inflammatory phenotype in the murine I/R injury model (137). Additionally, several papers have already indicated cytoprotective and pro-angiogenic effects of MSC-EVs, including murine (138) and rat model of myocardial infarction (127). In another study, administration of EVs secreted by the murine iPSCs improved heart function in vivo in the infarction-reperfusion model, without any signs of teratoma, in contrary to the injection of whole cells. Additionally, the therapeutic effect of those EVs was higher when compared to the group of animals treated with iPSCs, resulting in the greater improvement in left ventricle systolic function (128). Promising results of EV use in the small animal models encouraged scientists to follow attempts to test their efficacy also in a large animal models, which are an important step toward translating basic research into clinical practice. Porcine model seems to be the most optimal for the purpose of CVDs due to the several similarities in heart size and coronary circulation to the human heart (139). One of the first studies on the porcine model of AMI have demonstrated that the intracoronary injection of conditioned medium (CM) obtained from the MSCs culture significantly increased left ventricular ejection fraction (LVEF), decreasing the size of infarct zone and reducing the oxidative stress in the residual cells (140). Few years later similar results were also presented for the CM collected from porcine EPCs (141). MSCs-EVs were also used in the nonhuman primate AMI model, demonstrating improved cardiac functions and angiogenesis following vesicle administration, pointing out an important involvement of miR-486 signaling in those processes (142).

Therapeutic effects of EVs were also demonstrated in the CMD model studies, dedicated for an investigation of approaches that would primarily reduce chronic inflammatory state, scar fibrosis and cardiac tissue remodelling, which are a major hallmark of chronic cardiac disfunctions that lead to adverse clinical outcome (143). Cardiac fibrosis is a consequence of differentiation of cardiac fibroblasts into myofibroblasts and their excessive ECM deposition to replace dead cardiomyocytes following an acute injury and inflammatory signaling (144). However, fibrosis-related chronic disfunction of the cardiovascular system may be also a consequence of other factors, such as aging, diabetes mellitus or other metabolic disfunctions with an inflammatory background (145). In the context of EV-based CMD therapeutic approaches, EVs from cardiosphere-derived cells were shown to prevent cardiac remodelling and improve survival in murine non-ischaemic dilated cardiomyopathy model (146), as well as in the rat model of myocarditis (147).

Atherosclerosis is also one of common CVDs that has a strong inflammatory background. It results from the plaque formation inside the large arteries that narrow the vessel lumen. Chronic inflammation plays a pivotal role in the development and progression of atherosclerosis, starting from the activation of resident endothelial cells. Subsequently, it leads to the monocyte and leukocyte recruitment into atheroma, followed by the upregulation of pro-inflammatory cytokines, production of reactive oxygen species (ROS) and matrix metalloproteinases, consequently triggering thrombotic cascade which may lead to the AMI (148). SCs-EVs display a beneficial effect in the context of atherosclerosis treatment. As an example, administration of BM-MSCs-derived EVs into high-fat diet ApoE^-/- mice stimulated M2 polarization of residual macrophages, which led to the decrease in the inflammation and reduction of atherosclerotic plaque area. The mechanism of EV action was possibly related to the transfer of miR-let7 family that regulated activity of downstream signaling pathways, such as NF-κB and PTEN (130). Similar immunomodulatory effect was also shown for AT-MSC-EVs, that diminished inflammatory activation of both aortic endothelial cells stimulated with tumor necrosis factor alpha (TNF-α), as well as LPS-stimulated macrophages in vitro, reducing atherosclerotic plaque in vivo in low-density lipoprotein (LDL) receptor deficient (Ldlr^-/-) mice fed with a high-fat diet (131). In another study, EVs from UC-MSCs inhibited activation of eosinophils treated with oxidized LDL and promoted their apoptosis. This effect was even greater for EVs secreted by UC-MSCs overexpressing miR-100, with indicated role of frizzled 5 (FZD5)/Wnt/β-catenin pathway downregulation involved in this process. Decreased inflammation and atherosclerotic plaque following EVs treatment was also confirmed in this study in the murine in vivo model (149).

Taking together, SCs-EVs may be a promising factors for CVDs treatment, relying on their immunomodulatory and pro-regenerative activity.

The central nervous system (CNS)- associated disorders are one of the leading causes of disability and death worldwide. Apart from malfunctions associated with either cancerous processes or acute injuries such as traumatic brain and spinal cord injury or ischemic stroke, neurodegenerative diseases are common feature among CNS pathologies, with prognosed rise in their frequency caused by the increasing life expectancy. They include the most commonly recognized malfunctions such as PD, AD, Huntington’s disease or multiple sclerosis (150).

The molecular mechanisms underlying CNS-associated disorders are still poorly understood, but several studies indicate that inflammatory processes play an essential role in their development and progression (151). Thus, further studies are required to fully delineate and develop new approaches of their effective treatment. Among them, use of SCs and their EVs occurred to be a promising strategy (152), with the latter ones being of special focus due to their ability to overcome challenges associated with crossing the BBB. Thus, during a last decade EV-based treatments of CNS-associated malfunctions have emerged as potential therapeutic candidates, with several studies reporting neuroprotective effects of EVs secreted by the SCs ( Table 2 ) (160). In in vitro models, MSCs-derived EVs were demonstrated to reduce apoptosis, promote proliferation and stimulate secretion of pro-neurotrophic factors by neuroblastoma cell lines (161). On the other hand, EVs produced by AT-MSCs were shown to stimulate differentiation of neural progenitors, influencing miRNA and cytokine expression in the target cells (162). Comparative study demonstrated the ability of EVs derived from both MSCs and iPSCs cells to enhance the astrocyte recovery after irradiation, however vesicles obtained from MSCs exerted superior immunomodulatory effects (163). In another study, EVs secreted by iPSCs-derived neural stem cells were reported to be enriched in miRNAs and proteins known to be involved in neuroprotection, synaptogenesis and cytoprotection, possessing anti-inflammatory activity in vitro, as well as in vivo in the murine model of status epilepticus, following their intranasal administration (156). Improved recovery and angiogenesis together with reduced neuroinflammation were also reported following injection of EVs from BM-MSCs in rat models of spinal cord injury (164) and traumatic brain injury (165). Similarly, in murine model of focal cerebral ischemia MSCs-derived EVs exerted neuroprotection and neovascularisation, resulting from the regulation of the immune response in the site of injury (166). Apart from the rodent models, neuroprotective activity of human neural stem cell-derived EVs was also reported in porcine model of ischemic brain stroke, where authors presented data confirming reduced edema and intracranial haemorrhage following intravenous administration of EVs (155).

Protective role of EVs was also shown in the several models of neurodegenerative diseases (167). As an example, administration of neuroblastoma-derived EVs lowered the level of amyloid-β peptide (Aβ) that is known to be elevated in AD (168). Similarly, intracerebral injection of MSC-derived EVs in the murine model of AD reduced the level of amyloid plaques, mediated by the transfer of neprilysin protein known as a endopeptidase able to degrade Aβ (154). EVs were also employed as a drug delivery system in murine model of PD, by their loading with antioxidative catalase followed by the EV intranasal delivery, exerting neuroprotective and anti-inflammatory effects in vitro and in vivo (91). Moreover, in rat model of PD animals treated intranasally with EVs secreted by human exfoliated deciduous teeth SCs exhibited improved gait parameters (169). In another study, MSCs-EVs were shown to cross BBB in rat PD model and lower dopaminergic neuron loss in substantia nigra, concomitantly with an increased level of striatum (153). Protective role of SCs-EVs was also reported for the treatment of multiple sclerosis (MS), as neurodegenerative disease of CNS with the inflammatory background related to the BBB dysfunction and chronic activation of lymphocytes against oligodendrocyte proteins, that leads to the demyelination and synaptopathy (170). As an example, EVs from placental MSCs improved motor function and spinal cord myelination in autoimmune encephalomyelitis murine MS model (158). In another approach, MSCs-EVs were combined with LJM-3064 aptamer with previously demonstrated ability to induce remyelination. Such engineered hybrid particles exhibited anti-inflammatory activity and protected against CNS demyelination in murine MS model in vivo (159). Altogether, there has been accumulating evidence on the role of EVs in the treatment of different types of CNS-associated disorders.

Proper functioning of kidneys is essential for the effective control of body fluids osmolarity, pH and removal of toxic metabolites. Thus, kidney injuries are life-threatening conditions resulting in the dysregulation of homeostasis (171). One of the most severe kidney disorders is acute kidney injury (AKI) that accompanied by the systemic inflammation leads to the rapid damage of organ structure followed by a loss of renal function, with the need of patient hospitalisation, high mortality rate and high risk of the development of chronic kidney dysfunction (172). Thus, the development of effective therapeutic approaches for the AKI treatment is an important challenge of the modern medicine. EVs play an important role not only as prognostic factors and biomarkers of renal disfunction, but have also been demonstrated as potential new-generation tools for the therapy of AKI ( Table 3 ) (180). Importantly, an inflammatory response accompanying AKI has been widely reported to be significantly ameliorated by EVs from MSCs via their immunomodulatory stimuli. Meta-analysis study collecting the data from 31 preclinical studies on rodents have confirmed the therapeutic efficacy of MSC-EVs in AKI treatment (181). As an example, in the rat renal ischemia-reperfusion injury model, BM-MSCs-derived EVs inhibited apoptosis and stimulated tubular epithelial cell proliferation (182). In other study, EVs derived from native, but not from interferon gamma (IFN-γ)- stimulated UC-MSCs were able to alleviate the effect of hypoxia-induced AKI in the rat model (183). As nephrotoxicity is an important issue in oncological patients, being caused by the widely-used chemotherapeutic agents such as cisplatin, there is a need for the new therapeutic strategies that would reduce severe side effects related to the chemotherapy and improve clinical outcomes of patients. In the studies where rat cisplatin-induced AKI model was used, EVs secreted by UC-MSCs (184) and AT-MSCs (185) were able to exhibit cytoprotective activity, reducing cell death and inflammatory response. Additionally, in the murine model of cisplatin-induced AKI, EVs secreted by BM-MSCs improved renal function, but the effect was dependent on the route of EV administration, with multiple injections being beneficial over the single dose (186). The pro-regenerative activity in the context of renal function was also demonstrated for EVs from different types of cells. For instance, EVs from amniotic epithelial cells were shown to reduce nephrotoxicity in the murine model of cisplatin-induced AKI (187). ESC-EVs were also demonstrated to exhibit pro-regenerative effect in the murine model of ischemia-reperfusion AKI, by stimulating angiogenesis and proliferation of renal epithelial cells, as well as reducing renal fibrosis. These observations correlated with the activation of the resident Sox9+ cells that are known to be involved in the processes of formation and regeneration of renal tubular epithelium (104).

Apart from AKI, EVs were shown to exhibit immunoregulatory, cytoprotective and pro-regenerative activity in a treatment of chronic kidney disease (CKD) that leads to the progressive nephropathy. One of the important causes of CKD is renal hypoxia and persistent inflammation, which lead to the kidney fibrosis (188). Due to the complexity of CKD pathogenesis, current pharmacological treatments are unsatisfactory (189). The therapeutic effect of EVs in CKD treatment was demonstrated in meta-analysis covering the results from 35 studies, that mostly based on the unilateral ureteral obstruction (UUO) model of this disease (190). Protective, anti-inflammatory and anti-fibrotic role of MSC-EVs in the chronic renal dysfunction was observed both in vitro and in vivo (176), with an indication on an important role of EV-based miRNA transfer involved in those processes ( Table 3 ) (179).

Liver disfunctions, including acute injuries and chronic diseases, are considered as a significant burden experienced by many individuals, that may consequently lead to the life-threatening conditions such as end-stage cirrhosis, fibrosis or liver malignancies. Still, one of the standard therapeutic approaches is a liver transplantation. However, due to the limited availability of donors, mortality from liver-related malfunctions continues to be a critical issue, that raises the urgent need for an effective, alternative therapies for the liver replacement (191).

Liver-related diseases may be caused by alcohol, drugs, metabolic diseases or viral hepatitis. In terms of the treatment of liver disfunctions, the major goal is to inhibit fibrosis related to the chronic liver disease, that causes hepatic dysfunction, activation of hepatic stellate cells, excessive deposition of ECM and immunological response to the local inflammation (192). Thus, anti-fibrotic and anti-inflammatory therapeutic strategies including treatment with EVs, are of current interest. Indeed, several experimental approaches have demonstrated the effectiveness of different types of SC-derived EVs in ameliorating liver disfunctions ( Table 4 ). As an example, iPSC-derived EVs were shown to supress fibrosis in two murine models of liver injury, caused by either treatment with CCl₄ or by bile duct ligation (193). EVs secreted by MSCs differentiated from ESCs were also reported to alleviate thioacetamide-induced chronic liver injury, reducing cirrhosis and pro-fibrotic production of collagen I and α-smooth muscle actin (αSMA), with simultaneous decrease in the pro-apoptotic and pro-inflammatory factors (198). Similar antifibrotic effect was also demonstrated in CCl₄-induced liver fibrosis for EVs isolated from UC-MSCs and the mechanism of their action was related to the inhibition of epithelial-to-mesenchymal transition (EMT) of hepatic cells (194). In another study, UC-MSCs-derived EVs were also demonstrated to ameliorate acute liver injury due to the antioxidative and antiapoptotic effect (199). It was also shown that hepatocyte-derived EVs are able to alleviate inflammatory response and pro-fibrotic activation of hepatic stellate cells, as well enhance proliferation of hepatocytes, both in vitro and in vivo in the murine model of CCl₄ injury (195).

Involvement of EVs in the respiratory system is also well documented, with their role not only as potential biomarkers, but also as therapeutic agents, regulating the immune cell functions during airway inflammatory diseases ( Table 5 ) (207). Particularly, MSCs-EVs hold a great promise as factors mimicking beneficial immunomodulatory properties of their parental cells, thus augmenting inflammatory response typically associated with the respiratory system malfunction and tissue damage (208). Additionally, possibility to administer EVs via inhalation facilitates their entry into pulmonary system and targeted delivery of their cargo into the site of interest.

Among the variety of lung diseases, one of the most severe conditions are related to the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) that carry high morbidity and mortality rates, resulting from the rapid respiratory failure (209). In porcine model of influenza-induced ALI, intratracheal administration of porcine MSC-EVs resulted in diminishing lung injury, inhibition of virus replication in lung epithelial cells in vitro and in vivo and reduction of inflammation within the lung tissue (202). MSCs-EVs were also shown to alleviate alveolar inflammation and pulmonary edema in E. coli endotoxin-induced ALI (90). Similarly, in ex vivo perfused human model of E. coli-driven pneumonia, MSCs-EVs increased alveolar fluid clearance and antimicrobial activity of macrophages. This effect was even enhanced by the pre-treatment of parental MSCs with Toll-like receptor 3 agonist (204). In the murine model of lung injury EVs secreted by BM-MSCs decreased the lung vascular endothelial permeability caused by the of haemorrhagic shock, with possible involvement of the mechanism related to the reduction of cytoskeletal RhoA signaling activity (203). In addition, anti-inflammatory, protective and/or regenerative properties of MSC-EVs have also been observed in rodent models of pulmonary hypertension (210), radiation-induced injury (211), bronchopulmonary dysplasia (212) and idiopathic pulmonary fibrosis (213). In the last one the regenerative effect has been also demonstrated for EVs secreted by iPSCs (214). Additionally, in the murine model of lung ischemia-reperfusion injury, administration EVs derived from MSCs attenuated inflammation and edema (201). Similar outcome was also reported in the rat model, indicating an influence of EVs on the expression of genes regulating inflammation and oxidative stress (215).

Beneficial effect was also shown for MSCs-EVs in rodent models of asthma as one of the common manifestations of immune system overactivation. As an example, it was demonstrated that vesicles secreted by UC-MSCs were able to reduce inflammatory response and airway remodelling. Importantly, this effect was boosted for animals that received EVs from hypoxia-stimulated cells (200). Similarly, MSCs-EVs were shown to inhibit group 2 innate lymphoid cells (ILC2s) that are known to be involved in the pathogenesis of airway allergy. Additionally, those EVs were able to reduce the level of pro-inflammatory cytokines and mucus production in the murine model of asthma, with the suggested role of miR-146a transfer involved in this effect (206).

Anti-inflammatory and immunomodulatory properties of EVs make them a promising option for the treatment of diseases associated with the digestive system that are typically related to the multimodal gut inflammation (216). Indeed, several attempts were performed in this field so far ( Table 6 ). As an example, in the murine in vivo model of ulcerative colitis induced by the dextran sulphate sodium treatment, EVs from BM-MSCs ameliorated disease symptoms, including colon mucosa damage, by stimulating polarization of macrophages into anti-inflammatory M2 phenotype through the modulation of JAK/STAT signaling pathway (217). In another study, a suppressive influence of BM-EVs on macrophage activity was also demonstrated in murine model of inflammatory bowel disease (IBD), resulting in an improved gut functions and decreased mucosal inflammation (222). From another point of view, EVs from M2 macrophages were also reported to attenuate colitis in mice and their mode of action was related to the stimulation of Tregs via CCL1 chemokine (218).

Skin as the largest organ in the body plays an important role in the maintenance of homeostasis and provides a protective barrier against external hazardous factors, thus, is constantly exposed to potential severe injuries, including thermal and chemical burns, chronic wounds or persistent microbial infections, that may lead to the fatal trauma (223). SCs-EVs were used for the treatment of inflammatory skin diseases ( Table 7 ). As an example, EVs from AT-MSCs diminished symptoms of atopic dermatitis in the murine in vivo model of this disease, induced by the dust mite treatment of animals. Following administration of EVs the number of eosinophils and serum IgE decreased, together with the reduction of pro-inflammatory cytokines levels in the skin lesions (230). Similarly, in the in vivo model of oxazolone-induced dermatitis, AT-MSCs-EVs reduced inflammation, as well as improved ceramide production and epidermal barrier, preventing skin water loss (231). In another study, EVs from UC-MSCs reduced excessive proliferation of epidermis cells, decreased expression of interleukin IL-17 and IL-23, as well as inhibited activation of DCs is the murine model of psoriasis (227).

Recent studies have also shown beneficial effect of EVs in skin regeneration (232). For instance, subcutaneously injected EVs isolated from iPSCs-derived MSCs enhanced angiogenesis and re-epithelialisation, leading to the wound closure. Additionally, they also stimulated proliferation of skin fibroblasts and ECM production (225). In another study of murine full-thickness skin wound model, EVs from UC-MSCs promoted proliferation and migrative capacity of both endothelial cells and skin fibroblast, as well as improved angiogenesis in vitro, with improved re-epithelialisation demonstrated in vivo (226). Similarly, in the context of chronic wound treatment, UC-MSCs-derived EVs applied in the hydrogel formulation onto the wound accelerated skin healing and regeneration in the diabetic rat model (233). Interestingly, EVs from AT, but not from BM were able to enhance skin healing in murine model of diabetic murine model. These differences corresponded to the differential cargo in both types of EVs, with predominant role of BM-MSCs-EVs and AT-MSCs in promotion of skin cells proliferation and angiogenesis, respectively (228). On the other hand, in another study there was no significant difference in the pro-regenerative potential of MSCs from both BM and AT in such model, which may indicate the variance in the mechanism of action between cells and their secretory vesicles (234). An importance of immunomodulatory signaling mediated by MSCs-EVs was also demonstrated for the skin damage treatment. As an example, EVs from melatonin-preconditioned BM-MSCs triggered macrophage M2 polarization, resulting in the decrease of pro-inflammatory cytokines and increase in the expression of anti-inflammatory IL-10, enhancing angiogenesis and healing in rat diabetic wound model (229).

EVs derived from iPSCs may be also used for the purpose of skin regeneration. Importantly, due to the higher “stemness” potential of iPSCs when compared to MSCs, scientist attempt to utilize these properties in the context of antiaging skin treatment. As an example, dermal fibroblasts treated with hiPSCs-EVs possessed higher proliferative capability and thus lowered senescence. Additionally, UVB-stimulated photoaging process in those cells was also decreased following hiPSCs-EVs treatment (224). Similar results were obtained by another group, which demonstrated that “cell-engineered nanovesicles” obtained by the serial membrane extrusion of human iPSCs augmented senescent alterations in skin fibroblasts (235). Nevertheless, EVs from MSCs were also used in several studies related to the protection against skin aging. In one of studies, AT-MSCs-derived EVs attenuated UVB-triggered photoaging both in vitro, as well as in the murine in vivo model, and their mechanism of action was related to the inhibition of inflammatory-induced macrophage differentiation and ROS production, resulting in lower wrinkle scoring (236). Interestingly, direct comparison study have revealed higher antiaging effect of EVs derived from hiPSCs than MSCs (110).

Fighting the chronic pain that accompanies several inflammatory-related diseases is still a challenging aspect of medicine. There are several attempts reporting the possible usage of EVs in the pain treatment ( Table 8 ) (242). In one of the studies, UC-MSCs-EVs were used as a therapeutic agents in the rat model of neuropathic pain caused by the nerve injury. Intrathecal administration of EVs resulted in the reduced symptoms of pain and lower hind paw hypersensitivity, decreasing the expression of pro-inflammatory factors in dorsal root ganglion in the site of injury (237). In another report, intra-articular administration of secretome obtained from BM-MSCs stimulated with TNF-α and IFN-γ and ameliorated pain in the murine model of osteoarthritis (238). Moreover, EVs secreted by iPSCs-derived MSCs decreased tendinopathy-related pain symptoms in rat model in vivo, alleviating inflammation and enhancing proliferation of tenocytes (239). Not only EVs from SCs, but also immune cells may have the ability to reduce inflammation-related pain symptoms. For example, in the murine inflammatory pain model EVs from M2 macrophages were able to transfer miR-23a to microglia, increasing threshold of mechanical allodynia and thermal hyperalgesia via regulation of NF-E2-related factor 2 (NRF2) (241). Altogether these reports indicate that EVs may serve as a potential factors for the anti-pain treatment approaches.

Coronavirus infectious disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), was first time reported in Wuhan, China in a late 2021 and has rapidly spread over the world, emerging as a global pandemic issue. Till August 2022, COVID-19 affected more than half billion of people worldwide, causing death of more than 6 million (243). SARS-CoV-2 infects host cells by interaction of its spike protein with angiotensin converting enzyme 2 (ACE2) receptor, present on several types of epithelial and endothelial cells (244). Main clinical manifestations of this disease are related to the respiratory system, including strong cough, hypoxia, pneumonia and ARDS. However, it may also manifest by multiorgan disfunction, including cardiovascular, nervous or gastrointestinal system. COVID-19 is typically accompanied by mild to moderate flu-like inflammatory symptoms such as fever, muscle ache and general weakness, but in many individuals may lead to the acute cytokine storm, sepsis and in a consequence death (245). Long-term post-COVID complications were also widely reported, with multiple health issues that may last for several months from the moment of infection (246). COVID-19 outbreak has not only caused a death of many people, but also dramatically affected international economy, impacted global healthcare and negatively influenced a social life (247). Thus, increasing number of cases has raised a global pressure to find effective ways of COVID-19 prevention and effective treatment. Despite the rapid development of emergency vaccination, still its accessibility is not uniform, with accompanied hesitancy of the part of the society against the common vaccination. Additionally, there’s a lack of specific and highly effective treatment against COVID. One of the crucial issues is to inhibit uncontrolled hyperactivation of immune system that leads to the cytokine storm and consequently to the multiorgan damage (248).

It was shown that EVs may be considered not only as biomarkers of COVID-19 outcome (249), but also as immunomodulatory agents that may ameliorate inflammatory complications and improve the clinical outcome of patients ( Table 9 ) (254). In this respect, MSCs-EVs are predominantly tested as cell-free alternatives mimicking immunosuppressive properties of their cells of their origin. As an example, the potential of EVs from UC-MSCs to decrease the release of pro-inflammatory cytokines was demonstrated in vitro on human lung adenocarcinoma epithelial cells stimulated with SARS-CoV-2 peptides (250). Another study has demonstrated safety and efficacy of intravenous administration of BM-MSCs-derived EVs to 24 COVID-19-positive patients with moderate or acute ARDS. Additionally, following EV treatment an improved oxygenation ratio and decreased inflammatory status was also reported, which opened a possibility for the further studies, including clinical trials on the higher number of patients (251). Interestingly, elevated number of EVs possessing ACE2 receptor were found in the plasma of COVID-19 patients and were shown to inhibit binding of viruses and their spike protein to HEK cells in vitro, as well as to ameliorate severity of this disease in the rodent model (252). There are also attempts to use EVs as vaccines against SARS-CoV-2 infection. As an example, EVs derived from Salmonella typhimurium decorated by spike receptor-binding domain were used as immunization factors in syrian hamster COVID-19 model, exerting the effective production on neutralizing antibodies against few variants of SARS-CoV-2 (253). Altogether, use of EVs as immunoregulatory factors may open a new perspectives of COVID-19 treatment and prevention.

Osteoarthritis (OA) is a type of chronic degenerative disease of an articular cartilage. Consequently, it leads to the progressive inflammation, pain and joint dysfunction, predominantly in the knees, but also hips and fingers. It has been indicated as one of the ten most disabling disorders in the developed countries, with about 10% of men and close to 20% of women aged over 60 years to have symptomatic OA. Apart from age, major risk factors associated with OA are joint injuries and obesity (255). Currently available therapeutic approaches are limited and concentrate mainly either on temporal, pharmacological pain relief and reduction of inflammation, or on the invasive surgical interventions and joint replacement (256).

SCs-EVs were proven to support OA treatment, with the special regard to those secreted by MSCs ( Table 10 ). As an example, EVs from BM-MSCs were reported to increase the expression of type II collagen and aggrecan, with reduction of metalloproteinase 13 and iNOS, in OA-like chondrocytes in vitro. Additionally, they exhibited anti-inflammatory and cytoprotective effect in vivo, decreasing cartilage and bone degeneration in the knee joint in collagenase-induced murine OA model (257). In another study, BM-MSCs-EVs reduced expression of pro-inflammatory cyclooxygenase 2 (COX2) and NFκB signaling, with simultaneous enhancement of the proteoglycan and type II collagen level in TNF-α-stimulated chondrocytes derived from OA patients (258). Similarly, EVs isolated from AT-MSCs exhibited chondroprotective effect on IL-1β-stimulated OA chondrocytes in vitro, diminishing secretion of pro-inflammatory factors (TNF-α, IL-6, prostaglandin E2, nitric oxide, COX2) and increasing level of IL-10 and type II collagen (259). Furthermore, UC-MSCs-EVs had immunomodulatory effect in OA model in vitro and in vivo, promoting M2 macrophage polarization and secretion of anti-inflammatory IL-10, as well as inhibiting cartilage degradation. The mechanism of their action was related to miRNA cargo known to regulate PI3K pathway in targeted cells (260). Altogether, these data demonstrate the chondroprotective and immunomodulatory activity of EVs in the context of potential OA treatment.

Immunoregulatory capability of EVs makes them an attractive option for the treatment of cancer, as one of the leading causes of death worldwide. Oncological immunotherapy is one of the rapidly developing treatments, targeted to stimulate immune system toward anti-cancer defence, that includes checkpoint blockade therapies, use of chimeric antigen receptor (CAR) T-cells and cancer vaccines. Currently, there are attempts to use preparations containing EVs as anti-cancer vaccines ( Table 11 ) (264). This strategy relies on the use of EVs secreted by the cancer cells or by APCs, with the special focus on DCs. The latter ones were shown to contain functional MHC class I and II antigens, as well as co-stimulatory molecules capable to activate the anti-tumor response of cytotoxic T cells (265). Moreover, utilization of autologous tumor-derived EVs harbouring cancer-specific antigens as nanovaccines opens new possibilities of the development of personalized anti-cancer treatment. However, due to the low immunogenicity of autologous EVs from cancer cells, there are attempts to combine them with other factors that would enhance anti-tumor response of immune system. As an example, researchers created hybrid nanoparticles by combining EVs of tumor and E. coli origin, that were able to stimulate maturation of DCs and trigger strong anti-tumor immune response in colon, melanoma and breast cancer murine models (262). In another study, cell membrane vesicles from melanoma cells were combined with CpG oligonucleotides, TLR-9 agonist and DCs-targeting aptamer, enabling specific activation of immune system against cancer, together with a long-term immune memory effect (263).

Additionally, SCs-EVs were also shown to exhibit anti-cancer activity. In particular, EVs isolated from BM-MSCs inhibited proliferation of HepG2 hepatoma, Kaposi’s sarcoma, and ovarian tumor cell lines, inducing cancer cell death in vitro, as well as exhibiting anti-tumor activity following subcutaneous injection of EVs in the in vivo experiments (261). Similar results were also demonstrated for UC-MSCs-derived EVs in the model of bladder tumor (266). Thus, EV-based approaches may be a novel, promising strategy for the anti-cancer therapy.

Despite significant progress in the field, there are still several limitations of broader use of EVs in biomedical sciences. Translation of the basic science into the clinics encounters critical challenges and obtained EV preparations have to fulfil several stringent, but still not fully defined criteria, that include variety of quantitative and qualitative properties. Importantly, constantly increasing knowledge on EV biology raises new questions and doubts on their identity, optimal methods of isolation, as well as methodological barriers of their characterization (274). So far, several key aspects have been recognized as potential hindrances of EV utilization in pre-clinical and clinical studies.

One of the pitfalls is to obtain a pure EV fraction without accompanying non-vesicular entities such as protein complexes, lipoproteins or extracellular RNA, that are typically co-isolated by commonly used isolation methods such as ultracentrifugation (275). On the other hand, other methods that include elimination of concomitant impurities may cause significant reduction of EV yield, which is an important hindrance in terms of the medical use of EVs, where high amounts of EV preparations are required (276). Additionally, recent findings have demonstrated that the “protein corona” which surrounds EVs may be also needed for their biological activity and its removal by additional steps of EV purification may not be beneficial (277). Nevertheless, isolation method is one of the crucial factors that may influence functional properties of EVs and affect their downstream applications.

Another important difficulties to be overcome is a rapid macrophage-dependent clearance of EVs from the circulation (278) and their off-target biodistribution that lowers the level of EV accumulation in the site of interest (36). There are several factors influencing distribution of EVs after their in vivo uptake, including route of administration, dosing, cell source (279) and the size of EVs (280), that should be taken under the consideration during the design of EV-related studies.

Moreover, one of the critical bottlenecks in the clinical application of EVs is a lack of unified protocols of their isolation and characterization. Thus, there is also an urgent need for the development of reliable standarization and validation approaches, that would implement rigorous, complementary characterisation methods and would assure no batch-to-batch variation (271). However, due to the extreme complexity and variety of EV-related biological systems, it seems to be a huge challenge to find an optimal and universal experimental layout. As an example, based on the worldwide survey, there are several different isolation methods with ultracentrifugation being the most commonly used. However, the choice of EV isolation method will also vary depending on a type of the starting material, compromise between the purity and yield of obtained EV preparations, as well as their downstream application (281). Another difficulty is a standardized and controlled long-term storage of EV preparations, that would also allow to preserve their biological activity after thawing (282).

One of the critical hallmarks is also a scale-up production, that would not only ensure the sufficient quantity of EVs produced in a good manufacturing practice (GMP) standards, but would also not affect their quality (283). Several groups work on the development of bioreactor-based approaches for the bulk EV production (284). Additionally, scientists try to modify culture conditions of the donor cells, stimulating them physically or chemically in order to significantly increase the yield of secreted EVs (285). Despite existing challenges, several methodological approaches fulfilling GMP standard requirements were reported so far, including e.g. preparation of EVs from BM-MSCs (286) or UC-MSCs (287).

Despite several encountered difficulties to be overcome to facilitate common use of EVs in the tissue regeneration, the promising results of preclinical studies have become the basis for the attempts on using EV preparations in a medical practice. Currently, there are several clinical trials conducted around the world with the use of EV preparations (288). According to the ClinicalTrials.gov website, on October 2022 there were 84 interventional clinical trials for “extracellular vesicles” inquiry, with 15 of them being already completed. Among the top ones, 25 studies were related to the respiratory tract diseases, 16 to graft versus host disease (GvHD) and 10 to CNS diseases, with majority of them being related to the biomarker studies. Still, the clinical use of EVs for the therapeutic purposes is limited to ongoing early-phase studies, but initial results indicate no significant side effects following EVs administration, indicating their safety and therapeutic potential (289). As an example, in a recently reported case study, EVs derived from UC-MSCs were used for the intracochlear administration in the 55-year old patient suffering from Menière’s disease, who required an insertion of a cochlear implant, that typically causes inflammatory response and local fibrosis that may lead to the hearing loss. Obtained results demonstrated safety of EV injection, attenuation of inflammation and improvement of hearing capacity and speech perception parameter (290). Promising results have led to the preparation of the phase 1 clinical study. In another report, based on the previous data, including those obtained for the nonrandomized open-label cohort study related to the effect of EVs from BM-MSCs in COVID-19 associated ARDS treatment (251), randomized phase 2 clinical study “EXIT-COVID19” has been also conducted, but without already published results. Several other trials are still on the “recruiting” or “not yet recruiting” stage. Thus, direct indication on the effectiveness of EVs in the clinical practice should be expected within the upcoming years, which will allow not only to confirm safety of EV administration, but also to compare efficacy of EVs with the currently available treatments. Based on that it will be possible to indicate the most promising areas of EV-based therapeutic applications as alternatives to the currently utilized approaches.