Therapeutic Application of Mesenchymal Stem Cells Derived Extracellular Vesicles for Immunomodulation

The immunosuppressive potential of mesenchymal stem cells has been extensively investigated in many studies in vivo and in vitro. In recent years, a variety preclinical and clinical studies have demonstrated that mesenchymal stem cells ameliorate immune-mediated disorders, including autoimmune diseases. However, to date mesenchymal stem cells have not become a widely used therapeutic agent due to safety challenges, high cost and difficulties in providing long term production. A key mechanism underpinning the immunomodulatory effect of MSCs is the production of paracrine factors including growth factors, cytokines, chemokines, and extracellular vesicles (EVs). MSCs derived EVs have become an attractive therapeutic agent for immunomodulation and treatment of immune-mediated disorders. In addition to many preclinical studies of MSCs derived EVs, their beneficial effects have been observed in patients with both acute graft-vs.-host disease and chronic kidney disease. In this review, we discuss the current findings in the field of MSCs derived EVs-based therapies in immune-mediated disorders and approaches to scale EV production for clinical use.


INTRODUCTION
Mesenchymal stem cells (MSCs) bear great potential not only in regenerative medicine, but they also interfere with different pathways of the immune response and exhibit immunomodulatory activity (1,2). Preclinical and clinical studies both indicate that MSCs have immunosuppressive activity, including suppression of T-and B-cell proliferation, modulation of regulatory T cell function maturation and activation, antigen presentation by dendritic cells, decrease secretion of proinflammatory cytokines and cytotoxicity (3). Up to now MSCs have been exploited in 67 clinical trials (http://clinicaltrials.gov/; accessed April 2019) of inflammation-associated diseases, autoimmune diseases and transplant rejection (Supplementary Table 1).
The accumulating evidence supports MSCs producing a strong paracrine action on neighboring cells through a broad range of growth factors, chemokines, cytokines and extracellular vesicles (EVs) (4). EVs are a heterogeneous population of spherical membrane vesicles, containing biologically active cargo of molecules (proteins, lipids, mRNA, microRNA, siRNA, miRNA, ssDNA, dsDNA) deliverable to target cells (5,6). Due to the fundamental function of EV-mediation of intercellular communication, they are involved in numerous physiological and pathological processes and pathways (7). MSCs derived EVs have been used to recapitulate some of the biological activity of parent cells, including stimulation of regeneration in models of renal, heart, liver and nervous tissue injury (8-12) a function which is arguable the most prominent function of MSCs.
Due to the genetic instability (13), undesired differentiation (14, 15), and pulmonary embolism risks (16), the application of MSCs in a clinical setting is restricted. Cell-free therapy based on EVs offers a promising alternative to stem cell-based therapy for inflammation-associated diseases, autoimmune diseases, and transplant rejection (17)(18)(19).
A caveat in pursuing EVs for clinical therapeutic applications has been the difficulty of producing sufficient yield. Therefore, in parallel with investigation of biological activity and therapeutic efficacy, approaches to scale EV production are also underway (17, 20, 21). Large scale EV production research aims to generate an industrially feasible approach that retains clinicalgrade purity. As well as alternative approaches to isolate other EV-like and larger particles (21-23).
MSCs derived EVs are a promising therapeutic instrument which have advantages over cell therapy. In this review, we focus on the recent findings of therapeutic application of MSCs derived EVs for the treatment of immune-mediated disorders and the perspective application of different methods for large scale EVs production.

MSCS-BASED IMMUNOSUPPRESSIVE THERAPY
In vitro and in vivo studies have been used to demonstrate the MSCs anti-inflammatory and immunomodulatory properties on both innate (macrophages, NK cells, dendritic cells) and adaptive immune cells (T-cells, B-cells) (24).
The first clear evidence of MSCs immunosuppression of immune cells was produced in vitro by Aggarwal and Pittenger (30) by demonstrating human MSCs altered the cytokine secretion profile of co-cultured dendritic cells (DCs), T cells (TH1 and TH2), and natural killer (NK) cells to induce a more anti-inflammatory phenotype. In addition, the number of regulatory T cells (T-regs) was also increased (30). Successful immunosuppression of immune cells by MSCs in vitro lead to increasing the number of in vivo research and preclinical trials.
In more recent studies, MSCs isolated from human umbilical have been shown to inhibit inflammation in a rodent model of acute allergic rhinitis (31). Reduced expression of interleukin 4, tumor necrosis factor alpha (TNF-α), and immunoglobulin E were detected in the serum of animals treated with MSCs (31).
The effects of MSCs has also been tested in autoimmune encephalomyelitis (EAE) (the animal model of multiple sclerosis). Treatment with embryonic stem cell-derived MSCs in the cynomolgus monkey EAE model reduced the clinical symptoms of brain lesions and neuronal demyelination (34).
The first clinical trial using bone marrow derived MSCs was conducted in 2006 at the University of Cambridge (Supplementary Table 1). As a result of the trial the authors suggested improvements for the design of future studies to increase the efficacy of evaluation (35).
These recommendations have led to a growth in clinical trials in the area of MSCs based treatment of autoimmune disorders (Supplementary Table 1 Whilst the comparison of the data produced in these trials is complicated by variations in design (MSCs administration, dose and regimens). Importantly, to date there have been no reports of tumor development following MSCs infusion (36, 37).
Despite promising clinical studies, MSCs have not become a universal therapeutic agent for the treatment of immunemediated disorders (38). In the main, this is due to concerns over safety (transformation, undesired differentiation and blood vessels occlusion), and the labor intensive, industrially inapplicable procedure of preparation. It is known that the beneficial effects of MSCs are largely mediated by paracrine factors. Cell-free therapy based on mediators of cell-cell communication, of which EVs are considered a promising approach, are now considered to be a safer alternative to entire stem cell therapy (17). MSCs derived EVs offer compelling advantages over cell therapies in terms of improved biodistribution, lower toxicity, higher stability in the circulation and scalable production (39). Autologous and allogeneic MSCs can be used for the production of EVs-based therapeutics. The biocompatibility of autologous MSCs offers an attractive therapeutic approach. However, from a manufacturing perspective EVs derived from allogeneic MSCs can be obtained in higher concentrations and isolated from MSCs obtained from younger donors which are more biologically active and potentially more readily available (40).

MESENCHYMAL STEM CELLS DERIVED EXTRACELLULAR VESICLES
Originally it was thought the beneficial effects of MSCs based therapy in tissue regeneration was due to the engraftment and differentiation of MSCs within damaged tissues. Subsequently, it was found that relatively few transplanted MSCs engraft in host tissues (41, 42). Together with evidence that media conditioned by MSCs is sufficient to stimulate regeneration (43, 44), the development of the paracrine hypothesis of the therapeutic effects of MSCs was formed (45), the hypothesis that is now widely accepted. Within the broad spectrum of factors secreted by MSCs, EVs have been highlighted as a potential therapeutic alternative to MSCs use (46).
EVs are a heterogeneous population of vesicles, thought to contain two predominant subtypes-exosomes (40-150 nm) and microvesicles (400-2,000 nm) (17). Exosomes are vesicles of endosomal origin formed by invagination of the endosomal membrane inside of multi-vesicular bodies (MVBs) with subsequent release of exosomes as a result of fusion of the MVBs with the plasma membrane. Microvesicles are released by budding directly from the plasma membrane (17). EVs biogenesis and purification strategies have been described in several published reviews (64).
As both subpopulations of EVs overlap in both size and density, separation of the populations within a biological sample is difficult. Therefore, EVs is used as collective descriptive term to describe the population of small vesicles released by most types of cells including MSCs (17).
Evidence also demonstrates that the biological activity of EVs is also similar to that of parental MSCs. EVs possess angiogenic, anti-apoptotic, and immunomodulatory properties similar to parental MSCs (67). It was shown that mouse MSCs derived EVs are enriched with VEGF protein and miR-210-3p via which they stimulate angiogenesis in ischemic limbs (68). In a rat model of spinal cord injury, administration of MSCs derived EVs resulted in a decrease in cellular apoptosis and inflammation at the injured site. This was accompanied by a decrease in the expression levels of proinflammatory cytokines (TNF-α and IL-1β) and an increase in anti-inflammatory cytokines IL-10 (69). In a model of traumatic acute lung injury (ALI) an increase of the survival rate and suppression of inflammatory response in experimental animals was also demonstrated (70). BM-MSCs derived exosomes reduced infarct size and improved cardiac function in rats after the acute myocardial infarction (71).
These results demonstrate the potential promise of MSCs derived EVs as an alternative to whole cell therapy. EVs efficiently mimic therapeutic effects of MSCs, whilst negating some of the safety concerns related to the use of MSCs, such as tumorigenicity and blood capillary embolism specific for MSCs based therapy (72). Currently, due to the lack of a nucleus, EVs do not fall into the category of advanced therapy medicinal products (ATMP) according to the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) (73). Unlike gene therapy and cells-based approaches, EVs are not currently considered to be high-risk biologic drugs, but further conventional demonstrations of safety and efficacy in preclinical and clinical trials are essential (74).

IMMUNOMODULATORY ACTIVITY OF EXTRACELLULAR VESICLES
MSCs derived EVs have shown immunosuppressive effects on many types of immune cells: dendritic cells, T cells, B cells and macrophages (75) (Figure 1B). It was shown that MSCs-derived exosomes and microvesicles exert similar immunosuppressive functions (72). However, it should be noticed that exosomes were more efficient in suppressing inflammation in vivo in inflammatory arthritis (76).
Immunoregulation of T-cell mediated responses is an important tool to control autoimmune or inflammatory diseases.  immune activity in vivo enhancing the survival of allogenic skin grafts in mice (79) (Figure 1B). Morrison et al. demonstrated that MSC derived EVs promote an anti-inflammatory and highly phagocytic macrophage phenotype through mitochondrial delivery (80). Taken together, these studies suggest that MSCs derived EVs retain the biological activity of the parental cells and are promising immunosuppressive instrument.
A number of different animal models have been used to study the immunomodulating activity of MSCs derived EVs (Table 1), demonstrating immunological activity and modification of the expression of both anti-inflammatory and pro-inflammatory cytokines. The immunomodulatory effect of EVs derived from human MSCs within animal model of pathogen/antigen induced tissue injury demonstrated a range of effects, including increases in survival in induced lung injury (81), decreased synovial lymphocyte counts and lowered TNF-α mRNA expression in synovitis joints (48), and increased regulatory T cells following concanavalin A induced liver injury (82). Additional preclinical studies in autoimmune uveoretinitis have shown MSCs derived EVs reduce the infiltration of inflammatory T cells in the eyes reducing the intensity of symptoms (83). Moreover, Shigemoto-Kuroda et al. demonstrated that MSC derived EVs suppressed Th1 and Th17 development, inhibited activation of APCs and T cells, increased expression of the immunosuppressive cytokine IL-10 and prevented development of uveoretinitis (84). Table 1 summarizes some more recent findings in the field of preclinical studies of EVs-MSCs based therapy of immune-mediated disorders. As seen from Table 1 EVs demonstrate the immunosuppression activity regardless of MSCs source. EVs derived from adipose-derived -MSCs, amnion-derived MSCs, and umbilical cord-MSCs are as effective as BM-MSCs ( Table 1).
EVs circulate in body fluids disseminating throughout the body. To direct the immunosuppressive action of EVs, targeting strategies are actively being developed. Shamili et al. conjugated a myelin specific aptamer to the exosome surface and showed differences in the suppression of inflammatory response, demyelination process and severity of multiple sclerosis (95).
Clinical case studies to investigate the therapeutic potential of MSCs derived EVs have been conducted in patients with steroid-refractory acute graft-vs.-host disease (acute GvHD) (96). Treatment with EVs was reported to significantly improve GvHD symptoms in a 22-years female patient with severe cutaneous and intestinal GvHD, accompanied by a decrease in the level of IL-1b, TNF-a, IFN-γ secreted by patient-derived PBMCs (96).
Phase II/III clinical study on 20 patients with chronic kidney disease (CKD) showed that treatment with MSCs derived EVs improved the glomerular filtration rate (eGFR), serum creatinine level, blood urea and urinary albumin creatinine ratio (UACR) (97).
Despite these promising clinical studies, there are still limitations that need to be overcome in order to develop EVsbased medicines: (1) establishment of a recommended isolation protocol for large-scale preparation, purification and storage of EVs; (2) standardized protocols of EVs quantification, molecular, and physical EV characterization; and (3) defined quality control (QC) criteria for clinical use (74).

APPROACHES FOR INCREASING PLASMA MEMBRANE-DERIVED VESICLES YIELD
Limited yield and labor intensive procedures for EV isolation has made large-scale production by pharmaceutical companies

Source of EVs
Model of disease Observed effect References

Rat BM-MSCs Cardiac ischemia Inhibition of proliferation of T-cells in vitro, reduction of infarct size. (85)
Pig AD-MSCs Metabolic syndrome and renal artery stenosis Attenuation of renal inflammation and fibrosis, improving of medullary oxygenation, renal blood flow, and glomerular filtration rate.
Human BM-MSCs Local ischemic stroke (rat model) Immune suppression 1 week after the injury, regeneration of blood vessels and nervous tissues.
Human BM-MSCs Preterm brain injury (rat model) Amelioration of inflammation, neuronal degeneration, reduction of microgliosis and prevention of astrogliosis.
Human BM-MSCs Acute spinal cord injury Attenuation of microglia activation, improving of locomotor recovery and mechanical sensitivity.

Autoimmune diseases
Human AD-MSCs Multiple sclerosis (murine encephalomyelitis virus induced demyelinization) Immunomodulation, decrease of inflammatory infiltrates, reducing of brain atrophy, increase of cell proliferation in the subventricular zone. Plasma membrane-derived vesicles may be promising vector for the delivery of therapeutics. However, due to sporadic, unselective packing of cytoplasmic content into the plasma membrane-derived vesicles, these vesicles are not equal to the natural EVs, and the biological activity of such vesicles remains unknown and needs to be verified.
A more moderate approach to increase membrane vesicle production is treatment of cells with cytochalasin B (99). Cytochalasin B is a drug that causes disorganization of actin cytoskeleton (100). Treatment of cells with cytochalasin B resemble the disorganization of actin cytoskeleton by activated protease calpain during the natural process of microvesicle release (17, 101). Up to now EVs have been derived from HEK293 (99,102,103), 3T3 fibroblast (102), HUVECs (104), MDCKII-MDR1 (105), SH-SY5Y (22), and PC3 cells (106). Cytochalasin B-induced membrane vesicle (CIMVs) were used as vectors for nanoparticles and drugs delivery (102), decreasing the toxicity of chemotherapy in vivo by encapsulating doxorubicin (104). Cytochalasin B application to human cells promotes an increase in membrane vesicles yield by >100 fold (17). It was calculated that 17 ± 6% of the cell membrane transformed to CIMVs (99). The CIMVs released have a diameter of 100-1,000 nm (96%) which is comparable with naturally occurring EVs (22). Our studies of CIMVs have shown that their uptake by target cells is via heterophilic interaction of CIMVs membrane receptors with the surface proteins of target cells, this has a greater impact on CIMVs entry into target cells (106). Moreover, the biological activity of CIMVs is sufficient to stimulate capillary tube formation in vitro and angiogenesis in vivo by delivering growth factors (22). The use of Cytochalasin B to induced membrane vesicles (CIMVs) has advantages over endogenous EV production, including easier procedure of isolation, increased yield, more homogeneous composition due to the unselective mechanism of cytoplasmic content enclosing. The advantages of CIMVs production together with the evidence of retention of biological activity (22) strongly indicates that CIMVs may represent the next step toward the clinical use of EVs as therapeutic tools. However, the full immunomodulating properties of MSCs derived CIMVs remains to be fully evaluated.

CONCLUSION
MSCs derived EVs retain the biological activity of parental MSCs and demonstrate a similar therapeutic potential. EVs stimulate the viability and proliferation of target cells and modulate the immune microenvironment. Therefore, the EVs of MSCs are potential therapeutic tools, which have advantages over cell therapy in terms of safety, ease of storage/transportation and clinical use. However, one of the major limitations of clinical adaption of EVs is the poor scalability of production. However, improved understanding of the physical properties of EVs and the mechanism of their biogenesis is leading to improved approaches to increase yield and uniformity of their production. Taken together, these data suggest that MSC derived EVs are a promising therapeutic tool for the treatment of immune-mediated disorders, including autoimmune diseases.