Stem Cell-Derived Microvesicles: A Cell Free Therapy Approach to the
Regenerative Medicine
Tatiana Lopatina
1,2, Maria Chiara Deregibus
1, Vincenzo Cantaluppi
1and Giovanni Camussi
*,11Department of Internal Medicine, Centre for Molecular Biotechnology and Centre for Experimental Research in Medicine (CeRMS), Torino, Italy
2Faculty of Fundamental Medicine, Moscow State University, Moscow, Russia
Abstract: Microvesicles (MVs) include a heterogeneous population of vesicles released as exosomes from the endosomal compartment or as shedding vesicles from the cell surface of different cell types. The broad spectrum of biological activities displayed by MVs candidate them to a pivotal role in cell-to-cell communication. It is now recognized that they constitute an integral part of the intercellular microenvironment acting as vehicle for information transfer. After receptor-ligand interaction with target cells, MVs may directly stimulate the cells or may transfer from the cell of origin various bioactive molecules including membrane receptors, bioactive lipids and proteins. In addition, MVs may induce epigenetic changes in target cells by delivering specific subsets of mRNA and microRNA associated with different cell functions such as differentiation of blood cells, metabolic pathways and modulation of immune response. In vivo, MVs released from mesenchymal stem cells may account for the described paracrine action of these cells in tissue regeneration. A bi-directional exchange of genetic information between stem and injured cells could be envisaged: i) transcripts delivered by MVs from injured cells may reprogram the phenotype of stem cells to acquire specific features of the tissue; ii) transcripts delivered by MVs from stem cells may limit tissue injury and induce cell cycle re-entry of resident cells leading to tissue self-repair.
This review presents an overview of the many biological actions of MVs produced by stem cells that may be exploited in regenerative medicine to repair damaged tissues as an alternative to stem cell-based therapy.
Keywords: Angiogenesis, apoptosis, cell communication, exosomes, inflammation, kidney repair, liver repair, mesenchymal
stem cells, microvesicles, signal transduction, stem cells, transport of genetic information, tissue regeneration.
INTRODUCTION
The understanding of the cellular and molecular mechanisms involved in tissue repair provides new insights for regenerative medicine. A number of acute and chronic diseases may benefit from therapeutic strategies aimed to favor repair and regeneration of injured tissues. Therefore, on this perspective it is important to learn how we can use the reparative resource of the organism to activate its own protective mechanisms. At present, most regenerative processes are thought to be performed by stem/progenitor cells. These cells are virtually present in all adult tissues and organs and they may participate in the physiological cell turnover, growth, homeostasis and repair of limited injuries of many tissues.
Stem/progenitor cell transplantation has been suggested as an approach for treating many types of diseases. It has been shown that mesenchymal stem cells (MSCs) can stimulate regeneration of several tissues /organs after injury [1, 2].
In preclinical studies different types of stem cells isolated from adult tissues have been used for regeneration of
*Address correspondence to this author at the Dipartimento di Medicina Interna, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14, 10126, Torino, Italy; Tel: +39-011-6336708; Fax: +39-011-6631184; E-mail: giovanni.camussi@unito.it
damaged organs such as heart, liver, kidney, etc. [3-9]. These include bone marrow derived mononuclear cells, hematopoietic stem cells, MSCs, skeletal muscle myoblasts and the so-called cardiac stem cells. To date stem cell therapy in clinical settings has been employed for the treatment of a variety of severe pathologic conditions, such as cardiovascular, neurodegenerative, metabolic and immuno-mediated diseases. [5, 10] Several clinical trials are ongoing in different countries in particular with MSCs [3-6] (for the clinical trials see: www.clinicaltrial.gov). Despite the observed beneficial effects, there is no consistent evidence that the cells employed generate organ-specific cell population, able to replace the cell loss after injury. Indeed, transdifferentiation has not consistently been proved in many experimental models, and the phenomenon of cell fusion is considered a rare event. Therefore, it has been suggested that stem cells may act as producers of bioactive molecules (growth factors, cytokines, etc.) that account for the observed beneficial effects [1, 11]. It has been shown that MSCs produce and secrete a broad variety of cytokines, chemokines, and growth factors that may potentially be involved in regeneration, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and other angiogenic factors, neurotrophic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF) [1, 2, 12, 13]. Furthermore, hypoxic stress increases the production of
several of these factors. Indeed, tissue concentrations of these growth factors were found to be significantly increased in injured tissue after treatment with MSCs [14, 15]. A strong support for a paracrine mechanism in tissue repair comes from experiments in which the administration of conditioned medium from MSCs was shown to mimic the beneficial effects observed after stem cell therapy [16, 17]. Gnecchi and colleagues [18]demonstrated that conditioned medium from MSCs, particularly from genetically modified MSCs overexpressing Akt-1, exerts cardiomyocyte protection from ischemia. Takahashi et al. [19] injected conditioned medium from MSCs into acutely infarcted hearts and observed increased capillary density, decreased infarct size, and improved cardiac function compared with controls. These observations stimulated research aimed to identify the factors that mediate the biological effect of MSCs. On the other hand, a major limit for MSC clinical application is their inherent heterogeneity and variation associated with cell expansion. Additionaly, in vitro cell processing and expansion could induce changes that increase the risk of their therapeutic applications. Moreover, MSCs may undergo unwanted differentiation in vivo such as adipogenic differentiation in the kidney [7] or the osteogenic differentiation in the heart [20]. Therefore, identification of the molecules involved in the paracine action of stem cells may non only provide information on the mechanism of action, but also open new therapeutic options. We recently found that microvesicles (MVs) derived from adult stem cells may be an integral component of the paracrine mechanism involved in tissue regeneration [21]. MVs participate in cell-to-cell communications and regulate many cellular processes including proliferation, migration and differentiation by transfer of proteins, bioactive lipids, nucleic acids and other molecules from one cell to another [22, 23]. Therefore, MVs may transfer from stem cells to target cells critical information that may serve for tissue regeneration after injury. Since the use of stem cells in medicine could lead to tumorigenesis and other negative consequences [24], MVs may constitute an alternative therapeutic tool in regenerative medicine. At variance of stem cells, MVs cannot proliferate but can deliver to recipient cells growth factors, receptors, ligands, and even genetic information. The rationale for MV employment in regenerative medicine is based on the evidence that MVs may stimulate the endogenous repair by inducing a stem cell-like phenotype in differentiated tissue resident cells or by activating tissue resident stem cells. Moreover, it may be envisaged that custom-engineered MVs may be used for delivery of high levels of regenerative growth factors or nucleic acids.
CHARACTERISTICS AND NATURE OF MVs
The term «microvesicles» includes all secreted or extracellular membrane vesicles such as microparticles, ectosomes, exosomes, apoptotic bodies and shedding vesicles, or vesicles designed by their functions such as argosomes, oncosomes, promininosomes, prostasomes, etc. [22, 25-27]. MVs contain membrane and soluble proteins, nucleic acids and other bioactive molecules. It is thought that MVs originate through two distinct mechanisms: 1) blebbing of the plasma membrane with production of shedding vesicles also defined as ectosomes or microparticles; 2) the
endosomal processing and release of cytosol and plasma membrane material as part of exosomes. Many (if not all) cell types may produce MVs [28]. Another types of shedding vesicles are the apoptotic bodies released from dying cells. Apoptotic MVs often contain DNA as remnant of nucleus [22, 25, 29].
Shedding Microvesicles or Ectosomes
Shedding microvesicles are membranous vesicles with a size greater than 100 nm in diameter, extruded from the plasma membrane of many types of cells [30-32]. These MVs are enriched of lipid raft-associated molecules including tissue factor (TF) and flotillin-1 [33]. Formation of shedding MVs occurs through budding of plasma membrane, collecting specific transmembrane and cytosol proteins. This phenomenon is characterized by the exposure of phosphatidylserine (PS) residues on the outer surfaces of MVs [34]. The process happens under the control of several enzymes such as calpain, flippase, floppase, scramblase and gelsolin [35]. MVs carry specific marker molecules from the donor cells, for example platelet-derived MVs contain platelet/endothelial cell adhesion molecule (PECAM-1), while MVs from gliomas carry oncogenic growth factor receptors (e.g., EGFRvIII) [25]. Other MVs were shown to contain cytokines and chemokines (e.g., IL-1, and IL-8), VEGF and fibroblast growth factor 2 (FGF2) derived from donor cells [31, 35-38].
The mechanisms of MV release have been described in different types of cells. Firstly, the microparticles released from activated platelets were investigated. It was shown that blebbing of plasma membrane is associated with changes in membrane lipid asymmetry. Each of the two leaflets of the membrane bilayer has a specific composition. Acidic aminophospholipids (PS, phosphatidylethanolamine) are concentrated specifically in the inner side, whereas neutral phospholipids (phosphatidylcholine, sphingomyelin) are enriched in the external one. The transbilayer lipid distribution is under the control of several enzymes like flippase, floppase, and a lipid scramblase [39]. The increase of cytosolic Ca2+ following cell stimulation leads to activation of these enzymes resulting to surface exposure of PS. The subsequent cytoskeleton degradation by a Ca 2+-dependent proteolysis induces shedding of MVs from the cell surface [40, 41]. PS in the outer membrane acts as a template for the prothrombinase complex assembly and for coagulation activation [42, 43].
Endothelial cells, monocytes, vascular smooth muscle cells, and hepatocytes can also release MVs after activation by bacterial lipopolysaccharides, inflammatory cytokines (tumor necrosis factor- (TNF), interleukin 1 (IL-1)), aggregated lowdensity lipoproteins, or reactive oxygen species [44-47]. All these stimuli increase the level of intracellular Ca2+ that is crucial for MV release [48].
Other molecular mechanisms were described in neural cells (CNS phagocytes, microglia). The key players in these cells are acidic sphingomyelinase (aSMase) and P2X7 receptor. The sequence of events is: 1) ATP (dying cells release ATP) activates P2X7 receptor; 2) receptor stimulation leads to the activation of aSMase with rapid sphingomyelin hydrolysis; 3) sphingomyelin hydrolysis
results in increased membrane fluidity [49, 50], leading to membrane blebbing and MV shedding [36, 51, 52]. Dying cells by releasing ATP, may stimulate other cells to release MVs containing proinflammatory cytokines (IL-1) and may regulate the clearance of cellular debris.
Exosomes
Exosomes are MVs of 40–100 nm size with an endosomal origin. Exosomes originate upon fusion of multivesicular bodies with the plasma membrane. Exocytosis of multivesicular bodies has been described for various cell types, including reticulocytes [53-55], B-lymphocytes, dendritic cells [58], mast cells [59], platelets [32], and others. Exosomes share the biochemical characteristics with the vesicles of the multivesicular bodies. The biogenesis of exosomes is regulated by distinct cellular pathways [22, 60, 61]. The release of exosomes occurs by exocytosis and may be either constitutive triggered by a Ca2+-independent mechanism or consequent to stimulation by a Ca2+-regulated mechanism. The constitutive exocytosis occurs in almost all cell types and it is involved in the secretion of newly synthesized proteins. Exocytosis dependent on cell activation is initiated by an endosomal formation following the invagination of endocytic vesicles, that fuse with the early endosomes [62]. The initial steps of exosomes secretion is controlled by the endosomal sorting complex required for transport (ESCRT) [63]. Inward invaginations of plasma membrane lead to formation intracellular vacuoles (early endosomes) that, under control of ESCRT, mature into the late endosome/multivesicular bodies that can give rise to released exosomes following the redirection and fusion of multivesicular bodies with the plasma membrane. There is no clear evidence that ESCRT takes part in the molecular sorting of exosomal contents. However, some components of ESCRT complex such as Alix are found in exosomes [64, 65]. Alix is a protein that associates with the ESCRT machinery known to be required for sorting of the transferrin receptor [66]. An other molecular mechanism of exosome release includes ceramide [67, 68] that promotes membrane invaginations [69].
Exosomes contain not only cytosol constituents but also the extracellular domains of transferring receptors [62]. Therefore, exosomes are the result of the secretion process of the endosomal components of the cells.
When compared with shedding microvesicles, exosomes contain different set of molecules [25, 27, 70, 71]. Exosomes contain some marker proteins such as Alix, TSG101, HSC70, CD63, CD81, CD9. Moreover, the lipid content of exosomes include low phosphatidylserine exposure, ceramide, lipid rafts and sphingomyelin, whereas shedding microvesicles expose high level of phosphatidylserine [28]. The nucleic acid content is presented in both structures. The biological fluid as well as MVs released in vitro by cells contain a mixture of exosomes and shedding vesicles and the collection process by differential ultracentrifugation does not allow a complete separation. For this reason frequently the term MVs is used to indicate both exosomes and shedding microvesicles. At present, functional differences between exosomes and microvesicles have not been extensively investigated.
CHEMICAL COMPOSITION OF MVs
The function of MVs depends on their chemical composition. Usually MVs carry some specific lipids, membrane proteins, which help them to interact with defined cell, some soluble proteins (growth factors, RNA-binding proteins) RNA, sometimes DNA and other bioactive molecules. There are molecules that are common for every type of MVs and some that are specific for the cells of origin, the type of MVs, the culture conditions etc.
The protein content of MVs has been analyzed from various cell types and body fluids by several methods such as Western blot, Mass Spectrometry, fluorescence-activated cell sorting and immunoelectron microscopy. Proteomic analysis was performed on MVs from many different types of cells or body fluids [72-90]. Some proteins (HSPA8, CD63, ACTB, GAPDH, ENO1, HSP90AA1, CD9, CD81, YWHAZ, PKM2) were found in most of MVs [91], suggesting that they are necessary for MV function. However, the majority of MV proteins varies depending on the cell type and on the tissue of origin [79]. In addition, some proteins are specifically accumulated in MVs [28].
MVs do not transport only proteins and lipids. They carry also different RNA species [77]. Indeed, it has been shown that MVs contain mRNA and microRNA (miRNA), but a very little amount of ribosomal RNA. Microarray analysis revealed that some RNAs are specifically concentrated in MVs. In the same manner as proteins, RNAs from MVs include some common for most MVs and others specific for the cell- and tissue-of origin. Experiments of in vitro translation proved that the mRNAs shuttled by MVs are functional [77]. These different RNA species are likely to be specifically packed into the MVs by active sorting mechanisms that at present are largely unknown. Several RNA-binding proteins are detectable in MVs including HNRNPA2B1, HNRPAB, ILF2, NCL, NPM1, RPL10A, RPL5, RPLP1, RPS12, RPS19, SNRPG, TROVE2 [92], ESCRT-II [93]. Some of these RNA-binding proteins play a role in RNA stabilization, others are involved in its fate as in the case of Staufen protein family that plays a critical role in miRNA maturation [94]. MVs are considered to be a unique mechanism for transport of nucleic acids at long distance within the body as they provide a protection from nucleases. The process of RNA secretion has been shown to be largely energy dependent [92].
Since MVs can interact and be internalized within target cells, they may act as a widespread mechanism for horizontal cell-to-cell transfer of genetic information. MV-mediated delivery of mRNA and miRNA from donor to recipient cell has been shown in several laboratories [77, 95-99]. MVs can enter into target cells and induce specific transcription of delivered mRNA [97]. The patterns of secreted RNAs may vary in changing the conditions suggesting that secretion of RNAs via MVs is a regulated and adaptive process.
MV biological action may result from the complex of specific and synergic molecular combinations with a pre-programmed composition of proteins, lipids, and nucleic acids [100]. In this regard, the physiological functions of MVs may differ from the sum of predicted effects of their transported molecules. In addition, the biologic effects may vary depending on the metabolic state of the recipient cell.
Table 1 presents some molecules contained in MVs
derived from human MSCs that may take part in tissue regeneration.
PLEYOTROPIC BIOLOGICAL ACTIVITIES OF MVs
MVs contain a plenty of biological active molecules, which are not a random sample of the cytosol of the cell of origin. This chemical composition resulting from an energy-depended and highly controlled process suggests that MVs play an important biological role (Fig. 1). The first hypothesis was that MVs could be a mechanism of molecular throw-out [101]. Exocytose-like mechanism for removal of superfluous or harmful molecules is well described. This is the case of transferrin receptors that must be rapidly removed from reticulocytes to allow their differentiation into mature red blood cells that are dismissed through exocytosis [26]. However, more recently, it is becoming clear that MVs act as an intercellular mediator of communication able to influence several processes such as migration, proliferation, differentiation, angiogenesis, chemotherapy resistance, immunoregulation, inflammation,
coagulation, and cancer metastasis [22, 27, 28, 34, 63, 102, 103]
A proper coordination of different tissues in the organism requires communication among cells. Such communication is guaranteed not only by membrane surface molecules (e.g., Notch, EGFR) and soluble secreted proteins (e.g., growth factors, interleukins) but also by MVs. In this context, MVs can deliver within a defined microenvironment as well as at long distances proteins and nucleic acids. MVs could be a unique way for the transport of soluble molecules, that have not an export signal peptide in their sequences (IL-1 or basic FGF) [36, 104] or for nucleic acids, which are very sensitive to nucleases present in the extracellular fluids. In addition, MV transport prolongs the extracellular life of secreted molecules (e.g., VEGF), alters their gradient (Wnt and MMPs) and concentrates and integrates molecular partners at specific sites [34, 105, 106]. All types of MVs carry adhesion molecules on their surface, which are necessary for their entry into recipient cells. Blocking antibodies for various integrins, adhesion molecules or tetraspannins significantly reduced MV capture [21, 107, 108]. The mechanisms possibly involved in the interaction of
Table 1. Molecules Involved in Tissue Regeneration which are Expressed byMSC-Derived MVs
Molecule Function References
Proteins Growth factors and cytokines
(VEGF, FGF, PDGF, leptin, TNF, TGF-, IL-8, IL-6, IL-1, NGF, BDNF, NT3, NT4, GDNF)
Activation of blood vessel and axonal growth and survival; leukocyte
recruitment and activation [118-121] Matrix metalloproteases
MMP9, MMP2, MT1-MMP, EMMPRIN (MMP activator) and TIMPs (MMP inhibitors)
Proteolysis of extracellular matrix, cell migration [122] [123]
Membrane proteins and receptors
Dll4, Tspan8, Notch Navigation molecules for vessels and axons [113] [124, 125]
mRNA
IL1RN
MT1X Immune regulation [21]
CRLF1 Promote survival of neuronal cells [21]
COL4A2, IBSP Extracellular matrix proteins [21]
MAGED2, CEACAM5, COL4A2, SCNN1G, PKD2L2 Endothelial cell differentiation [21]
RAX2, OR11H12, OR2M3, DDN, GRIN3A Neural cells differentiation [21]
VEGF, HGF, IL-8 Angiogenic factors [77, 136]
Cyclin D1, Cyclin A2, Bcl-xl, Bcl-2 Cell cycle regulation, proliferation [134]
Oct-4, GATA-2, GATA-4, SCL, Nanog, Rex-1, HoxB4 Stem cell expansion, pluripotency [134]
microRNA
miR-21, miR-22 differentiation of stem cells [162-164]
miR-21, miR-17-3p Proangiogenic action [96]
miR-222 Antiangiogenic action [96]
lipids
Sphingomyelin Lipid involved in cell signaling, angiogenesis, nerve growth [165] Phosphatidylserine Template for other proteins such as prothrombinase complex assembly [42, 43]
MVs with target cells are: 1) MV membrane proteins can interact with receptors in a target cell and activate intracellular signaling (direct stimulation of target cells); 2) MVs can fuse with the target cell membrane and release their contents inside the recipient target cell. This leads to modification of the recipient cell membrane with the addition of new membrane receptors and changes of lipid compositions. Moreover, MV inner molecules (protein, mRNA and miRNA) can activate a cascade of signaling events in the recipient target cells.
MVs may Act as Signaling Complexes by Direct Stimulation of Target Cells
MVs could carry several types of receptors and surface molecules including TF, TNF, MHC Class I/II, CCR5 chemokine receptor [100, 109]. Through these molecules MVs can interact with the target cell in a juxtacrine fashion, thereby activating the target cell. For example, MVs carrying inter cellular adhesion molecule 1 (ICAM1) can be captured
by lymphocyte function-associated antigen 1 (lFA1; a ligand for ICAM1), on the surface of CD8+ dendritic cells and activate T cells [110, 111]. One of the most important step in the development pathway which involves Notch signaling can be performed through MVs, because MVs express delta-like 4 (Dll4), a transmembrane Notch ligand. Therefore, MVs may activate angiogenesis and axon growth by interacting with Notch receptors expressed by endothelial or nerve cells respectively [112, 113]. MV receptors can determine immunotolerance of cancer cells: cell death ligand (FasL) exposed on tumour cell-derived MVs is lethal for Fas-expressing lymphoid cytotoxic effector cells [114]. Another possible mechanism of target cell activation includes enzyme cleavage of MV membrane proteins and following action of resulting molecules as ligands for receptors expressed by target cells. Interestingly, some of the exosomal membrane proteins are not identified in the cell surface of the originating cell (e.g., LAMP-2) [72] suggesting that MVs may act as a carrier for specific proteins to be delivered to target cells.
Fig. (1). Pleyotropic biologic effects of MVs and potential involvement in tissue repair. The physiological response to injury involves several overlapping phases including inflammatory response, new vessel and tissue formation and finally remodeling. Inflammation is aimed to remove damaged cells whereas regeneration is aimed to replace the cell loss. Finally remodeling is required for the achievement of the original tissue architecture. In this contest, MVs secreted by MSCs may contribute to coordinate the regenerative response as they contain molecules that may stimulate angiogenesis, axon growth, protect cells from apoptosis and act as immunoregulators. This cocktail may enhance tissue repair after injury. Some of these molecules take part in multiple processes, for example, VEGF has proangiogenic and neurotrophic function, Dll4 and EPHB3 regulate directional growth of vessels and axons and whereas some of the proteins induce proliferaion others start differentiation. ANGPT, angiopoietin; BDNF, brain derived neurotrophic factor, bFGF, basic fibroblast growth factor; Dll4, Delta like ligand 4; EMMPRIN, extracellular matrix metalloproteinase inducer; EPHB3, Ephrin type-B receptor 3; GDNF, glial cell derived neurotrophic factor; ILs, interleukins; Mif, macrophage migration inhibitory factor; MMPs, Matrix metalloproteinases; MSC, mesenchymal stem cell; MVs, microvesicles; NGF nerve growth factor, Notch, neurogenic locus notch homolog protein; NT3 neurotrophic factor 3; NT4, neurotrophic factor 4; PDGF, platelet derived growth factor; TGF-, Transforming growth factor beta; TIMPs, tissue inhibitors of metalloproteinases; TNF, tumor necrosis factor-alpha, Tspan8, tetraspanin 8; VEGF, vascular endothelial growth factor; Wnt3, wingless-type MMTV integration site family, member 3.
MVs may Act by Transferring Receptors Among Cells
MV fusion with the target cell leads to transfer of membrane proteins and lipids. For example, chemokine receptors, especially CCR5 or CXCR4 [115], which are necessary for HIV infection, are transferred via MVs to different types of cells and induce susceptibility to viral infection in these cells [109, 115-117]. MVs from tumor cells contain and transfer the oncogenic form of epidermal growth factor receptor (EGFR), which induces an oncotransformation of recipient cells [31]. The previously mentioned Notch ligand Dll4 can be transferred from MVs to endothelial cells and incorporated into their membrane resulting in inhibition of Notch signaling. Transfer of Dll4 has been shown in vivo from tumor cells to host endothelium [113].
MVs may Deliver Proteins to the Target Cells
MVs carry also soluble proteins that can be transferred into recipient cells resulting in changes in cellular responses. For instance, endothelial cell-derived MVs can activate angiogenesis through the transfer of pro-angiogenic molecules such as growth factors (VEGF, bFGF, PDGF, leptin, FGFa, TNF, and TGF- and others [118-121]), proteases (e.g., MMP9, MMP2 and MT1-MMP) [122] and their activator (EMMPRIN) [123], navigation molecules Dll4 [113]or Tspan8 [124, 125]. Moreover, MVs also influence gene transcription by transferring transcription factors, such as peroxisome proliferator-activated receptor gamma (PPAR) and retinoid receptor (RXR) [126]. MVs are able to induce apoptosis in target cells by transferring
caspase-1 from lipopolysaccharyde-activated monocytes [127, 128] or promote cell infection by transferring from infected cells the immunodeficiency virus or prions [85, 129].
This MV-depended transport of growth factors, their receptors, anti- or pro-apoptotic or toxic molecules may promote cooperative events and activate defined processes within a heterogeneous cellular population.
MVs may Mediate a Horizontal Transfer of Genetic Information
As mentioned above, MVs may transfer among cells mRNA [102, 130], microRNA [77, 96, 131] and DNA [132] MVs are enriched of RNA molecules specific for the cell of origin. The fact that MVs contain specific patterns of RNA that may change under different conditions suggests that secreted RNA plays an important biological role in the organism. Indeed, the RNA content of MVs may vary in different cell culture conditions; for instance, exosomes released by mouse mast cells exposed to oxidative stress, can influence the response of other cells to the oxidative stress [133].
The relevance of this transfer of genetic information among cells has been shown during differentiation [134], inflammation [77], regeneration [135] and cancer [136]. It has been shown that embryonic stem cells through MV-secreted RNAs are able to promote clonogenicity of hematopoietic stem cells and induce the expression of Oct4, Nanog, Rex and other genes associated with stemness [134].
MVs released from endothelial progenitor cells activate an angiogenic program in quiescent endothelial cells by a horizontal transfer of selected patterns of pro-angiogenic mRNA that are translated into proteins within the recipient cells [102, 130]. It has been also shown that RNA shuttled by MVs secreted from endothelial cells promotes angiogenesis in tumor [136]. Tissue-specific mRNA and miRNA can be transferred between cells and induce tissue-specific gene expression in other cells as recently shown by Aliotta and co-workers [97].
ROLE OF MV-MEDIATED CELL-TO-CELL INTERACTION IN STEM CELL BIOLOGY
Like other cell types also stem cells may release MVs. MVs derived from stem cells are able to reprogram target cells, for example hematopoietic progenitors [134], endothelial cells [130], kidney cells [137] and liver cells [138] suggesting that MVs are a stem cell instrument for tissue development regulation, regeneration, and cell differentiation. It is known that resident stem cells are present in all adult tissues [139] and that they participate in development and repair processes. They are situated in specific niches with differentiated cells. Stem cells and differentiated tissue cells are in strong dependence on each other [140, 141]. The stem/differentiated cell communication regulates the balance between self-renewal and differentiation, that helps tissue to develop, to adapt and to repair [140]. MVs derived from stem cells may be one of the crucial components supporting self-renewal and expansion of stem cells in the niche [102, 134]. Ratajczak et al. [134] showed that MVs released from embryonic stem cells are capable to increase pluripotency of hematopoietic progenitors by delivering RNA and proteins. Indeed, mRNA of Oct-4, Rex-1, Nanog, SCL and GATA-2 genes are highly enriched in MVs in respect to donor embryonic stem cells and MVs contain Wnt3 and Oct4 proteins. These results indicate that MVs are selectively enriched in mRNA during their formation. MVs adhere to target cells, fuse with them, deliver their content into the cytoplasm and the carried mRNAs are translated into proteins by the recipient cells [134]. Recently, Quesenberry and Aliotta proposed that MVs are an integral component of the network of environmental signals that allow the differentiation decision of stem cells in the continuum model of stem cell biology [97, 142].
MVs Derived from Injured Tissue may Reprogram the Phenotype of Bone Marrow or Resident Stem Cells
The participation of stem cells in the reparative processes of organs/tissues includes a bi-directional communication between stem and differentiated cells.
Stem cells can receive signals from injured cells able to induce their recruitment and differentiation. Several studies demonstrated that injured tissues release an increased number of MVs and that mRNAs specific of the damaged tissue are up-regulated in MVs present in blood [143-145]. Quesenberry and Aliotta [146] showed that injured lung cells are able to induce differentiation of bone marrow stem cells trough delivering MVs containing high levels of lung-specific mRNAs. MVs evoke in bone marrow cells the
expression of lung-specific genes such as Clara cell-specific protein, surfactant B, and surfactant C.
We can hypothesize that the MVs derived from injured tissues may organize resident stem cells to tissue repair by a paracrine effect.
MVs Derived from Stem Cells may Reprogram Cells Survived to Injury and Favor Tissue Regeneration
Stem cells stimulate tissue-specific cells to proliferate, protect them from apoptosis and induce progenitor cell differentiation. It has been shown that MSCs stimulate kidney [147], myocardial and vascular regeneration [17, 18, 148, 149], reduce inflammation and promote lung repair [150], enhance nerve and vessels growth mostly through a paracrine mechanism [2, 13]. In fact, MSCs enhance resident cell proliferation after injury and decrease cell apoptosis, but do not usually incorporate into the tissues [147]. It has been shown that even conditioned media from MSCs stimulated migration and proliferation of resident cells, inhibited apoptosis, increased survival, and limited tissue injury [147, 151]. These results suggest that the permanent engraftment of MSCs is not necessary for tissue repair and support the contention that they act by a paracrine/endocrine mechanism.
Plenty of paracrine factors have been involved in stem cell-dependent regeneration. They include not only soluble factors, but also the proteins and RNA carried by MVs released by these cells. Molecules delivered by MVs may induce differentiation of resident stem cells, viability support of differentiated cells survived to injury, and may activate tissue remodeling. It has been shown that MVs released from MSCs are able to enter in the epithelial cells of kidney [21] or hepatocytes [138], delivering their contents. This stimulates in vitro proliferation and apoptosis resistance of tissue-specific cells, and in vivo, accelerates the functional and morphological recovery of kidney and liver tissues [21, 138]. RNA plays the key role in this action. Indeed, stem cell-derived MVs contain specific sets of RNA that can reprogram differentiated cells to a more pluripotent stage. RNA destruction in MVs significantly reduced both the in
vitro and the in vivo effects of MVs, suggesting a mechanism
dependent mainly on RNA delivery.
Based on these data, one can speculate that MVs may be a prospect therapeutic tool free of possible tumorigenicity or stem cell-elicited immune response.
THERAPEUTIC POTENTIAL OF STEM CELL-DERIVED MVs IN TISSUE REPAIR
It has been shown that MVs may regulate angiogenesis by promoting endothelial cell migration, proliferation, invasion, and tube formation [100, 121, 130, 152]. Molecular mechanisms include different types of biological active molecules carried by MVs (Fig. 1). Some of them are lipids such as sphingomyelin and arachidonic acid [153]. Most of the angiogenic stimulators are proteins such as matrix metalloprotease (MMP)-2 and MMP-9 [122], TF [154], leptin, TNF, acidic fibroblast growth factor (FGFa), VEGF, and TGF- [121]. In addition, MVs can enhance angiogenesis both in vitro and in vivo after transfer into recipient cells of pro-angiogenic RNA [130]. It has been
shown that MVs from endothelial precursor cells contain mRNA associated with the PI3K/AKT and eNOS-signaling pathway [130] that play a critical role in angiogenesis [155]. Therefore, MVs may activate angiogenesis either by means of transfer of RNA or by delivering pro-angiogenic factors.
Based on the pleyotropic effects of MVs derived from stem cells, one may envisage that the administration of MVs favors tissue regeneration by inducing changes in the phenotype of tissue resident cells survived to injury (Fig. 2). Indeed, stem cell derived MVs retain several biological activities of the stem cells of origin that are relevant for tissue regeneration and repair. In particular, stem cell derived MVs contain transcripts involved in the control of cell survival, proliferation and differentiation. In vitro and in vivo experiments provide evidence that stem cell derived MVs may counteract apoptosis, inhibit inflammatory and immune response, and stimulate cell proliferation.
It has been shown that MSCs stimulate the regeneration of kidney tissue after acute kidney injury and help to improve functional recovery in chronic kidney diseases [156-158]. Similar studies indicate that MSC may improve the recovery of liver tissue after damage [8, 159].
Bruno et al. [21] showed that MVs secreted by MSCs
stimulate the kidney tissue repair in a manner comparable to MSCs. These MVs stimulate proliferation and protect tubular epithelial cells against apoptosis both in vitro and in
vivo. Moreover, MSC-derived MVs protect kidney from the
development of chronic damage following an acute injury [137]. Herrera et al. [138] showed that MVs derived from human liver stem cells increased in vitro proliferation and apoptosis resistance of human and rat hepatocytes and favor functional and morphological recovery after 70% hepatectomy. This effect was shown to be associated with specific translation of the MV-shuttled mRNA into the hepatocytes of treated rats [138]. Moreover, it was proved that the transferred RNA plays the main role in these actions, because the destruction of RNA in MVs significantly decreased their regenerative effect on kidney and liver. Of course these data do not exclude the participation of MV-shuttled proteins in the regeneration process [21].
CONCLUSIONS
MVs released from stem cells retain several biological properties of the cell of origin. This can be exploited in regenerative medicine for designing therapeutic strategies that avoid the injection of stem cells.
The safety of adult human MSCs has been well documented in several clinical trials. There are no reports of relevant adverse effects of autologous or allogeneic MSC infusion in humans even in a limited number of long-term studies [3-6]. However, based on preclinical studies, some concern remains for development of organ fibrosis or tumorigenesis [160]. Stem cells once injected may in some cases undergo unwanted differentiation. In experimental animal models it has been shown that MSCs injected in the ischemic heart may in the long-term differentiate into bone [20] and in the kidney into adipocytes favoring development of a chronic renal failure [7]. After lung irradiation MSC
administration was shown to favor fibroblast and myofibroblast accumulation in focal pulmonary areas [161]. In addition, the intravenous administration of MSCs may induce formation of pulmonary emboli or infarctions. Moreover, one of the problems of MSC clinical application is the inherent heterogeneity of MSC population that may render difficult the evaluation of the potency of different MSC preparations as well as the comparison of different clinical trials. Estimation of the biological safety and potency of MVs should be easier than for cells, because they
contain well defined patterns of molecules. In addition, allogeneic stem cells may elicit an immune response. MVs derived from adult stem cells do not express HLA class I or II antigens and, at least, in the experimental animals do not activate immunity [21]. The mechanism of action is mainly related to the delivery of proteins and mRNAs and miRNAs that limit the injury and activate regenerative programs in tissue resident cells suggesting that changes induced in the recipient cells are transient. The preliminary experiments in animals indicate MV long-term safety. Ongoing studies are
Fig. (2). Role of MVs released from stem cells in tissue regeneration. A. MVs released from MSCs or from resident stem cells may reprogram tissue injured cells by delivering proteins, mRNA and/or miRNA that induce cell cycle re-entry of these cells thus favoring tissue regeneration. B. The therapeutic use of stem cell-derived MVs would imply the in vitro expansion of relevant stem cells, the collection of cell-free supernatant and the concentration of MVs ether by ultracentrifugation or ultrafiltration techniques. Cultured stem cells may provide a continuous source for MVs. Once isolated, the molecular content of MVs can be defined by proteomic and gene array analysis to evaluate whether they carry the relevant molecules for the tissue regeneration before the injection in patients.
evaluating manufacturing techniques suitable for a large-scale production of MVs from cultured stem cells based on ultrafiltration technologies. However, further experiments are needed to define disease specificity, bio-distribution and persistency of the biologic effects of MVs as well as the long-term safety before MVs may find clinical applications.
ACKNOWLEDGMENTS
Funded by Regione Piemonte, Piattaforme Biotecnolo-giche, Pi-Stem project and by grant of the Ministry of Education and Science, Russian Federation 16.512.12. 2005. LT is a fellow of UNESCO/L’ORÉAL Co-Sponsored Fellowships for Young Women in Life Sciences 2011.
CONFLICT OF INTEREST
MCD, VC, and GC are named inventors in related patents on microvesicles.
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