Extracellular Vesicles as an Emerging Paradigm of Cell-to-Cell Communication in Stem Cell Biology

Cell-to-cell communication is the fundamental mechanism that enables multicellular organisms to maintain tissue homeostasis and normal cellular functions. Recent studies have demonstrated that extracellular vesicles (EVs), including exosomes and microvesicles, may act as a crucial mediator of intercellular communication. It is well-established that EVs are small membrane vesicles secreted from numerous cell types, including immune cells, tumor cells, and stem cells [1-3]. In addition, EVs have been found in various body fluids, such as blood, saliva, and urine [4,5]. EVs are secreted either in a constitutive or regulated manner. For instance, a number of tumor cells release EVs constitutively [6], whereas primary B cells secrete EVs when stimulated with potent activation signals, such as cytokine [7].


Introduction
Cell-to-cell communication is the fundamental mechanism that enables multicellular organisms to maintain tissue homeostasis and normal cellular functions. Recent studies have demonstrated that extracellular vesicles (EVs), including exosomes and microvesicles, may act as a crucial mediator of intercellular communication. It is well-established that EVs are small membrane vesicles secreted from numerous cell types, including immune cells, tumor cells, and stem cells [1][2][3]. In addition, EVs have been found in various body fluids, such as blood, saliva, and urine [4,5]. EVs are secreted either in a constitutive or regulated manner. For instance, a number of tumor cells release EVs constitutively [6], whereas primary B cells secrete EVs when stimulated with potent activation signals, such as cytokine [7].
Initially, EVs became of interest because they are implicated in antigen presentation [8]. Thus, many studies have focused on the potential therapeutic effect of EVs as a cell-free vaccine for human malignancies [9]. More recently, the findings that EVs harbor bioactive molecules, such as proteins, lipids, and nucleic acids have shed new light on the role of EVs as a paracrine mediator of cell-to-cell communication. In particular, EVs contain genetic materials, such as mRNAs and microRNAs (miRNAs), enabling exchange of information between cells [10]. It has been documented that a number of cell types can epigenetically modulate their neighboring cells by transferring genetic information via EVs [11]. The message delivered by EVs varies, depending on the pathophysiological state of the cell of origin [12]. A recent study showed that hepatocellular carcinoma cell (HCC)-derived EVs contained a selected group of miRNAs, altering the behavior of recipient HCC cells [13]. In stem cell biology, the discovery of EVmediated intercellular communication has spurred research on the therapeutic opportunities of stem cell-derived EVs in regenerative medicine.

Biogenesis of EVs
There is accumulating evidence that vesicles released from cells are heterogeneous in terms of biogenesis and size [14]. The first type of vesicles, known as exosomes, originates from multivesicular bodies and fuse with the plasma membrane, which leads to secretion to the extracellular space. Exosomes range from 40 nm to 100 nm in size and can be characterized by the expression of tetraspanins such as CD9, CD63, and CD81 [15]. Another class of vesicles is referred to as microvesicles which are distinguished from exosomes by the mechanisms of biogenesis. Microvesicles are produced by direct budding of the plasma membrane and this relies on dynamic interplay between phospholipid redistribution and cytoskeleton activation. These shedding vesicles are known to range from 100 nm to 1 μm in size [12]. However, the exact biogenesis and characterization of two different types of EVs remain to be explored. The traditional method employed to purify EVs is ultracentrifugation combined with sucrose density gradients. In addition, EVs isolation kits using EVs precipitation solutions have been developed. More detailed review has recently been published [16].
Exosomes secreted by the prostate was used as reservoirs of tumorassociated proteins for prostate cancer detection and progression [20]. Moreover, it has been documented that EVs harbor lipids, such as eicosanoids, fatty acids, cholesterol, and lipid-related enzymes [21]. Recent studies reported that EVs also contain DNA. Glioblastoma and astrocyte cells secreted microvesicles carrying mitochondrial DNA [22]. In addition, a set of mRNAs and miRNAs have been identified in EVs derived from numerous cell types, such as human renal cancer stem cells [23], tumor-associated macrophages [24], and adipocytes [25]. Based on these findings, it has been suggested that circulating miRNAs probably transported by EVs in cancer patients can serve as novel diagnostic markers. EV-encapsulated miRNAs are relatively stable since they are protected from extracellular degrading enzymes [26,27].

Cell-to-cell communication through EVs
The discoveries that EVs harbor bioactive contents, such as proteins and nucleic acids raise the possibility that EVs might play a significant role in cell-to-cell communication. Recent works indicated that EVs are able to convey proteins to the recipient cells. Active Wnt proteins secreted on exosomes activated the Wnt signaling pathway in target cells [28]. EGFR-bound exosomes induced tumor antigenspecific regulatory T cells [29]. More intriguingly, one elegant study elucidated the role of exosomes as a vehicle for exchange of genetic information [30]. The author's isolated exosomes from a mouse mast cell line MC/9 and a human mast cell line HMC-1, and primary bone marrow-derived mouse mast cells. Using microarray assessments, they identified 1,300 mRNAs and 120 miRNAs in these mast cell-derived exosomes. Surprisingly, many of them were exosome-specific as they were not detectable in the cytoplasm of the donor cell. The results proved that mRNAs in exosomes were intact and functional and they were transferable to other mouse and human mast cells. More importantly, their data showed that once mouse exosomal mRNAs were transferred to human mast cells, new mouse protein could be synthesized in human mast cells, suggesting that genetic materials shuttled by vesicles modify the behavior of the surrounding cells. Subsequently, a number of studies investigated the role of EVs in the context of immune responses, tumor development, and stem cell biology. Indeed, significant evidence has demonstrated that tumorderived EVs have detrimental effects on the immune response, thus promoting the immunosuppressive microenvironment for their survival [31].

Stem-Derived EVs as a Paracrine Mediator Embryonic stem cell-derived EVs
Ratajczak et al. [3] were the first to suggest that EVs derived from stem cells exert profound effects on the microenvironment by transferring stem cell-specific proteins and mRNAs. In their study, the authors demonstrated that microvesicles derived from embryonic stem cells (ESCs) contained Wnt-3 and mRNAs implicated in pluripotent transcription factors. These molecular components were transferred to the neighboring cells, thus reprogramming hematopoietic progenitors. In another study, ESC-derived microvesicles were engineered to carry green fluorescent protein (GFP) and these modified microvesicles fused with other ESCs, shuttling their GFP [32]. In addition, it was found that miRNAs were enriched in ESC-derived microvesicles and a subset of miRNAs was transferred to mouse embryonic fibroblasts. Recently, Katsman et al. [33] reported that microvesicles derived from ESCs induced de-differentiation and alterations in gene expression of Müller cells of the retina. They performed microarrays of Müller cells treated with ESC-derived microvesicles compared to untreated Müller cells. Müller cells incubated with ESC-derived microvesicles showed the up-regulation of genes and miRNAs associated with cellular proliferation and induction of pluripotency and the down-regulation of genes important to differentiation and cell cycle arrest.

Mesenchymal stem cell-derived EVs
Collino et al. [34] found that microvesicles generated by human mesenchymal stem cells (MSCs) and human liver stem cells harbored unique patterns of miRNAs associated with ribonucleoproteins known to be responsible for the intracellular trafficking of RNAs. They also contained proteins involved in the transport and stability of mRNAs such as Staufen1, Staufen2. In another study, it was found that specific miRNAs, such as hsa-let-7b and hsa-let-7g were present as their precursor forms in MSC-derived microvesicles [35]. These studies suggest that a dynamic regulation of RNA compartmentalization occurs during the biogenesis of stem cell-derived EVs and stem cells may modulate their neighboring cells by delivering RNA contents.

Endothelial progenitor cell-derived EVs
It has been suggested that the molecular contents present in EVs are specific to the donor cells. Deregibus et al. [36] reported that microvesicles secreted from endothelial progenitor cells (EPCs) enhanced angiogenesis. The data indicated that EPC-derived microvesicles were taken up by endothelial cells, which resulted in enhancement of endothelial cell survival, proliferation and tube formation. Microarray analysis and quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) showed that EPC-derived microvesicles conveyed mRNAs involved in the PI3K/AKT signaling pathway, triggering an angiogenic program in endothelial cells. More recently, the same group demonstrated that miR-126 and miR-296, known as pro-angiogenic miRNAs, were enriched in EPC-derived microvesicles and these might contribute to the up-regulation of proangiogenic pathways in the recipient cells [37,38].

Cancer stem cell-derived EVs
A recent work showed that microvesicles derived from cancer stem cells (CSCs) act as a transporter for exchange of information between tumors and their surrounding cells, thus engendering a favorable microenvironment for cancer progression [23]. In this study, the authors found that only CD105-positive CSC-derived microvesicles activated an angiogenic program in normal human endothelial cells, stimulating their growth and vessel formation. Moreover, treating SCID mice with CSC-derived microvesicles significantly enhanced lung metastases. The molecular characterization of CSC-derived microvesicles displayed a set of pro-angiogenic mRNAs and miRNAs implicated in tumor development and metastases.

Application of Stem Cell-Derived EVs Stem cell-derived EVs and tissue repair
There is increasing evidence that stem cell-derived EVs contribute to tissue remodeling and have profound effects on the recipient cells in a paracrine manner (Figure 1). In this context, it has been suggested that EVs released from stem cells play a critical role in exchange of information between stem cells and tissue-injured cells [12]. Thus, the potential application of stem cell-derived EVs in regenerative medicine has been tested in a variety of experimental models. EVs secreted from tissue resident stem cells alter the behavior of the target cells. Herrera et al. [39] demonstrated that microvesicles derived from human liver stem cells facilitated hepatic regeneration after hepatectomy in rats by activating proliferation and apoptosis resistance of hepatocytes. In this study, the authors indicated that human liver stem cell-derived microvesicles shuttled a subset of mRNAs implicated in the control of proliferation and apoptosis. Over the past decade, the role of MSCs in regenerative medicine and their potential use as vehicles for gene delivery have been intensely investigated since it is well-established that MSCs migrate to injured tissues and participate in wound healing and tissue repair [40,41]. Accumulating evidence supports the notion that MSC-derived EVs help to repair tissue damage. For instance, purified exosomes from MSCs reduced infarct size in a myocardial ischemia/reperfusion injury mouse model [42]. Furthermore, using an acute myocardial infarction rat model, Bian et al. [43] demonstrated that EVs secreted from human bone marrow MSCs enhanced proliferation, migration, and tube formation of endothelial cells in a dose-dependent manner. Several studies reported that administration of MSC-derived microvesicles improved the recovery from acute kidney injury by stimulating proliferation of tubular cells in different renal injury models [44][45][46]. In addition, it has been suggested that MSC-derived EVs exert therapeutic effects on neurological diseases [47]. A recent study indicated that microvesicles produced by MSCs promoted sciatic nerve regeneration in rats, suggesting MSC-derived microvesicles as a novel approach to peripheral nerve cell therapy [48]. Also, MSC-derived exosomes delivered miR-133b to neural cells, which resulted in enhancement of neurite outgrowth [49]. In another study, systemic injection of MSC-derived exosomes improved neurovascular remodeling and neurogenesis after stroke in rats, implying that MSC-derived EVs may provide a potential therapeutic benefit for the treatment of neurological diseases [50].

MSC-derived EVs as a vehicle for gene delivery
Recently, several studies evaluated MSC-derived EVs as a potential vehicle for gene delivery. Katakowski et al. [51] isolated exosomes released by the MSCs transfected with a miR-146b, known as an antitumor miRNA, expressing vector. The authors showed that injection of miR-146b-expressing exosomes derived from the transfected MSCs significantly inhibited glioma growth in a rat model. Furthermore, Munoz et al. [52] reported that the delivery of synthetic anti-miR-9 by MSC-derived exosomes to the Glioblastoma Multiforme (GBM) cells reversed the chemoresistance of GBM cells. The data showed that anti-miR-9 shuttled by MSC-derived exosomes downregulated the expression of the multidrug transporter, thus sensitizing the GBM cells to temozolomide. Since EVs are bi-lipid and nonsynthetic structure that protects molecules from degradation, they are regarded as an ideal gene delivery vector. However, it is challenging to purify uniform EVs because EVs are heterogeneous population and a subset of molecular contents transported by EVs may vary in a contextdependent manner.

Stem cell-derived EVs and tumor
More recently, the effect of stem cell-derived EVs on tumor growth has been explored. Human liver stem cellderived microvesicles suppressed hepatoma growth in SCID mice by transferring tumor suppressor miRNAs [53]. In addition, we demonstrated that MSC-derived exosomes significantly downregulated the expression of vascular endothelial growth factor (VEGF) in breast cancer cells, thus suppressing angiogenesis in vitro and in vivo [54]. The results indicated that MSC-derived exosomes delivered miR-16, a miRNA known to target VEGF, and miR-16 was involved in the anti-tumor effect of MSC-derived exosomes. Furthermore, Bruno et al. [55] reported that MSC-derived microvesicles suppressed different types of tumor progression in vitro and in vivo. The authors treated MSC-derived microvesicles with HepG2 hepatoma, Kaposi's sarcoma, and Skov-3 ovarian tumor cell lines. The data demonstrated that MSC-derived microvesicles promoted cell cycle arrest in all cell lines and induced apoptosis in HepG2 and Kaposi's cells and necrosis in Skov-3. In contrast, a recent study found that exosomes from human bone marrow MSCs promoted angiogenesis in tumors by activating the extracellular signal-regulated kinase1/2 (ERK1/2) pathway in vivo [56]. Thus, whether stem cell-derived EVs are pro-or anti-tumorigenic has been a matter of debate. Nevertheless, these observations suggest that stem cell-derived EVs serve as an important mediator of intercellular communication in the tumor microenvironment.

Conclusions
Based on the accumulating evidence that EVs secreted from stem cells convey bioactive components to the recipient cells, EVs have emerged as a key player of cell-to-cell communication in stem cell biology. The molecular contents delivered by EVs may differ, depending on the state of the cells in the microenvironment. Since stem cell-derived EVs can transport stem-cell specific genetic materials, including mRNAs and miRNAs, they may trigger a regenerative program in injured cells in a paracrine manner. Conversely, EVs released from injured cells may induce stem cell differentiation. However, the exact characteristics and biological functions of stem cell-derived EVs are not fully elucidated. In addition, the effect of stem cell-derived EVs on tumor development is currently controversial. Thus, in order to harness stem cell-derived EVs as a therapeutic option, further studies are required.