The Potential Role of Microvesicles in Mesenchymal Stem Cell-Based Therapy

A s plasma membrane-derived particles for intercellular communication, microvesicles (MVs) released from various cell types have increasingly become a focus of interest in stem cell-based therapy. MVs can be harvested by ultracentrifugation at 100,000 g from the supernatants of cells and have a diameter of 100–1000 nm [1,2]. They originate from multivesicular bodies and comprise of 2 regions: the membrane and the internal cargo. The membrane (consisting of proteins and lipids) packages the bioactive molecules, such as lipids, proteins, DNA, mRNA, and micro-RNA [2,3]. The components of MVs are dependent on the parental cells from which they originate and on the mechanisms by which they released them; thus, the function of MVs from different cells may vary. For instance, MVs from tumor cells regulated extracellular matrix degradation, whereas those from endothelial progenitor cells activated the angiogenesis [4,5]. The cargo inside the MVs determines the function after targeting the neighboring cells and the remote ones by fusion or interaction [2,3]. Therefore, the striking effect of MVs on cell-to-cell interaction and tissue regeneration cannot be ignored.

Currently, MVs have been isolated from different cells [4 –6], as well as from mesenchymal stem cells (MSCs) [7 –9]. MSCs, which have been obtained from a variety of tissues, have been extensively investigated for regenerative strategies, and those from bone marrow (BM-MSCs) are the most widely studied [10 –14]. The regenerative mechanisms ascribed to MSCs can be classified into 3 categories: differentiating toward reparative or replacement cell types, enhancing the nutrient supply, and improving the survival and function of the endogenous cells via paracrine actions [15 –17]. Because of the inhospitable microenvironment of the injured or degenerating tissues, a large proportion of the implanted MSCs may die or undergo apoptosis in a short period post-transplantation [18,19]. Only a rare proportion of transplanted MSCs remained therefore alive, and evidence of their differentiation in vivo toward mature phenotypes of resident cells is very scanty [18], suggesting that mechanisms other than differentiation of transplanted cells replacing endogenous damaged cells are invoked. Therefore, it was suggested that the paracrine signaling partly evoked the repair [17]. This notion was further supported by experimentation where the MSC-conditioned medium rather than cells themselves and therefore the secreted products of the cells acted as paracrine modulators of tissue regeneration [20 –22]. Definitely, most of these secreted molecules from MSCs included cytokines, growth factors, and nucleic acids that cannot span the membranes freely and a vehicle should be involved to facilitate the crossing. Although the underlying mechanisms of transport are not well elucidated, MSC-derived MVs have been supposed as shuttles for the functional components for MSC paracrine action. The following 2 reasons would provide a better interpretation.

First, MVs are secreted to the extracellular space throughout budding and fission of the membrane [2,7]; thus, they possessed several characteristics of the parental MSCs. For example, MVs isolated from human BM-MSCs were positive for CD29, CD44, and CD73 [23]. In addition, MVs can be labeled by the commonly used fluorescent dyes such as PKH26, so that it is possible to observe the membrane fusion of MVs with the recipient cells in which the phenotypes may change accordingly [23]. These structural patterns are similar with that of MSCs.

On the other hand, the containing cargo exerts function similar to those of MSCs. It has been found that human BM-MSC-derived MVs were enriched of mature micro-RNA that may be transferred to the recipient cells; in other words, MVs may have a functional impact on the target cells through micro-RNA delivery [24]. Additionally, the transfer of membrane-related proteins and the modulation of immune responses by MSC-derived MVs were recorded [25,26]. Interestingly, the targeted cells would regulate MSCs in the same way that could be determined by the bidirectional intercellular MV transfer between MSCs and nucleus pulposus cells during direct coculture [27]. The above evidence further broadened our current understanding regarding the mechanisms of MSC paracrine actions. Furthermore, like MSCs, MVs demonstrated therapeutic potency. Bruno and colleagues reported that human BM-MSC-derived MVs enhance the proliferation of tubular epithelial cells and protect them from apoptosis induced by serum deprivation, vincristine, and cis-platinum; in vivo studies suggested that MVs could accelerate the recovery from acute kidney injury (AKI) induced by glycerol in SCID mice [23]. The therapeutic effect by MVs was further determined in the rats with AKI induced by ischemia–reperfusion injury and subsequent chronic kidney disease [28]. Beyond in these 2 nonlethal AKI animal models, more recently, the same authors described that MV-based therapy still worked in the lethal cisplatin-induced AKI [29]. In view of the above studies, it was suggested that the therapeutic potential of MSC-derived MVs for kidney regeneration was similar to that of MSCs.

Although a lot of further studies regarding the safety and effectiveness of MSC-derived MVs will need to be done, it is anticipated that MVs could become an alternative therapeutic approach to the MSC-based ones, with several advantages. In vitro extensive expansion methods, cryopreservation techniques, and complications, maldifferentiation [30 –32] induced by the use of replicating MSCs can be avoided. The most important advantage should be the escape from the risk of malignancies of MSCs, although it is still a subject of debate [33]. MSC transplantation in humans has been considered to be safe so far, and currently, there are more than 260 ongoing MSC-based clinical trials that are listed in http://clinicaltrials.gov/. Nevertheless, the safety and especially the relation between MSC transplantation and tumor growth should be taken into consideration before an extensively clinical application. According to the evidence in literature, the tumor growth-promoting effect of MSCs was investigated in a xenograft mouse model after MSCs were cotransplanted with human cancer cells [33]. This potential was related to the capacity of differentiation into tumor-like cells and enhancement of neovascularization in tumor and paracrine signaling [33 –37]. Nevertheless, the protumorigenic effect of MSCs was not observed in human clinical studies with a follow-up period ranging from 1 month to 6.8 years [33]. The discrepancies between results from the experimental models and from the clinical trials may be attributed to the fact that the animal models cannot truly mimic the tumor microenvironment established in humans; furthermore, the short follow-up period and the characteristics of recruited unhealthy subjects should also be seriously considered. Hence, this significant safety concern of MSCs makes it reasonable to develop a new stem cell-free therapeutic approach. Moreover, MSC-derived MV-based therapy becomes the potential one, especially after the antitumor growth effect was found.

In a recent article published in this journal by Bruno and colleagues, MVs derived from human BM-MSCs were found to inhibit tumor growth [38]. The isolated MVs were positive for BM-MSC markers (ie, CD29, CD44, CD73, and CD105) and LAMP-1, which is an exosomal marker. After exposure to MVs, the proliferation and viability of HepG2, Kaposi, and Skov-3 cancer cells were significantly inhibited, and these effects were associated with the deregulation of the cell cycle progression. This conclusion was further validated by an in vivo study after weekly intratumor injection of MVs into the established tumors. The growth of these tumors was diminished, and extensive apoptosis and necrosis inside the tumor were illustrated. Although these 3 kinds of tumor may not represent all the potential human malignancies, and their formation process is not strictly consistent with that in humans, these conclusions provide us new understanding of MV-based therapy.

Since MVs possessed some structural and functional properties of MSCs for tissue regeneration, and since MVs inhibited the established tumor growth, it would appear reasonable to assume that MSC-derived MV-based therapy may become a new therapeutic approach in regenerative medicine. Nevertheless, several research gaps need to be addressed before clinical application. First, the precise definition of MVs needs to be clarified, because the sediment obtained from the supernatants by ultracentrifugation likely contains a mixture of MVs and exosomes, although MVs and exosomes are distinct in morphology and release mechanism [2]. Interestingly, the therapeutic potential of MSC-derived exosomes has also been determined [39]. Second, the exact mechanisms regarding the interaction of MVs and the injured tissues, the homing and biodistribution of MVs, all need further investigation. Third, the regenerative potential of MSC-derived MVs was clearly demonstrated in renal-related diseases; the therapeutic potential should be therefore extensively investigated in other tissues to expand the knowledge on this issue and see if MV beneficial effects can be exerted also into other injured/diseased tissues/organs. Furthermore, because the function of MVs is determined by the cell of origin, there is no doubt that the integral component of MVs that are isolated from MSCs obtained from different tissues should be different, thereby doing also varies the MV functions. This difference may provide a novel approach for stem cell selection in the regeneration of specific tissues, especially those depending on the paracrine action of implanted MSCs. Undoubtedly, safety is always the most important issue for every new approach. Last but not the least, similar with the development of stem cell-based therapy, the criteria for production, identification, applicability, and contraindications should be validated.

At this time, the study of Bruno and colleagues extends current knowledge regarding the role of MVs in MSC-based therapy and provides new insights into the contribution of MSC-derived MV-based therapy.