Open Access

Extracellular vesicles in mesenchymal stromal cells: A novel therapeutic strategy for stroke (Review)

  • Authors:
    • Yingchen Li
    • Qilai Cheng
    • Guoheng Hu
    • Tianhao Deng
    • Qimei Wang
    • Jianda Zhou
    • Xinping Su
  • View Affiliations

  • Published online on: March 22, 2018     https://doi.org/10.3892/etm.2018.5993
  • Pages: 4067-4079
  • Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

A stroke is a focal cerebral insult that frequently causes severe neurological deficit and mortality. Recent studies have demonstrated that multipotent mesenchymal stromal cells (MSCs) hold great promise for neurovascular remodeling and neurological function recovery following a stroke. Rather than a direct replacement of parenchymal brain cells, the therapeutic mechanism of MSCs is suggested to be the secretion of soluble factors. Specifically, emerging data described MSCs as being able to release extracellular vesicles (EVs), which contain a variety of cargo including proteins, lipids, DNA and various RNA species. The released EVs can target neurocytes and vascular cells and modify the cell's functions by delivering the cargo, which are considered to mediate the neural restoration effects of MSCs. Therefore, EVs may be developed as a novel cell‑free therapy for neurological disorders. In the present review, the current advances regarding the components, functions and therapeutic potential of EVs are summarized and the use of MSC‑derived EVs as a promising approach in the treatment of stroke are highlighted.

Introduction

Stroke is one of the leading causes of neurological dysfunction and mortality worldwide (1). Intravenous thrombolytic therapy has been demonstrated to effectively restore cerebral blood flow; however, the narrow therapeutic window (within 3–4.5 h following stroke onset) and serious hemorrhagic complications limit this treatment to only a small proportion of stroke sufferers (2). Endovascular intracranial thrombectomy allows an extension of this therapeutic window and potentially diminishes the risk of intracranial bleeding; however, mechanical recanalization devices also exhibit limitations due to vessel tortuosity, arterial stenosis and inaccessibility of the thrombus (3). Therefore, there is an urgent need to investigate safer and more effective treatment to restore brain function. In recent years, multipotent mesenchymal stromal cells (MSCs) have emerged as promising candidates among the innovative treatment options, which may significantly promote the recovery of neural function following a stroke (4,5). However, rather than directly replacing parenchymal brain cells, the secretion of extracellular vesicles (EVs) is suggested to be the primary therapeutic mechanism of MSC therapy (6,7). EV-based therapy circumvents the disadvantages and limitations of MSC transplantation, including embolization and possible tumor differentiation, therefore it may be an alternative to MSCs in restoring neurological function (8). In the present review, recent advances regarding the components, functions and therapeutic potential of EVs have been summarized and the future of MSC-derived EVs as a promising cell-free therapeutic approach for stroke has been discussed.

Therapeutic potential of MSCs in the treatment of stroke

MSC are multipotent adult progenitor cells, which can be isolated from various sources, such as bone marrow, placenta, umbilical cord, umbilical cord blood and adipose tissues (9,10). MSCs have the unique abilities of self-renewal, proliferation and multipotent differentiation, and can differentiate into the different cell types of the three germ layers; neurons, adipocytes, osteoblasts, hepatocytes, endothelial cells and islet b cells (1113). In recent years, MSCs have received attention from researchers worldwide and several features have identified them as promising candidates for the treatment of diseases of the central nervous system. For instance, MSCs may be derived, cultured and expanded with no serious ethical issues, and they can migrate into the brain parenchyma via the circulatory system (14). It has also been determined that MSCs exhibit immunosuppressive properties and immune privilege and therefore may be applied in allogeneic cellular transplantation (15,16). In addition, they release a number of anti-inflammatory factors, including indoleamine 2,3-dioxygenase, prostaglandin E2 and interleukin-10 following transplantation and reduce the inflammatory immune response following cerebral ischemia injury (17).

A number of previous studies have confirmed the remarkable neuroregenerative ability of MSCs. MSC infusion in animal models of stroke have led to reduced infarct volume, enhanced synaptogenesis, increased vessel density and improved neurological function (9). In clinical trials, transplantation of MSCs was demonstrated to be a safe and feasible therapeutic strategy for stroke patients (Table I) (1824).

Table I.

Clinical trials of MSC therapy in patients with stroke.

Table I.

Clinical trials of MSC therapy in patients with stroke.

Lead author, yearStudy designBrain infarctCells usedRoute of applicationCell doseAdverse effectsOutcome(Refs.)
Bang, 2005Control, n=25; treatment, n=5; 1-year follow upAcute MCA infarctAutologous BM-MSCsIV5×107 cells in two dosesNoneImproved BI; decreased mRS(18)
Lee, 2010Control, n=36; treatment, n=16; 5-year follow upAcute MCA infarctAutologous BM-MSCsIV5×107 cells in two dosesNoneDecreased mRS(19)
Honmou, 2011No control group; treatment, n=12; 1-year follow upChronic ischemicAutologous BM-MSCsIVSingle dose of 0.6–1.6×108 cellsNoneDecreased NIHSS, reduced lesion volume(20)
Bhasin, 2011Control, n=6; treatment, n=6; 6-month follow upChronic MCA infarctAutologous BM-MSCsIV5–6×107 cellsNoneIncreased F-M score and mBI(21)
Jiang, 2013No control group; treatment, n=3; 6-month follow upAcute MCA infarctAllogeneic UC-MSCsIASingle dose of 2×107 cellsNoneImproved muscle strength and mRS(22)
Bhasin, 2013Control, n=20; treatment, n=6; 6-month follow upChronic ischemicAutologous BM-MSCsIV5–6×107 cellsNoneIncreased mBI(23)
Steinberg, 2016No control group; treatment, n=16; 1-year follow upChronic Ischemic BM-MSCsModified allogeneicICSingle doses of 2.5×106, 5.0×106, or 10×106 cellsNoneIncreased ESS, F-M score; decreased NIHSS(24)

[i] BI, Barthel index; BM-MSC, bone marrow mesenchymal stem cell; ESS, European Stroke Scale; F-M score, Fugl-Meyer score; IA, Intra-artery; IC, intracerebral; ICH, intracerebral hemorrhage; IV, intra

Following cell transplantation in a previous study, MSCs were hypothesized to integrate and differentiate into neural cells and replace damaged brain tissue, however, the majority of grafted cells became mechanically entrapped at the precapillary level and, ultimately, long-term survival was only observed in a limited number of delivered MSCs in the brain (25). Previous data have suggested that the therapeutic effects are attributed to the secretion of paracrine factors by MSCs rather than the differentiation of the administered cells (2628). A previous study has identified a class of MSC-released EVs, which may be associated with the brain restoration and repair effects of MSC therapy (29). EVs serve an important role in intercellular communication by transferring protein and RNA cargo between parent and target cells. The EVs released by MSCs function in the brain and vessels and induce neurovascular remodeling, anti-apoptosis and anti-inflammatory effects, which are considered to be novel molecular mechanisms of MSC therapy (6,3032).

Constitution and characteristics of extracellular vesicles

Aside from the biologically active cytokines or factors being important components of cell secretions, it seems that the majority of cells are able to release EVs constitutively or following stimulation (33). EVs are cell-derived membrane-bound entities that contain cytoplasmic components. Structural analyses have revealed that EVs are surrounded by phospholipid bilayers, which are predominantly composed of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine (PS) (34). In particular, the structure of the bi-lipid membrane provides an efficient platform for the interaction of EVs and target cells through vesicle-cell channels (35). With an endosomal origin, EVs contain various types of cellular components, such as cytoskeleton proteins, signaling proteins, lipids, RNA and DNA. The cargos of EVs are dependent on the original cell types and may be affected by the conditions of cell culture (36). Furthermore, EV proteins and RNAs typically participate in cell-cell communication to determine the biological effects of EVs on recipient cells (37,38). Present in all biological fluids in the body, such as serum, urine and cerebrospinal fluid, EVs may contain certain specific biomarkers of diseases, thus they are promising candidates in the diagnosis of different disorders (39,40). The abilities of transporting biomolecules and targeting specific cell populations further raise the possibility of EVs as therapeutics (41).

To date, the biochemical structure, composition, biological characteristics and physiological effects of EVs have only been partially elucidated. Nevertheless, the important role of EVs in intercellular communication has received much attention in the last 10 years, as these particles are capable of transferring biological molecules such as proteins, lipids, mRNAs and microRNAs to target cells (42). The timeline (1969–2016) of articles referring to extracellular vesicles, microvesicles and exosomes are presented in Fig. 1. The present study conducted a search for literature in the PubMed database (https://www.ncbi.nlm.nih.gov/pubmed/) and the following search terms were utilized: ‘extracellular vesicles’, ‘microvesicles’ and ‘exosomes’. The original studies published in various languages were retrieved, while the reviews, editorials and articles investigating irrelevant objects were excluded.

Types of EVs

EVs can be categorized into three major types based on size and intracellular origin; exosomes, microvesicles (MVs) and apoptotic bodies. Exosomes originate from multivesicular bodies (MVBs) and are discharged via p53-regulated exocytosis, which is dependent on cytoskeleton reorganization but independent of intracellular calcium concentration (43). When MVBs fuse with cell membranes, the intraluminal particles are released from the cell and these particles are referred to as exosomes. Typically, exosomes are reported to be homogenous in size (30–120 nm) and have a density in sucrose of 1.13–1.19 g/ml (44). The most common markers of exosomes are tetraspanins [cluster of differentiation (CD)63, CD81 and CD9], heat-shock protein (Hsp)60, Hsp70, Hsp90, ALG-2-interacting protein X (Alix) and the origin-cell-specific markers (45). In addition, exosomes are rich in annexins, clathrin, lipid raft markers (flotillin-1 and flotillin-2), tumor susceptibility gene 101 (Tsg101), major histocompatibility complex (MHC) molecules and Rab family proteins, whereas PS is expressed at a low level in exosomes (45).

MVs, also known as microparticles, nanoparticles, ectosomes and shedding vesicles, are formed in response to specific stimuli (46). MVs have a diameter of 100–1,000 nm and a density in sucrose of 1.04–1.07 g/ml. They are released via direct budding and shedding from cytomembranes and this process is dependent on activation of calpain, calcium influx and the cytoskeleton (8,47). Large amounts of PS and lipid raft-associated molecules are characteristic markers of MVs and the membranes of MVs are rich in cholesterol, sphingomyelin and ceramide (43).

Apoptotic bodies, which are derived from cells undergoing apoptosis, represent the last kind of EVs. They are typically >1,000 nm and contain membrane contents, tightly packed organelles and fragmented DNA (48). Apoptotic bodies are exocytosed under the regulation of adenosine triphosphate-dependent enzymes from the cytomembrane, they express PS on the surface and have permeable membranes (45). Apoptotic bodies have been suggested to induce anti-inflammatory or tolerogenic responses when taken in by nearby cells; however, their specific role remains undefined (49).

The electron micrographs of exosomes and MVs released by human umbilical cord-MSCs are presented in Fig. 2. The ultrastructure of human umbilical cord-MSCs was visualized using transmission electron microscopy. The umbilical cord tissue was obtained in April 2017 from a patient at the Obstetrical department of the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine (Changsa, China). Briefly, MSCs were washed with PBS three times and digested with 0.25% trypsin-EDTA (cat. no. 25200-056; Invitrogen; Thermo Fisher scientific, Inc., Waltham, MA, USA) at 37°C for 2 min. Following trypsinization with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), cells were pelleted via centrifugation at 200 × g for 5 min at 4°C. The supernatant was removed and the cells were fixed with 1% glutaraldehyde solution at 4°C overnight. Subsequently, the glutaraldehyde was discarded and 1% osmium tetroxide solution was added to the pellets and incubated for 24 h at 4°C. The sample was dehydrated via graded ethanol washes and embedded in Durcupan resin for 6 h at room temperature. Sections (thickness, 60 nm) were cut using an ultramicrotome and placed on copper grids. A solution of 3% Uuranyl acetate and 2% lead citrate was used to counterstain the sections for 1 h at 37°C. The sample was observed with a transmission electron microscope (Hitachi, Ltd., Tokyo, Japan, HT7800) at 80 kV. The key characteristics of the three types of EVs are summarized in Table II.

Table II.

Key characteristics of extracellular vesicles.

Table II.

Key characteristics of extracellular vesicles.

CharacteristicExosomesMicrovesiclesApoptotic bodies
Size30–120 nm100–1,000 nm≥1,000 nm
Density in sucrose1.13–1.19 g/ml1.04–1.07 g/ml1.16–1.28 g/ml
OriginBy exocytosis of MVB; process dependent on cytoskeleton reorganization but independent on Ca2+Outward budding of plasma membrane; process dependent on Ca2+, calpain and cytoskeleton activationOutward budding of apoptotic cell membrane
MarkersTetraspanins, Tsg101, Alix, Hsp, annexins, Low exposure of PS, the origin-cell-specific markersLipid raft-associated molecules, high expression of PSExpression of PS
ContentProteins, lipids, mRNA and miRNA, rare DNAProteins, lipids, mRNA, miRNA, plasmid DNAIntracellular fragments and cellular organelles
Isolation technique Ultracentrifugation, electron microscopy Ultracentrifugation, electron microscopyFlow cytometry, electron microscopy
Storage−80°C−80°CNot available

[i] Hsp, heat-shock protein; MVB, multivesicular body; PS, phosphatidylserine; Tsg101, tumor susceptibility gene 101; Alix, ALG-2-interacting protein X; miRNA, microRNA.

The strict separation of these vesicles by size or biogenesis has not yet been established. As all of these vesicle types are present in vitro and in vivo, the mixed vesicles cannot be dissected from each other once they have left the cell and in the present review, the term EVs is used to collectively describe the extracellular membrane vesicles.

Isolation of EVs

At present, there are a number of approaches used to isolate EVs, including ultracentrifugation (UC), polymeric precipitation, the use of size exclusions and aqueous two phase system (ATPS) (50).

UC is the most accepted method, which involves a series of centrifugation steps (8). Cell fragments and large vesicles are separated from EV-containing fluid by gradually raising the centrifugal force (from 2,500–10,000 × g) and EVs are precipitated with two rounds of centrifugation at 100,000 × g for 2–3 h each time. This method is reliable, inexpensive and highly reproducible, but is lengthy and requires specialized laboratory apparatus (30).

Another isolation method is polymeric precipitation (e.g., ExoQuick-TC™; System Biosciences, LLC, Palo Alto, CA, USA), which can decrease the solubility of MVs and form precipitation (51,52). This approach has the advantages of a high yield and efficiency, but the poor purity obtained reduces the reliability of the results (53).

Size exclusions (e.g., filters) are applied by passing the sample through 0.8-µm pore filters to remove large particles and followed by centrifugation to concentrate the eluted EVs (30,54). It's a simple and low-cost technique, which can be used alone or in conjunction with UC, but this method has a low throughput as lots of EVs may adhere to the filters (55). Furthermore, the products may be contaminated by other vesicles of the same size (53).

Recently, Shin et al (55) demonstrated a simple and efficient method to isolate EVs from saliva using an ATPS. ATPS rapidly separated EVs from a mixture of vesicles and proteins by partitioning different types of vesicles into different phases; this method achieved a high yield and purity within a short time. However, the application of ATPS has not yet been tested extensively and a standard isolation method is still utilized in EV research. A schematic diagram for separation of EVs is represented in Fig. 3.

Characterization of EVs

Due to the ability of fluorescence activated cell sorting (FACS) to identify the same individual vesicles by different parameters, is the most commonly used method for the study of EV. The operational process of FACS is simple and the results can be quantified to a high level of quality (56). The main disadvantage is that a flow cytometer has poor discrimination under 500 nm and is only fit for detection of big vesicles. Previously, high-definition cytometers have appeared on the market and they are capable of detecting nanoparticles as small as 0.1–0.2 µm in diameter (57). Electron microscopy is typically combined with FACS to provide morphological information about the EVs (54). However, this technique has limitations in quantitative examination and the process is complicated and costly (58). Nanoparticle tracking analysis (NTA) is typically applied to analyze the mean size, modal value and size distribution of EVs (50). The vesicles with a diameter from 30–10,000 nm can be precisely and easily detected in only 5 min. The main limitation of this technique is that the quality of NTA is influenced by particle concentration and the samples need to be diluted properly to obtain reliable results (59). Additionally, enzyme linked ELISA and western blotting (WB) are used to analyze the proteins associated with EVs (60,61). Commonly used markers include tetraspanins, Tsg101, Alix, annexins, lipid raft-associated molecules and the origin-cell-specific surface proteins (62). However, both ELISAs and WB are unsuitable to quantify the protein expression level in EVs and can only be employed to demonstrate the presence of proteins (50). Commonly used methods for characterization of EVs are summarized in Table III.

Table III.

Common methods for extracellular vesicle characterization.

Table III.

Common methods for extracellular vesicle characterization.

TechniqueInformation acquiredLimitations
Electron microscopyMorphology, sizeUnquantifiable, complicated and costly
Fluorescence activated cell sortingPhenotype, numberLimited working range
Nanoparticle tracking analysisSize, concentration, size distributionDilution needed
ELISA and western blottingPhenotypeUnquantifiable
Storage of isolated EVs

Reservoir vessels, buffers, storage temperature and programs have specific marked impacts on the results of EV experiments. It is advisable to store EVs in silicified vessels to prevent the adhesion of EVs to the vessel surfaces (50). PBS is considered to be the optimal choice for EV resuspension and the standard temperature for MV storage is suggested to be −80°C (63). As for any bioactive substances, it is recommended to have the samples frozen and thawed quickly and to minimize the cycles of freezing and thawing, although it has previously been suggested that freeze/thaw cycles have no negative effect on EVs (58,64). In general, further study is required to determine the optimal storage conditions.

Biological functions of extracellular vesicles

EVs are released from cell membranes when the cells are stimulated by biological agonists (e.g. interleukin, endotoxin and apoptosis factors) or chemical stress (oxidative or hypoxic stress) (8,40). EVs were previously treated as only cell fragments or debris and their biological activities were ignored (35); however, an increasing number of studies have demonstrated that these tiny vesicles are important messengers in cell-to-cell communication as they can preserve and deliver cellular signals (6567). EVs contain various types of proteins (cytoskeleton, surface molecules, receptors and enzymes), lipids similar to the cytomembrane and nucleic acids [mRNA, microRNA (miRNA), long non-coding RNA and DNA] (6870). As multicomponent lipid vesicles, EVs have the ability to transfer cellular cargos to target cells and induce alterations of phenotype and behavior. Furthermore, they can protect the cytoplasmic components from damage or degradation by chemicals and enzymes (71,72). In general, EVs are able to mediate cellular interaction and exchange of information primarily through the pathways illustrated in Fig. 4.

Exosomes originate from MVBs and are discharged by membrane fusion, whereas MVs are released by direct budding from the cytomembrane. EVs may mediate cell-cell communication mechanisms including: i) Stimulation of recipient cells by functioning as signal complexes; ii) transfer of surface receptors or lipids into recipient cells; iii) delivery of cytoplasmic proteins and nucleic acids by the endocytic pathway; and iv) delivery of cytoplasmic proteins and nucleic acids by the membrane fusion.

EVs may directly stimulate the recipient cells by functioning as signal complexes. Previous studies have demonstrated that EVs contain several types of surface molecules and receptor proteins including Fas-L protein, co-stimulator factors, adhesion molecules, MHC I and MHC II molecules (43,73,74). Therefore, cells with various receptors may be activated by EV ligands, e.g., exosomes expressing delta-like ligand 4, a membrane-spanning Notch ligand, which interacts with Notch receptors presented on endotheliocytes or neurocytes and causes angiogenesis and neurogenesis, respectively (75).

EVs may transfer surface receptors or lipids into recipient cells. For example, microparticles from platelets can deliver CD41 to endotheliocytes via membrane fusion, resulting in the proadhesive abilities of these cells (76). In addition, EVs are also able to act as vehicles for spreading infective agents between cells. The chemokine receptor 5 or C-X-C chemokine receptor type 4 from T lymphocytes can be diverted to non-lymphoid cells and this microparticle-mediated transfer may render these cells susceptible to human immunodeficiency virus infection (77,78).

Furthermore as part of the ligand and receptor interaction, EVs may also affect the function of target cells via the delivery of cytoplasmic proteins (79). EVs released by endotheliocytes can promote angiogenesis through the secretion of angiogenic stimulators, e.g., growth factors, proteases and their activators (8082). In addition, Mitra et al (83) recently demonstrated that endotoxin-activated mononuclear cells induced cell apoptosis by transporting caspase-1 in microparticles. As a consequence, EV-mediated transfer of cellular factors and bioactive molecules may support the interaction effects between cell populations.

Finally, growing evidence indicates that EVs contribute to the transfer of nucleic acids and genetic information may be delivered to target cells, causing alterations of cellular activities and functions. Katsman et al (84) previously demonstrated that MVs released from embryonic stem cells (ESCs) contained a large amount of miRNAs, which may be transferred to human Müller cells in vitro. In addition, it was demonstrated that ESC-released MVs increased the pluripotency and proliferation of hematopoietic progenitor cells (HPC) via delivery of ESC-derived mRNAs (85). The pretreatment of MVs with RNase may eliminate the biological effects on HPCs and this observation further confirmed the important role of mRNA transfer via MVs (85).

Extracellular vesicles as an alternative to MSCs

MSCs were demonstrated to release the highest amount of EVs out of a group of different cell lines, e.g., the human acute monocytic leukemia cell line, the primary human small airway epithelial cells and the mouse embryonic stem cell-derived insulin-producing cell line (86). There is no difference in morphological characteristics between the EVs released by MSCs and other cell types. In addition, aside from the common biomarkers, MSC-released EVs express certain specific surface antigens (including CD44, CD73, CD90 and CD105), which can be detected for their identification (30). As to the contents of the vesicles, the protein components of MSC-released EVs do not remain unchanged. In three independent batches of exosomes obtained from conditioned medium (CM) of MSCs, 379, 432 and 420 proteins were identified respectively, and only 154 unique proteins (~20%) were demonstrated to be in present in all (87). Functional clustering of those proteins was also performed in this study, which revealed that exosomes exhibit potential to initiate many biological processes, which is in accordance with the multiple therapeutic efficacies of MSCs (87).

Conversely, it has recently been demonstrated that MSCs produce many EVs that contain selected classes of mRNAs and miRNAs with specific functions (67,88,89). Based on gene ontology analysis, Eirin et al (66) demonstrated that EVs from adipose-derived MSC (AD-MSCs) were rich in different types of RNAs and their microRNA cargos (miRNA148a, miRNA 532–5p, miRNA 378) could target transcription factors or genes to induce angiogenesis, adipogenesis, apoptosis and proteolysis in recipient cells. In addition, it was reported that human bone marrow (BM)-MSCs are able to release exosomes containing mRNA for insulin-like growth factor 1 receptor (IGF-1R) and the transfer of IGF-1R mRNA may ameliorate cisplatin-induced renal dysfunction by increasing the proliferation of proximal tubular cells (90). Together, this suggested the possibility that MSCs may alter the expression of gene products and regulate the features and action of neighboring cells by EV-mediated delivery of nucleic acids, e.g., mRNA, small interfering RNA and miRNA.

As a consequence, EVs, by transferring specific types of biological molecules and genetic information, are suggested to be notable paracrine factors associated with signaling between MSCs and target cells and they may be an alternative to MSCs to restore organism function. A previous study demonstrated that the injured lung cells may deliver EVs containing lung-specific genes to BM cells, causing the expression of lung-specific proteins on MSCs (91). As a result, BM-MSCs were able to convert into cells with a pulmonary epithelial cell phenotype following transplantation into lethally X-ray-irradiated mice (91). Therefore, MSCs and injured cells may have bi-directional cell-cell communication during the process of tissue reparation. EVs secreted by injured cells may trigger the physiological activities of MSCs and MSC-derived EVs may induce the reparative and proliferative alterations of tissue cells (92,93).

Therapeutic effects of MSC-released extracellular vesicles on stroke

Although the application of MSC transplantation has attracted attention in neural regeneration research, the use of MSC-released EVs has also received intense scholarly interest. EVs may regulate neural survival, apoptosis, proliferation and regeneration following brain damage (Table IV) (32). MSC-secreted EVs have been demonstrated to promote neural repair and functional recovery in animal models of ischemic stroke. It was indicated that intravenous delivery of exosome-enriched EVs generated from BM-MSCs significantly improved axonal plasticity and neurite remodeling in the ischemic cortex of middle cerebral artery occlusion (MCAO) rats (94). In addition, a similar neurorestorative effect of MSC-derived EVs was exhibited by cortical neuron models of glutamate excitotoxicity (95). Following exposure to glutamate for 15 min, neurons were co-cultured with CM containing EVs of AD-MSC for 18 h. Compared with the control groups, MSC-CM reduced LDH release, inhibited neuron apoptosis and promoted neuronal regeneration and bioenergy restoration (95).

Table IV.

Therapeutic Effects of MSC-released extracellular vesicles on stroke.

Table IV.

Therapeutic Effects of MSC-released extracellular vesicles on stroke.

ModelName/size (nm)IsolationIdentifyOriginAdministrationBiological function(Refs.)
Mice/MCAOExtracellular vesicles/not shownPEG plus UCMicro BCA, WBBM-MSCsIVImproved neurological impairment and angioneuro-genesis, suppressed immune responses(31)
Cortical neurons; glutamate excite-toxicityMSC conditioned medium/-− -− -Human AD-MSCsIn vitro co-cultureInhibited neuronal cell apoptosis, promoted nerve regeneration and repair, restored bioenergy(95)
PC12 cells; glutamate excitotoxicityMicrovesicles/not shownUCFlow cytometry, Bradford method, TEMBM-MSCsIn vitro co-cultureActivated the phosphoinositide 3-kinase/protein kinase B signaling pathway(96)
Cortical neurons; not shownExosomes/40–100 nmUCTEMBM-MSCsIn vitro co-cultureIncreased neurite branch number, total neurite length and microRNA-133b levels(97)
Rats/MCAOExosomes/not shownUCMicro BCA assayBM-MSCsIVImproved functional recovery, neurogenesis, neurite remodeling and angiogenesis(98)
Rats; subcortical infarct modelExtracellular vesicles/50–100 nmExosome extraction kitElectron microscopy, Nano Sight,AD-MSCsIVImproved functional recovery, fiber tract integrity, axonal sprouting WB and immunofluorescence and white matter repair markers and restored white matter integrity(109)
Rats; MCAOExosomes/not shownUCWB, TEM, micro BCA assay, qNano particle analysisBM-MSCsIntra-arterial injectionIncreased functional improvement, neurite remodeling and brain plasticity(110)

[i] AD-MSCs, adipose derived mesenchymal stem cells; BCA, bicinchoninic acid; BM-MSC, bone marrow mesenchymal stem cell; IV, intravenous; MCAO, middle cerebral artery occlusion; PC12 cells, rat adrenal pheochromocytoma cells; UC, ultracentrifugation; PEG, polyethene glycol; Akt, protein kinase B; WB, western blotting; TEM, transmission electron microscopy.

Furthermore, EVs from MSCs were reported to modulate signaling pathways to treat ischemic stroke. Lin et al noted that MVs released by BM-MSCs were able to protect PC12 cells from glutamate-induced damage (96). In that study, MVs enhanced protein kinase B (Akt) phosphorylation and B-cell lymphoma-2 (Bcl-2) expression and reduced Bcl-2-associated X protein and caspase-3 expression, and these effects were removed by inhibition of phosphoinositide 3-kinase (PI3K). This suggested that MSC-MVs acted as neural protective agents via activation of the PI3K/Akt pathway. In addition, Xin et al (97) previously observed that the expression of miRNA-133b in MSC-released exosomes increased when MSCs were cultured with ischemic brain extracts from rats subjected to MCAO. Further research demonstrated that EVs from BM-MSCs were able to transfer miRNA-133b to astrocytes and neurons in the ischemic boundary zone of rats, resulting in promotion of neural plasticity and functional recovery (94). These studies indicated that EV-mediated secretion of miRNAs contributed to the neuroprotective effects of MSCs on stroke.

With the exception of the beneficial effects on neurogenesis, EVs can improve angiogenesis following cerebral ischemia. It was demonstrated that intravenous administration of MSC-generated exosomes significantly increased the percentage of newly formed von Willebrand factor-positive cells in the ischemic zone (98). Compared with PBS-treated controls, rats receiving exosome treatment also demonstrated an improvement in neurovascular plasticity in the stroke affected hemisphere and promotion of neurological function recovery. Finally, it is important to emphasize that EVs may have immunosuppressive effects on cerebral inflammatory-related diseases. Doeppner et al (31) proposed that EVs from BM-derived MSC lineages were able to modify immune reactions and restore the reshaping ability of the injured brain following focal cerebral ischemia. Alongside the normalization of B lymphocytes, natural killer cells and T lymphocytes, a deactivation of dendritic cells was observed in the peripheral blood of MSC-EV-treated mice and the suppression of immune responses provided an appropriate extracellular environment for neurovascular remodeling and contributed to the functional recovery following a stroke.

Advantages and challenges of MSC-EV therapy

In spite of the indicated promised of MSC-based therapy in regenerative medicine, EVs demonstrate a number of advantages over MSCs in clinical applications. First, exogenous administration of MSCs may cause some serious side effects, e.g., malignant transformation, tumor generation or microvascular obstruction (99101). In contrast, EVs, given their nanometer dimensions, have no vascular obstructive effects or apparent adverse effects following the in vivo allogeneic administration. Compared to the direct delivery of cells, EVs have a unique capability to easily cross the blood brain barrier, which is very important in the treatment of neurological disorders (102,103). Additionally, EVs can be stored at −80°C for 6 months without degradation of their contents and this is important for the protection of soluble molecules including biological factors and nucleic acids (63). Notably, MSC-derived EVs can be modified to express a high level of biological factors, surface proteins, mRNAs and miRNAs that promote tissue repair and functional recovery and these engineered EVs may be utilized as a novel class of cell-based therapeutics (40,104).

Nevertheless, specific problems must be resolved if EVs are to be suitable for clinical applications. First, it must be determined to what extent EVs contribute to the therapeutic benefits of MSC-administration. Second, novel techniques are required to obtain large-scale production of EVs. Chen et al (105) previously suggested that transformation of the Myc gene may be a useful strategy to ensure an infinite supply of MSCs for the production of EVs and the high proliferative rate of MSCs may reduce the time and economic costs, and increase the output of EV-production. Third, further studies should look into the detailed mechanisms of the interaction between EVs and target cells and the contents of EVs, i.e., proteins, lipids and nucleic acids, must be intensively researched. Finally, it's imperative to investigate the potential side effects of EVs in therapy. Salido-Guadarrama et al (106) reported that tumor cells could achieve cell-cell communication by the release and transfer of miRNAs packed into tumor-secreted exosomes and, tumor-secreted exosomes served an important role in the establishment, maintenance and enhancement of tumor microenvironment, and they may act as mediators in cancer metastasis. Although EVs have no potential to directly form tumors, this does not mean that MSC-EV application has no risk of tumor promotion. Therefore, further studies are required to evaluate the long-term biological safety of EV administration. Table V exhibits the advantages and challenges of MSC based therapy and MSC-EV based therapy.

Table V.

Advantages and challenges of MSC based therapy and MSC-EV based therapy.

Table V.

Advantages and challenges of MSC based therapy and MSC-EV based therapy.

Type of therapyAdvantagesChallenges
MSC based therapyPotential of proliferation and differentiation, release of MVs and other biological factorsMalignant transformation, tumor generation, microvascular obstruction
MSC-EV based therapyNo apparent adverse effects, capability to cross the blood brain barrier, no vascular obstructive effects, easy to be stored and engineeredDetermine the specific benefits and mechanisms of MV administration, in-depth study of MV contents, potential side effects: tumor promotion

[i] MSC, mesenchymal stem cells; EV, extracellular vesicles; MV, microvesicles.

Conclusion

In recent years, the therapeutic potential of MSC-derived EVs has attracted a lot of attention. EVs should not be regarded as mere cell fragments or cellular waste and evidence has demonstrated that they are associated with the regulation of cell-cell communication and have significant biological effects on recipient cells. Remarkably, the idea of using EVs as a cell-free vaccine to eradicate tumors was conceived ~20 years ago (107) and clinical trials of EV-based therapy in cancer patients were initially conducted in the early 2000s (108). Currently, no clinical trials have been applied for an MSC-EV administration to treat strokes (http://clinicaltrials.gov) and it is too early to claim that MSC-derived EVs could be clinically used for functional recovery following cerebral ischemia. The specific mechanisms and the potential side effects of EV administration need to be fully investigated and novel techniques are required to obtain large-scale production of EVs. In addition, there is currently no internationally recognized standard for clinical level production and quality control of EV-based therapeutics. These unresolved problems are the remaining impediments to this therapy and, therefore, more animal experiments and clinical tests are required prior to MSC-EV application as a conventional treatment.

Acknowledgements

Not applicable.

Funding

The present review was supported by the National Natural Science Foundation of China (grant nos. 81273751, 81372140, 81360627, 81572689 and 81573941) and the China Postdoctoral Science Foundation (grant no. 2017M610509).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

Dr YL, Professor QC and Professor GH wrote the draft; Dr TD and Dr QW assisted with the literature search; and Professor JZ, and Professor XS revised and proofed the manuscript, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The protocol for analysis of human umbilical cord was approved by the ethics committee of the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine (Changsa, China). Donors of human umbilical cord provided informed written consent prior to their inclusion in the present study.

Consent for publication

The patients have provided written informed consent for the publication of any associated data and accompanying images.

Competing interests

The authors declare that they have no competing interests and they have no financial relationships to disclose.

Glossary

Abbreviations

Abbreviations:

AD-MSCs

adipose-derived mesenchymal stromal cells

ATPS

aqueous two phase system

BI

barthel index

BM-MSC

bone marrow mesenchymal stem cell

CM

conditioned media

CNS

central nervous system

DEX

dextran

ESC

embryonic stem cells

EVs

extracellular vesicles

FACS

fluorescence activated cell sorting

FBS

fetal bovine serum

F-M Score

Fugl-Meyer score

HPCs

hematopoietic progenitor cells

Hsp

heat-shock protein

IGF-1R

insulin-like growth factor 1 receptor

LDH

lactate dehydrogenase

MCA

middle cerebral artery

MSCs

mesenchymal stromal cells

MVs

microvesicles

MVBs

multivesicular bodies

NIHSS

National Institutes of Health Stroke Scale

PBS

phosphate buffer solution

PC12 cells

rat adrenal pheochromocytoma cells

PEG

polyethylene glycol

PS

phosphatidylserine

RT

room temperature

SN

supernatant

Tsg101

tumor susceptibility gene 101

UC

ultracentrifugation

WB

western-blotting

References

1 

Moskowitz MA, Lo EH and Iadecola C: The science of stroke: Mechanisms in search of treatments. Neuron. 67:181–198. 2010. View Article : Google Scholar : PubMed/NCBI

2 

Demaerschalk BM, Kleindorfer DO, Adeoye OM, Demchuk AM, Fugate JE, Grotta JC, Khalessi AA, Levy EI, Palesch YY, Prabhakaran S, et al: Scientific rationale for the inclusion and exclusion criteria for intravenous alteplase in acute ischemic stroke: A statement for healthcare professionals from the american heart association/american stroke association. Stroke. 47:581–641. 2016. View Article : Google Scholar : PubMed/NCBI

3 

Grunwald IQ, Wakhloo AK, Walter S, Molyneux AJ, Byrne JV, Nagel S, Kühn AL, Papadakis M, Fassbender K, Balami JS, et al: Endovascular stroke treatment today. AJNR Am J Neuroradiol. 32:238–243. 2011. View Article : Google Scholar : PubMed/NCBI

4 

Tu J, Yang F, Wan J, Liu Y, Zhang J, Wu B, Liu Y, Zeng S and Wang L: Light-controlled astrocytes promote human mesenchymal stem cells toward neuronal differentiation and improve the neurological deficit in stroke rats. Glia. 62:106–121. 2014. View Article : Google Scholar : PubMed/NCBI

5 

Wei ZZ, Gu X, Ferdinand A, Lee JH, Ji X, Ji XM, Yu SP and Wei L: Intranasal delivery of bone marrow mesenchymal stem cells improved neurovascular regeneration and rescued neuropsychiatric deficits after neonatal stroke in rats. Cell Transplant. 24:391–402. 2015. View Article : Google Scholar : PubMed/NCBI

6 

Zhang ZG and Chopp M: Exosomes in stroke pathogenesis and therapy. J Clin Invest. 126:1190–1197. 2016. View Article : Google Scholar : PubMed/NCBI

7 

Lopatina T, Bruno S, Tetta C, Kalinina N, Porta M and Camussi G: Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential. Cell Commun Signal. 12:262014. View Article : Google Scholar : PubMed/NCBI

8 

Tan X, Gong YZ, Wu P, Liao DF and Zheng XL: Mesenchymal stem cell-derived microparticles: A promising therapeutic strategy. Int J Mol Sci. 15:14348–14363. 2014. View Article : Google Scholar : PubMed/NCBI

9 

Li Y, Hu G and Cheng Q: Implantation of human umbilical cord mesenchymal stem cells for ischemic stroke: Perspectives and challenges. Front Med. 9:20–29. 2015. View Article : Google Scholar : PubMed/NCBI

10 

Marquez-Curtis LA, Janowska-Wieczorek A, McGann LE and Elliott JA: Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects. Cryobiology. 71:181–197. 2015. View Article : Google Scholar : PubMed/NCBI

11 

Olson AL and McNiece IK: Novel clinical uses for cord blood derived mesenchymal stromal cells. Cytotherapy. 17:796–802. 2015. View Article : Google Scholar : PubMed/NCBI

12 

Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S and Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science. 284:143–147. 1999. View Article : Google Scholar : PubMed/NCBI

13 

Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, et al: Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 418:41–49. 2002. View Article : Google Scholar : PubMed/NCBI

14 

Hao L, Zou Z, Tian H, Zhang Y, Zhou H and Liu L: Stem cell-based therapies for ischemic stroke. Biomed Res Int. 2014:4687482014. View Article : Google Scholar : PubMed/NCBI

15 

Bernardo ME and Fibbe WE: Mesenchymal stromal cells: Sensors and switchers of inflammation. Cell Stem Cell. 13:392–402. 2013. View Article : Google Scholar : PubMed/NCBI

16 

Gornostaeva A, Andreeva E and Buravkova L: Factors governing the immunosuppressive effects of multipotent mesenchymal stromal cells in vitro. Cytotechnology. 68:565–577. 2016. View Article : Google Scholar : PubMed/NCBI

17 

Khubutiya MS, Vagabov AV, Temnov AA and Sklifas AN: Paracrine mechanisms of proliferative, anti-apoptotic and anti-inflammatory effects of mesenchymal stromal cells in models of acute organ injury. Cytotherapy. 16:579–585. 2014. View Article : Google Scholar : PubMed/NCBI

18 

Bang OY, Lee JS, Lee PH and Lee G: Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol. 57:874–882. 2005. View Article : Google Scholar : PubMed/NCBI

19 

Lee JS, Hong JM, Moon GJ, Lee PH, Ahn YH and Bang OY: STARTING collaborators: A long-term follow-up study of intravenous autologous mesenchymal stem cell transplantation in patients with ischemic stroke. Stem Cells. 28:1099–1106. 2010. View Article : Google Scholar : PubMed/NCBI

20 

Honmou O, Houkin K, Matsunaga T, Niitsu Y, Ishiai S, Onodera R, Waxman SG and Kocsis JD: Intravenous administration of auto serum-expanded autologous mesenchymal stem cells in stroke. Brain. 134:1790–1807. 2011. View Article : Google Scholar : PubMed/NCBI

21 

Bhasin A, Srivastava MV, Kumaran SS, Mohanty S, Bhatia R, Bose S, Gaikwad S, Garg A and Airan B: Autologous mesenchymal stem cells in chronic stroke. Cerebrovasc Dis Extra. 1:93–104. 2011. View Article : Google Scholar : PubMed/NCBI

22 

Jiang Y, Zhu W, Zhu J, Wu L, Xu G and Liu X: Feasibility of delivering mesenchymal stem cells via catheter to the proximal end of the lesion artery in patients with stroke in the territory of the middle cerebral artery. Cell Transplant. 22:2291–2298. 2013. View Article : Google Scholar : PubMed/NCBI

23 

Bhasin A, Srivastava MV, Mohanty S, Bhatia R, Kumaran SS and Bose S: Stem cell therapy: A clinical trial of stroke. Clin Neurol Neurosurg. 115:1003–1008. 2013. View Article : Google Scholar : PubMed/NCBI

24 

Steinberg GK, Kondziolka D, Wechsler LR, Lunsford LD, Coburn ML, Billigen JB, Kim AS, Johnson JN, Bates D, King B, et al: Clinical outcomes of transplanted modified bone marrow-derived mesenchymal stem cells in stroke: A phase 1/2a study. Stroke. 47:1817–1824. 2016. View Article : Google Scholar : PubMed/NCBI

25 

Toma C, Wagner WR, Bowry S, Schwartz A and Villanueva F: Fate of culture-expanded mesenchymal stem cells in the microvasculature: In vivo observations of cell kinetics. Circ Res. 104:398–402. 2009. View Article : Google Scholar : PubMed/NCBI

26 

Lin YC, Ko TL, Shih YH, Lin MY, Fu TW, Hsiao HS, Hsu JY and Fu YS: Human umbilical mesenchymal stem cells promote recovery after ischemic stroke. Stroke. 42:2045–2053. 2011. View Article : Google Scholar : PubMed/NCBI

27 

Vu Q, Xie K, Eckert M, Zhao W and Cramer SC: Meta-analysis of preclinical studies of mesenchymal stromal cells for ischemic stroke. Neurology. 82:1277–1286. 2014. View Article : Google Scholar : PubMed/NCBI

28 

Caplan AI and Correa D: The MSC: An injury drugstore. Cell Stem Cell. 9:11–15. 2011. View Article : Google Scholar : PubMed/NCBI

29 

Basso M and Bonetto V: Extracellular vesicles and a novel form of communication in the brain. Front Neurosci. 10:1272016. View Article : Google Scholar : PubMed/NCBI

30 

Yu B, Zhang X and Li X: Exosomes derived from mesenchymal stem cells. Int J Mol Sci. 15:4142–4157. 2014. View Article : Google Scholar : PubMed/NCBI

31 

Doeppner TR, Herz J, Görgens A, Schlechter J, Ludwig AK, Radtke S, de Miroschedji K, Horn PA, Giebel B and Hermann DM: Extracellular vesicles improve post-stroke neuroregeneration and prevent postischemic immunosuppression. Stem Cells Transl Med. 4:1131–1143. 2015. View Article : Google Scholar : PubMed/NCBI

32 

Xin H, Li Y and Chopp M: Exosomes/miRNAs as mediating cell-based therapy of stroke. Front Cell Neurosci. 8:3772014. View Article : Google Scholar : PubMed/NCBI

33 

Couzin J: Cell biology: The ins and outs of exosomes. Science. 308:1862–1863. 2005. View Article : Google Scholar : PubMed/NCBI

34 

van der Pol E, Boing AN, Harrison P, Sturk A and Nieuwland R: Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev. 64:676–705. 2012. View Article : Google Scholar : PubMed/NCBI

35 

Yáñez-Mó M, Siljander PR, Andreu Z, Zavec AB, Borràs FE, Buzas EI, Buzas K, Casal E, Cappello F, Carvalho J, et al: Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 4:270662015. View Article : Google Scholar : PubMed/NCBI

36 

Raposo G and Stoorvogel W: Extracellular vesicles: Exosomes, microvesicles, and friends. J Cell Biol. 200:373–383. 2013. View Article : Google Scholar : PubMed/NCBI

37 

Katakowski M, Buller B, Zheng X, Lu Y, Rogers T, Osobamiro O, Shu W, Jiang F and Chopp M: Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. Cancer Lett. 335:201–204. 2013. View Article : Google Scholar : PubMed/NCBI

38 

Zhang HG and Grizzle WE: Exosomes: A novel pathway of local and distant intercellular communication that facilitates the growth and metastasis of neoplastic lesions. Am J Pathol. 184:28–41. 2014. View Article : Google Scholar : PubMed/NCBI

39 

Fais S, O'Driscoll L, Borras FE, Buzas E, Camussi G, Cappello F, Carvalho J, da Silva Cordeiro A, Del Portillo H, El Andaloussi S, et al: Evidence-based clinical use of nanoscale extracellular vesicles in nanomedicine. ACS Nano. 10:3886–3899. 2016. View Article : Google Scholar : PubMed/NCBI

40 

Ratajczak MZ: The emerging role of microvesicles in cellular therapies for organ/tissue regeneration. Nephrol Dial Transplant. 26:1453–1456. 2011. View Article : Google Scholar : PubMed/NCBI

41 

Reiner AT, Witwer KW, van Balkom BWM, de Beer J, Brodie C, Corteling RL, Gabrielsson S, Gimona M, Ibrahim AG, de Kleijn D, et al: Concise review: Developing best-practice models for the therapeutic use of extracellular vesicles. Stem Cells Transl Med. 6:1730–1739. 2017. View Article : Google Scholar : PubMed/NCBI

42 

Antonyak MA and Cerione RA: Emerging picture of the distinct traits and functions of microvesicles and exosomes. Proc Natl Acad Sci USA. 112:3589–3590. 2015.PubMed/NCBI

43 

Biancone L, Bruno S, Deregibus MC, Tetta C and Camussi G: Therapeutic potential of mesenchymal stem cell-derived microvesicles. Nephrol Dial Transplant. 27:3037–3042. 2012. View Article : Google Scholar : PubMed/NCBI

44 

Lai CP and Breakefield XO: Role of exosomes/microvesicles in the nervous system and use in emerging therapies. Front Physiol. 3:2282012. View Article : Google Scholar : PubMed/NCBI

45 

Konala VB, Mamidi MK, Bhonde R, Das AK, Pochampally R and Pal R: The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration. Cytotherapy. 18:13–24. 2016. View Article : Google Scholar : PubMed/NCBI

46 

Gyorgy B, Hung ME, Breakefield XO and Leonard JN: Therapeutic applications of extracellular vesicles: Clinical promise and open questions. Annu Rev Pharmacol Toxicol. 55:439–464. 2015. View Article : Google Scholar : PubMed/NCBI

47 

Kanninen KM, Bister N, Koistinaho J and Malm T: Exosomes as new diagnostic tools in CNS diseases. Biochim Biophys Acta. 1862:403–410. 2016. View Article : Google Scholar : PubMed/NCBI

48 

Ban LA, Shackel NA and McLennan SV: Extracellular vesicles: A new frontier in biomarker discovery for non-alcoholic fatty liver disease. Int J Mol Sci. 17:3762016. View Article : Google Scholar : PubMed/NCBI

49 

Lai FW, Lichty BD and Bowdish DM: Microvesicles: Ubiquitous contributors to infection and immunity. J Leukoc Biol. 97:237–245. 2015. View Article : Google Scholar : PubMed/NCBI

50 

Witwer KW, Buzàs EI, Bemis LT, Bora A, Lässer C, Lötvall J, Hoen Nolte-'t EN, Piper MG, Sivaraman S, Skog J, et al: Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2:203602013. View Article : Google Scholar

51 

Zlotogorski-Hurvitz A, Dayan D, Chaushu G, Korvala J, Salo T, Sormunen R and Vered M: Human saliva-derived exosomes: Comparing methods of isolation. J Histochem Cytochem. 63:181–189. 2015. View Article : Google Scholar : PubMed/NCBI

52 

Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O and Hendrix A: The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles. 3:248582014. View Article : Google Scholar

53 

Sàenz-Cuesta M, Osorio-Querejeta I and Otaegui D: Extracellular vesicles in multiple sclerosis: What are they telling us? Front Cell Neurosci. 8:1002014. View Article : Google Scholar : PubMed/NCBI

54 

György B, Módos K, Pàllinger E, Pálóczi K, Pásztói M, Misják P, Deli MA, Sipos A, Szalai A, Voszka I, et al: Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters. Blood. 117:e39–e48. 2011. View Article : Google Scholar : PubMed/NCBI

55 

Shin H, Han C, Labuz JM, Kim J, Kim J, Cho S, Gho YS, Takayama S and Park J: High-yield isolation of extracellular vesicles using aqueous two-phase system. Sci Rep. 5:131032015. View Article : Google Scholar : PubMed/NCBI

56 

Lacroix R, Robert S, Poncelet P and Dignat-George F: Overcoming limitations of microparticle measurement by flow cytometry. Semin Thromb Hemost. 36:807–818. 2010. View Article : Google Scholar : PubMed/NCBI

57 

van der Vlist EJ, Hoen Nolte-'t EN, Stoorvogel W, Arkesteijn GJ and Wauben MH: Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protoc. 7:1311–1326. 2012. View Article : Google Scholar : PubMed/NCBI

58 

Sokolova V, Ludwig AK, Hornung S, Rotan O, Horn PA, Epple M and Giebel B: Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces. 87:146–150. 2011. View Article : Google Scholar : PubMed/NCBI

59 

Szatanek R, Baj-Krzyworzeka M, Zimoch J, Lekka M, Siedlar M and Baran J: The methods of choice for extracellular vesicles (EVs) characterization. Int J Mol Sci. 18:pii: E1153. 2017. View Article : Google Scholar : PubMed/NCBI

60 

Mokarizadeh A, Delirezh N, Morshedi A, Mosayebi G, Farshid AA and Mardani K: Microvesicles derived from mesenchymal stem cells: Potent organelles for induction of tolerogenic signaling. Immunol Lett. 147:47–54. 2012. View Article : Google Scholar : PubMed/NCBI

61 

Chen TS, Lai RC, Lee MM, Choo AB, Lee CN and Lim SK: Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs. Nucleic Acids Res. 38:215–224. 2010. View Article : Google Scholar : PubMed/NCBI

62 

Wang J, Zhong Y, Ma X, Xiao X, Cheng C, Chen Y, Iwuchukwu I, Gaines KJ, Zhao B, Liu S, et al: Analyses of endothelial cells and endothelial progenitor cells released Microvesicles by using microbead and Q-dot based nanoparticle tracking analysis. Sci Rep. 6:246792016. View Article : Google Scholar : PubMed/NCBI

63 

Théry C, Amigorena S, Raposo G and Clayton A: Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3: Unit 3.22. 2006. View Article : Google Scholar

64 

Jayachandran M, Miller VM, Heit JA and Owen WG: Methodology for isolation, identification and characterization of microvesicles in peripheral blood. J Immunol Methods. 375:207–214. 2012. View Article : Google Scholar : PubMed/NCBI

65 

Li Y, Liu Z, Xin H and Chopp M: The role of astrocytes in mediating exogenous cell-based restorative therapy for stroke. Glia. 62:1–16. 2014. View Article : Google Scholar : PubMed/NCBI

66 

Eirin A, Riester SM, Zhu XY, Tang H, Evans JM, O'Brien D, van Wijnen AJ and Lerman LO: MicroRNA and mRNA cargo of extracellular vesicles from porcine adipose tissue-derived mesenchymal stem cells. Gene. 551:55–64. 2014. View Article : Google Scholar : PubMed/NCBI

67 

Phinney DG, Di Giuseppe M, Njah J, Sala E, Shiva S, St Croix CM, Stolz DB, Watkins SC, Di YP, Leikauf GD, et al: Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nat Commun. 6:84722015. View Article : Google Scholar : PubMed/NCBI

68 

Fleury A, Martinez MC and Le Lay S: Extracellular vesicles as therapeutic tools in cardiovascular diseases. Front Immunol. 5:3702014. View Article : Google Scholar : PubMed/NCBI

69 

Taylor DD and Gercel-Taylor C: Exosomes/microvesicles: Mediators of cancer-associated immunosuppressive microenvironments. Semin Immunopathol. 33:441–454. 2011. View Article : Google Scholar : PubMed/NCBI

70 

Kanada M, Bachmann MH, Hardy JW, Frimannson DO, Bronsart L, Wang A, Sylvester MD, Schmidt TL, Kaspar RL, Butte MJ, et al: Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA. 112:E1433–E1442. 2015.PubMed/NCBI

71 

Turchinovich A and Cho WC: The origin, function and diagnostic potential of extracellular microRNA in human body fluids. Front Genet. 5:302014. View Article : Google Scholar : PubMed/NCBI

72 

Liu RT, Wang SW and Liu J: Exosomes: The novel vehicles for intercellular communication. Prog Biochem Biophys. 40:719–727. 2013.

73 

György B, Szabó TG, Pàsztói M, Pál Z, Misják P, Aradi B, László V, Pállinger E, Pap E, Kittel A, et al: Membrane vesicles, current state-of-the-art: Emerging role of extracellular vesicles. Cell Mol Life Sci. 68:2667–2688. 2011. View Article : Google Scholar : PubMed/NCBI

74 

Thery C, Ostrowski M and Segura E: Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 9:581–593. 2009. View Article : Google Scholar : PubMed/NCBI

75 

Sheldon H, Heikamp E, Turley H, Dragovic R, Thomas P, Oon CE, Leek R, Edelmann M, Kessler B, Sainson RC, et al: New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes. Blood. 116:2385–2394. 2010. View Article : Google Scholar : PubMed/NCBI

76 

Janowska-Wieczorek A, Majka M, Kijowski J, Baj-Krzyworzeka M, Reca R, Turner AR, Ratajczak J, Emerson SG, Kowalska MA and Ratajczak MZ: Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment. Blood. 98:3143–3149. 2001. View Article : Google Scholar : PubMed/NCBI

77 

Mause SF and Weber C: Microparticles: Protagonists of a novel communication network for intercellular information exchange. Circ Res. 107:1047–1057. 2010. View Article : Google Scholar : PubMed/NCBI

78 

Rozmyslowicz T, Majka M, Kijowski J, Murphy SL, Conover DO, Poncz M, Ratajczak J, Gaulton GN and Ratajczak MZ: Platelet- and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV. AIDS. 17:33–42. 2003. View Article : Google Scholar : PubMed/NCBI

79 

Desrochers LM, Bordeleau F, Reinhart-King CA, Cerione RA and Antonyak MA: Microvesicles provide a mechanism for intercellular communication by embryonic stem cells during embryo implantation. Nat Commun. 7:119582016. View Article : Google Scholar : PubMed/NCBI

80 

Yong PJ, Koh CH and Shim WS: Endothelial microparticles: Missing link in endothelial dysfunction? Eur J Prev Cardiol. 20:496–512. 2013. View Article : Google Scholar : PubMed/NCBI

81 

Liu Y, Huang W, Zhang R, Wu J, Li L and Tang Y: Proteomic analysis of TNF-α-activated endothelial cells and endothelial microparticles. Mol Med Rep. 7:318–326. 2013. View Article : Google Scholar : PubMed/NCBI

82 

de Jong OG, Verhaar MC, Chen Y, Vader P, Gremmels H, Posthuma G, Schiffelers RM, Gucek M and van Balkom BW: Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes. J Extracell Vesicles. 1:183962012. View Article : Google Scholar

83 

Mitra S, Wewers MD and Sarkar A: Mononuclear phagocyte-derived microparticulate caspase-1 induces pulmonary vascular endothelial cell injury. PLoS One. 10:e01456072015. View Article : Google Scholar : PubMed/NCBI

84 

Katsman D, Stackpole EJ, Domin DR and Farber DB: Embryonic stem cell-derived microvesicles induce gene expression changes in Muller cells of the retina. PLoS One. 7:e504172012. View Article : Google Scholar : PubMed/NCBI

85 

Ratajczak J, Miekus K, Kucia M, Zhang J, Reca R, Dvorak P and Ratajczak MZ: Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: Evidence for horizontal transfer of mRNA and protein delivery. Leukemia. 20:847–856. 2006. View Article : Google Scholar : PubMed/NCBI

86 

Yeo RW, Lai RC, Zhang B, Tan SS, Yin Y, Teh BJ and Lim SK: Mesenchymal stem cell: An efficient mass producer of exosomes for drug delivery. Adv Drug Deliv Rev. 65:336–341. 2013. View Article : Google Scholar : PubMed/NCBI

87 

Lai RC, Tan SS, Teh BJ, Sze SK, Arslan F, de Kleijn DP, Choo A and Lim SK: Proteolytic potential of the MSC exosome proteome: Implications for an exosome-mediated delivery of therapeutic proteasome. Int J Proteomics. 2012:9719072012. View Article : Google Scholar : PubMed/NCBI

88 

Collino F, Bruno S, Incarnato D, Dettori D, Neri F, Provero P, Pomatto M, Oliviero S, Tetta C, Quesenberry PJ and Camussi G: AKI recovery induced by mesenchymal stromal cell-derived extracellular vesicles carrying MicroRNAs. J Am Soc Nephrol. 26:2349–2360. 2015. View Article : Google Scholar : PubMed/NCBI

89 

Koniusz S, Andrzejewska A, Muraca M, Srivastava AK, Janowski M and Lukomska B: Extracellular vesicles in physiology, pathology, and therapy of the immune and central nervous system, with focus on extracellular vesicles derived from mesenchymal stem cells as therapeutic tools. Front Cell Neurosci. 10:1092016. View Article : Google Scholar : PubMed/NCBI

90 

Tomasoni S, Longaretti L, Rota C, Morigi M, Conti S, Gotti E, Capelli C, Introna M, Remuzzi G and Benigni A: Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem cells. Stem Cells Dev. 22:772–780. 2013. View Article : Google Scholar : PubMed/NCBI

91 

Dooner MS, Aliotta JM, Pimentel J, Dooner GJ, Abedi M, Colvin G, Liu Q, Weier HU, Johnson KW and Quesenberry PJ: Conversion potential of marrow cells into lung cells fluctuates with cytokine-induced cell cycle. Stem Cells Dev. 17:207–219. 2008. View Article : Google Scholar : PubMed/NCBI

92 

Pegtel DM, Peferoen L and Amor S: Extracellular vesicles as modulators of cell-to-cell communication in the healthy and diseased brain. Philos Trans R Soc Lond B Biol Sci. 369:pii: 20130516. 2014. View Article : Google Scholar : PubMed/NCBI

93 

Lozito TP and Tuan RS: Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors. J Cell Mol Med. 18:2372–2384. 2014. View Article : Google Scholar : PubMed/NCBI

94 

Xin H, Li Y, Liu Z, Wang X, Shang X, Cui Y, Zhang ZG and Chopp M: MiR-133b promotes neural plasticity and functional recovery after treatment of stroke with multipotent mesenchymal stromal cells in rats via transfer of exosome-enriched extracellular particles. Stem Cells. 31:2737–2746. 2013. View Article : Google Scholar : PubMed/NCBI

95 

Hao P, Liang Z, Piao H, Ji X, Wang Y, Liu Y, Liu R and Liu J: Conditioned medium of human adipose-derived mesenchymal stem cells mediates protection in neurons following glutamate excitotoxicity by regulating energy metabolism and GAP-43 expression. Metab Brain Dis. 29:193–205. 2014. View Article : Google Scholar : PubMed/NCBI

96 

Lin SS, Zhu B, Guo ZK, Huang GZ, Wang Z, Chen J, Wei XJ and Li Q: Bone marrow mesenchymal stem cell-derived microvesicles protect rat pheochromocytoma PC12 cells from glutamate-induced injury via a PI3K/Akt dependent pathway. Neurochem Res. 39:922–931. 2014. View Article : Google Scholar : PubMed/NCBI

97 

Xin H, Li Y, Buller B, Katakowski M, Zhang Y, Wang X, Shang X, Zhang ZG and Chopp M: Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth. Stem Cells. 30:1556–1564. 2012. View Article : Google Scholar : PubMed/NCBI

98 

Xin H, Li Y, Cui Y, Yang JJ, Zhang ZG and Chopp M: Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats. J Cereb Blood Flow Metab. 33:1711–1715. 2013. View Article : Google Scholar : PubMed/NCBI

99 

Mousavinejad M, Andrews PW and Shoraki EK: Current biosafety considerations in stem cell therapy. Cell J. 18:281–287. 2016.PubMed/NCBI

100 

Wong RS: Mesenchymal stem cells: Angels or demons? J Biomed Biotechnol. 2011:4595102011. View Article : Google Scholar : PubMed/NCBI

101 

Jeong JO, Han JW, Kim JM, Cho HJ, Park C, Lee N, Kim DW and Yoon YS: Malignant tumor formation after transplantation of short-term cultured bone marrow mesenchymal stem cells in experimental myocardial infarction and diabetic neuropathy. Circ Res. 108:1340–1347. 2011. View Article : Google Scholar : PubMed/NCBI

102 

Shao H, Chung J, Balaj L, Charest A, Bigner DD, Carter BS, Hochberg FH, Breakefield XO, Weissleder R and Lee H: Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy. Nat Med. 18:1835–1840. 2012. View Article : Google Scholar : PubMed/NCBI

103 

Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S and Wood MJ: Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 29:341–345. 2011. View Article : Google Scholar : PubMed/NCBI

104 

Lener T, Gimona M, Aigner L, Börger V, Buzas E, Camussi G, Chaput N, Chatterjee D, Court FA, Del Portillo HA, et al: Applying extracellular vesicles based therapeutics in clinical trials-an ISEV position paper. J Extracell Vesicles. 4:300872015. View Article : Google Scholar : PubMed/NCBI

105 

Chen TS, Arslan F, Yin Y, Tan SS, Lai RC, Choo AB, Padmanabhan J, Lee CN, de Kleijn DP and Lim SK: Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs. J Transl Med. 9:472011. View Article : Google Scholar : PubMed/NCBI

106 

Salido-Guadarrama I, Romero-Cordoba S, Peralta-Zaragoza O, Hidalgo-Miranda A and Rodríguez-Dorantes M: MicroRNAs transported by exosomes in body fluids as mediators of intercellular communication in cancer. Onco Targets Ther. 7:1327–1338. 2014.PubMed/NCBI

107 

Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, Ricciardi-Castagnoli P, Raposo G and Amigorena S: Eradication of established murine tumors using a novel cell-free vaccine: Dendritic cell-derived exosomes. Nat Med. 4:594–600. 1998. View Article : Google Scholar : PubMed/NCBI

108 

Escudier B, Dorval T, Chaput N, André F, Caby MP, Novault S, Flament C, Leboulaire C, Borg C, Amigorena S, et al: Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: Results of thefirst phase I clinical trial. J Transl Med. 3:102005. View Article : Google Scholar : PubMed/NCBI

109 

Otero-Ortega L, Laso-García F, Gómez-de Frutos MD, Rodríguez-Frutos B, Pascual-Guerra J, Fuentes B, Díez-Tejedor E and Gutiérrez-Fernández M: White matter repair after extracellular vesicles administration in an experimental animal model of subcortical stroke. Sci Rep. 7:444332017. View Article : Google Scholar : PubMed/NCBI

110 

Xin H, Wang F, Li Y, Lu QE, Cheung WL, Zhang Y, Zhang ZG and Chopp M: Secondary release of exosomes from astrocytes contributes to the increase in neural plasticity and improvement of functional recovery after stroke in rats treated with exosomes harvested from MicroRNA 133b-overexpressing multipotent mesenchymal stromal cells. Cell Transplant. 26:243–257. 2017. View Article : Google Scholar : PubMed/NCBI

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May-2018
Volume 15 Issue 5

Print ISSN: 1792-0981
Online ISSN:1792-1015

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Copy and paste a formatted citation
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Spandidos Publications style
Li Y, Cheng Q, Hu G, Deng T, Wang Q, Zhou J and Su X: Extracellular vesicles in mesenchymal stromal cells: A novel therapeutic strategy for stroke (Review). Exp Ther Med 15: 4067-4079, 2018.
APA
Li, Y., Cheng, Q., Hu, G., Deng, T., Wang, Q., Zhou, J., & Su, X. (2018). Extracellular vesicles in mesenchymal stromal cells: A novel therapeutic strategy for stroke (Review). Experimental and Therapeutic Medicine, 15, 4067-4079. https://doi.org/10.3892/etm.2018.5993
MLA
Li, Y., Cheng, Q., Hu, G., Deng, T., Wang, Q., Zhou, J., Su, X."Extracellular vesicles in mesenchymal stromal cells: A novel therapeutic strategy for stroke (Review)". Experimental and Therapeutic Medicine 15.5 (2018): 4067-4079.
Chicago
Li, Y., Cheng, Q., Hu, G., Deng, T., Wang, Q., Zhou, J., Su, X."Extracellular vesicles in mesenchymal stromal cells: A novel therapeutic strategy for stroke (Review)". Experimental and Therapeutic Medicine 15, no. 5 (2018): 4067-4079. https://doi.org/10.3892/etm.2018.5993