review http:/ /www.kidney-international.org
© 2010 International Society of Nephrology
Exosomes/microvesicles as a mechanism of
cell-to-cell communication
Giovanni Camussi1, Maria C. Deregibus 1, Stefania Bruno2, Vincenzo Cantaluppi 1 and Luigi Biancone 1
1 Department of Internal Medicine, Centre for Molecular Biotechnology and Centre for Research in Experimental Medicine (CeRMS),
Torino, Italy and 2Sis-Ter, Palazzo Pignano, Italy
Microvesicles (MVs) are circular fragments of membrane
released from the endosomal compartment as exosomes
or shed from the surface membranes of most cell types.
An increasing body of evidence indicates that they play
a pivotal role in cell-to-cell communication. Indeed, they
may directly stimulate target cells by receptor-mediated
interactions or may transfer from the cell of origin to various
bioactive molecules including membrane receptors, proteins,
mRNAs, microRNAs, and organelles. In this review we discuss
the pleiotropic biologic effects of MVs that are relevant for
communication among cells in physiological and
pathological conditions. In particular, we discuss their
potential involvement in inflammation, renal disease, and
tumor progression, and the evidence supporting a
bidirectional exchange of genetic information between stem
and injured cells. The transfer of gene products from injured
cells may explain stem cell functional and phenotypic
changes without the need of transdifferentiation into tissue
cells. On the other hand, transfer of gene products from stem
cells may reprogram injured cells to repair damaged tissues.
Kidney International (2010) 78, 838-848; doi:10.1038/ki.2010.278;
published online 11 August 2010
KEYWORDSe: xosomes; microvesicles; renal injury; stem cells
Correspondence: Giovanni Camussi, Dipartimento di Medicina lnterna,
Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14, Torino 10126,
Italy. E-mail: giovanni.camussi@unito.it
Received 9 April 2010; accepted 9 June 201 0; published on line 11
August 2010
838
Cell-to-cell communication is required to guarantee proper
coordination among different cell types within tissues. Cells
may communicate by soluble factors, 1 adhesion moleculemediated
cell-to-cell interactions including cytonemes that
connect neighboring cells enabling ligand-receptor-mediated
transfer of surface-associated molecules, or by tunneling
nanotubules that establish conduits between cells, allowing
the transfer of not only surface molecules but also cytoplasmic
components.2’3 Recent studies have suggested that cells
may also communicate by circular membrane fragments
named microvesicles (MVs).4 For a long time, MVs were
considered to be inert cellular debris, and the frequently
observed vesicles by electron microscopy in the interstitial
space of tissues or in blood were considered the consequence of
cell damage or the result of dynamic plasmamembrane
tumover.5 De Broe et al.6 first suggested that circular
plasmamembrane fragments released from human cells may
result from a specific process and showed that they may
carry functional membrane enzymes in the same ratio as the
membrane of the cells of origin. However, only recent studies
have assigned a defined function to the vesicles/exosomes
released in the microenvironment by various cell types.
Two distinct processes of vesicle release from the cells have
been described. MVs may derive from the endosomal
membrane compartment that after fusion with the plasma
membrane are extruded from the cell surface of activated
cells as exosomes.7’8 Otherwise, MVs may take origin by direct
budding from the cell plasma membrane as shedding vesicles9.
As the vesicle population detectable both in vitro and in viva is
a mixed population of exosomes and shedding vesicles, we will
refer to them collectively as MVs. Released MVs may remain in
the extracellular space in proximity of the place of origin or
may enter into the biological fluids reaching distant sites. This
may explain the presence of MVs in the plasma, urine, milk,
and cerebrospinal fluid. The bulk of MVs present in the
circulation is derived from platelets, 10 and in less extent from
other blood cells and endothelial cells.11 The MVs derived from
platelets are also designed as micro particles, 10 whereas those
derived from polymorphonuclear leukocytes are also named
ectosomes.12 Finally, MVs released during morphogenesis of
multicellular organisms are indicated as argosomes. 13 Besides
normal cells, tumor cells may also release MVs, and in patients
suffering from neoplastic diseases, tumor-derived MVs may be
Kidney lnternatianal (201 O) 78, 838-848
G Camussi et al.: Microvesicles and cell-to-cell communication
detected within the biological fluids.14’15 Therefore, MVs are an
assorted population, differing in cellular origin, number, size,
and antigenic composition, 16 that are shed by various cell types
in physiological and pathological conditions.
FORMATION OF MVs
The release of MVs may be constitutive or consequent to cell
activation by soluble agonists, by physical or chemical stress
such as the oxidative stress and hypoxia, and by shear stress.4
(i) Exosomes have an endosome origin and are a rather
homogenous population with a size ranging from
30 to 120 nm. 7 They are stored as intraluminal vesicles
within multivesicular bodies of the late endosome and
are released when these multivesicular bodies fuse with
the cell membrane (Figure la). Our knowledge on the
mechanism of assembly and sorting of the exosomes is
only partial, because of the fact that a common sorting
signal for all cell types has not so far been identified. 17
They are released by exocytosis through a mechanism
dependent on cytoskeleton activation and under the
regulation of p53 protein. 18
(ii) Shedding vesicles are usually larger than exosomes
with size ranging from 100 nm to 1 μm. Formation of
shedding vesicles takes place from the budding of small
cytoplasmic protrusions followed by their detachment
from the cell surface (Figure lb). This process is
dependent on calcium influx, calpain, and cytoskeleton
reorganization.9 Schara et al.19 describe two physical
mechanisms involved in the formation of MVs and
nanotubes: the curvature-mediated lateral redistribution
of membrane components with the formation of
membrane nanodomains and the plasma-mediated
attractive forces between membranes. The intracellular
levels of calcium ions modify the asymmetric phospholipid
distribution of plasmamembranes by specific
enzymes named flippase, floppase, and scramblase.20
The increase in calcium ions inhibits translocase and
induces activation of scramblase that translocates
phosphatydilserine from the inner leaflet of the cell
membrane bilayer to the outer. Therefore, MVs expose
on their surface large amounts of phosphatydilserine and
are enriched in proteins associated with membrane lipid
rafts.21 Moreover, the intracellular pathways that activate
reorganization of cytoskeleton induce the detachment of
plasmamembrane protrusions from the cortical actin.
Calcium ions by activation of calpain that cleaves tallin
and activin and of gelsolin that cleaves actin-capping
proteins also favor the reorganization of cytoskeleton.22
Therefore, depending on the cell of origin and on the
mechanism of formation, MVs vary on size and molecular
composition.
MV BIOLOGICAL ACTIVITIES
It is now recognized that MV s are an integral part of the
intercellular microenvironment and may act as regulators of
Kidney International (2010) 78, 838-848
a
Degradative
multivesicular bodies
Plasma
membrane
review
Exocytic
multivesicular bodies
Figure 1 I Schematic representation of exosome and shedding
vesicle formation. (a) Release of exosomes. Exosomes are
accumulated within the multivesicular bodies as a result of
endosome compartmentalization. The vesicles present in
multivesicular bodies may undergo degradation or exocytosis.
The exocytic multivesicular bodies fuse with membrane after cell
stimulation and release exosomes. (Upper inset) Representative
transmission electron microscopy showing exocytosis of
exosomes from the surface of a mesenchymal stem cell (original
magnification x 15,000). (Lower inset) Representative
transmission electron microscopy showing a multivesicular body
within a mesenchymal stem cell (original magnification x 10,000).
(b) Production of shedding vesicles from the cell surface.
Shedding vesicles are sorted out from cytoplasm by budding
of cell plasmamembrane in response to cell stimulation. (Left
micrograph) Transmission electron microscopy panel showing
vesicles shed from the surface of an endothelial progenitor cell
(original magnification x 10,000); the inset shows the high
magnification ultrastructure of a vesicle shed from an endothelial
progenitor (original magnification x 25,000). (Right micrograph)
Transmission electron microscopy panel showing an aspect of cell
membrane budding in an endothelial progenitor cell during
microvesicle (MV) formation (original magnification x 15,000).
The mechanisms involved in MV cargo as well as those involved in
membrane-sorting processes remain at present largely unknown.
cell-to-cell communication. This concept is based on the observation
that MVs released from a given cell type may interact
through specific receptor ligands with other cells, leading to
839
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b
Transfer of
membrane
receptors
G Camussi et al.: Microvesicles and cell-to-cell communication
Figure 21 Schematic representation of mechanisms involved in microvesicle (MV)-mediated cell-to-cell communication. (a) MVs may
act as a ‘signaling complex’ through surface-expressed ligands that directly stimulate the target cells. (b) MVs may transfer receptors
between cells. (c) MVs may deliver functional proteins or infectious particles to target cells. (d) MVs may transfer genetic information via
mRNA, microRNA (miRNA), or transcription factors from one cell to another.
target cell stimulation directly or by transferring surface
receptors.23•24 This implicates that MVs interact only with target
cells that specifically recognize rather than just with any cell
present in the microenvironment 25 This interaction may either
be limited to a receptor-mediated binding to the surface of target
cells forming a platform for assembly of multimolecular
complexes or leading to cell signaling, either to be followed by
internalization as a result of direct fusion or endocytic uptake by
target cells.9 Once internalized, MVs can fuse their membranes
with those of endosomes, thus leading to a horizontal transfer of
their content in the cytosol of target cells. Alternatively, they may
remain segregated within endosomes and be transferred to
lysosomes or dismissed by the cells following the fusion with the
plasmamembrane, thus leading to a process of transcytosis.9
Ratajczak et al.4 proposed that MY-mediated cell-to-cell
communication emerged very early during evolution as a
template for the development of further more refined
mechanisms of cell communication. MYs may influence the
behavior of target cells in multiple ways (Figure 2).
MVs may act as signaling complexes by direct stimulation of
target cells
MVs derived from platelets, for instance, have an important
role in coagulation as their phosphatydilserine-enriched
membranes provide a surface for assembly of clotting
factors.4•9•26 The coagulation defects seen in Scott syndrome
depend on defective scrambling of membrane phospholipids
with an impaired formation of MVs.26 After activation,
platelets shed MVs coated with tissue factor that may interact
with macrophages, neutrophils, and other platelets by ligation
with molecules expressed on the surface of these cells such as Pselectin.
27 On the other hand, MYs released from neutrophils
express activated leukocyte integrin alpha M beta2 (Mac-1)
that is able to induce platelet activation.28 Moreover, plateletderived
MVs, besides coagulation, trigger various cell responses
as they activate endothelial cells,29 polymorphonuclear neutrophils,
30 and monocytes,31 and influence the functions of
normal and malignant human hemopoietic cells.4
MVs may act by transferring receptors between cells
The transferring of receptors between cells is supported by
the observation that bystander B cells rapidly acquire antigen
840
receptors from activated B cells by a membrane transfer.32
This allows an amplified expansion of the antigen-binding
B cells with the ability to present a specific antigen to CD4
T cells. A number of other receptors were found to be
transferred from one to another cell type. For instance, MY s
can transfer the adhesion molecule CD41 from platelets
to endothelial cells33 or to tumor cells,23 conferring
pro-adhesive properties to them. MY-mediated transfer of
Fas ligand from tumor cells induces apoptosis of activated
T cells favoring tumor immune escape. 34 On the other hand,
formation of shedding vesicles may be protective for cells that
dismiss from their membranes to the extracellular compartment
the potentially harmful molecules such as Fas or the
membrane attack complex.35’36 It has also been postulated
that MYs may contribute in spreading certain infective agents
such as human immunodeficiency virus type 1.37 •38 Indeed,
the transfer by MVs of CXCR4 (chemokine (CXC motif)
receptor 4) and CCRS (chemokine (CC motif) receptor 5)
chemokine co-receptors for human immunodeficiency virus
type I may favor the entry of the virus in cells other than the
lympho-hemopoietic lineage.8•39 However, the viral transfer
by MYs may also occur by the so-called ‘Trojan exosome
hypothesis’ involving a direct delivery.40
MVs may deliver proteins within the target cells
An example of this mechanism is the recently reported
MY-mediated transfer of a cell death message via encapsulated
caspase-1.41 It has been found that endotoxin-stimulated
monocytes induce the cell death of vascular smooth
muscle cells by releasing MY s containing caspase-1. This
trans-cellular apoptosis induction pathway depends on the
function of the delivered caspase-1 within the target cells. It
has also been suggested that MYs may contribute to
dissemination of certain infective agents, such as human
immunodeficiency virus or prions. 42 •43
MVs may mediate a horizontal transfer of genetic
information
The occurrence of epigenetic changes has been frequently
reported in co-culture conditions. An explanation of this
phenomenon is the transfer of genetic information between
cells. It has been shown that tumor-derived MVs may transfer
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G Camussi et al.: Microvesicles and cell-to-cell communication
not only surface determinants but also mRNA of tumor
cells to monocytes. 44 Ratajczak et al.45 demonstrated that
MVs derived from murine embryonic stem cells (ESCs)
may induce an epigenetic reprogramming of target cells.
ES-derived MVs were shown to improve survival of
hematopoietic stem/progenitor cells, to induce upregulation
of early pluripotent and early hematopoietic markers, and to
induce phosphorylation of mitogen-activated protein kinase
p42/44 and Akt. In addition, ES-derived MVs were shown to
express mRNAs for several pluripotent transcription factors
that can be delivered to target cells and translated to the
corresponding proteins. As RNase inhibited MY-mediated
biological effect, the involvement of mRNA in the observed
biological effects was suggested.45 We demonstrated that MVs
derived from human endothelial progenitor cells can also
act as a vehicle for mRNA transport among cells.46 MVs
generated from endothelial progenitor cells were incorporated
in normal endothelial cells by interaction with cx4 and
~l integrins expressed on their surface and activated an
angiogenic program. 46 This effect was also proved in viva
in severe combined immunodeficient mice, where
MY-stimulated human endothelial cells subcutaneously
implanted within Matrigel organized in a patent vessel
network connected with the murine vasculature. RNase
pretreatment ofMVs abrogated their angiogenic activity even
though they were internalized by endothelial cells, suggesting
a critical role for RNA transfer following MV incorporation.
The molecular analysis of mRNA indicated that MVs derived
from endothelial progenitor cells were shuttling a specific
subset of cellular mRNA, including mRNA associated with
pathways relevant for angiogenesis such as the PI3K/AKT
and endothelial nitric oxide synthase signaling pathways.
Protein expression and functional studies demonstrated that
phosphatidylinositol 3-kinase and endothelial nitric oxide
synthase were upregulated in target cells after MV incorporation.
As a proof of transduction in target cells of mRNA
delivered from MVs, we used the green fluorescent protein
(GFP) mRNA as reporter. Endothelial cells targeted with
MVs carrying GFP mRNA produced the GFP proteins. 46
More recently, we demonstrated that MVs derived from
human stem cells may also deliver in viva human mRNA to
mouse cells, resulting in protein translation. 47 .48 Yuan et al.49
have recently shown that besides mRNA, MV s may transfer
in target cells microRNA. They demonstrated that MVs
derived from ESCs contain abundant microRNA and that
they can transfer a subset of microRNAs to mouse embryonic
fibroblasts in vitro. As microRNAs are naturally occurring
regulators of protein translation, this observation opens the
possibility that stem cells can alter the expression of genes
in neighboring cells by transferring microRNAs contained
in MVs.
ROLE OF MVs IN INFLAMMATION AND IN CARDIOVASCULAR
AND RENAL DISEASES
Inflammation is sustained by multiple interactions among
cells. In this context, MVs may act at different stages of the
Kidney International (2010) 78, 838-848
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process by carrying either anti-inflammatory or proinflammatory
factors.50 MVs derived from platelets and
macrophages were found to be accumulated in the lipid core
of the atherosclerotic plaques with the potential of triggering
pro-inflammatory, angiogenic, and thrombotic signals.51
These observations rise the possibility that targeting
MVs may be a therapeutic strategy in atherosclerosis.9•50
Indeed, increased levels of MVs of mainly endothelial
origin were observed in cardiovascular pathology.52 Endothelial
dysfunction is an initial event in the development
of atherosclerosis and correlate with an unfavorable
cardiovascular prognosis. 53 Injured endothelial cells may
release MVs, which are considered as markers of endothelial
dysfunction. 54 Moreover, MVs have been implicated
in the modulation of inflammation, as at early stages
neutrophil-derived MVs may stimulate the production of
anti-inflammatory cytokines55 ’56 and at later stages
MVs released from fibroblasts may induce the production
of pro-inflammatory cytokines such as interleukin-6 and
the monocyte chemotactic protein 1 and metalloproteinases.
57
In experimental membranous glomerulonephritis, we
found that the vesicular shedding of terminal components
of complement from the cell plasma membrane protect
podocytes from lyses.35 This, by reducing their surface and
activating the cytoskeleton, may favor retraction of foot
processes and disruption of the slit pore thus favoring
proteinuria. 35
Although in healthy subjects, circulating MVs are mainly
derived from platelets, in pathological conditions MVs may
derive from other cell types such as endothelial and
inflammatory cells and erythrocytes. Augmented blood levels
of MVs have been found in various diseases such as preeclampsia,
58 diabetes,59 acute coronary syndrome, 60 severe
hypertension, 61 multiple sclerosis,62 vasculitis,63 as well as in
patients with chronic renal failure.64 •65
Circulating levels of MVs derived from endothelial
cells correlate with arterial stiffness in hemodialysed
patients. 64 ’66 ’67
The recent discovery of exosomes/MV s in normal urine
opens the possibility of obtaining information on the cell
of origin in physiological and pathological conditions.
It is conceivable that the analysis of urinary MVs may
provide protein biomarkers for the involvement of different
cellular components of the nephron. 68 Indeed, it has been
recently shown that fetuin-A present in urine exosomes is a
novel biomarker of structural renal injury in experimental
models of cisplatin-induced nephrotoxicity and in intensive
care unit patients developing acute kidney injury (AKI).69
Moreover, a reduction in urinary exosomal levels of
aquaporin-1 has been associated with renal ischemia,eperfusion
injury in rats.70 Zhou et al.71 described the presence
of transcription factors in urinary exosomes in different
experimental models of AKI (cisplatin and ischemiareperfusion)
and of podocyte injury (puromycin-treated
rats or podocin-V transgenic mice). In particular, the
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transcription factor activating transcription factor 3 was
associated with AKI and the Wilms tumor 1 with an early
podocyte injury.71
In the setting of transplantation, it has been shown that
the exchange of exosomes between dendritic cells in
lymphoid organs may constitute a potential mechanism by
which passenger leukocytes transfer alloantigens to recipient
antigen-presenting cells, leading to an increased generation
of donor-reactive T cells.72 However, other studies showed
that dendritic cell-derived exosomes may induce tolerance
rather than immune stimulation. In particular, exosomes
isolated from bone marrow-derived dendritic cells administered
before transplantation can modulate heart allograft
rejection, prolonging survival. 73 Moreover, dendritic
a
b
Tissue injury
SM-derived or
tissue-resident
stem cells
~ ‘ erentiation
G Camussi et al.: Microvesicles and cell-to-cell communication
cell-derived exosomes administered after heart transplantation
in combination with short-term immunosuppression
can induce regulatory responses that are able to modulate
allograft rejection and to induce donor-specific allograft
tolerance. 74
On the other hand, MVs derived from cytomegalo
virus-infected endothelial cells can stimulate allogenic
CD4 + memory T cells, providing a new potential mechanism
by which cytomegalovirus can exacerbate allograft
rejection. 75
ROLE OF MVs IN TUMOR BIOLOGY
MVs derived from activated platelets were found to be able to
induce metastasis and angiogenesis in lung cancer.76 Tumor
EGF HGF MSP IGF-1 VEGF
Tissue
repair
~
SM-derived
stem cells
Tissue-resident
stem cells
Figure 3 I Schematic representation of bidirectional exchange of genetic information between stem cells and tissue-injured
cells mediated by microvesicles (MVs}. (a) MVs released from tissue-injured cells may reprogram the phenotype of stem cells to acquire
tissue-specific features by delivering to stem cells the mRNAs and/or microRNAs (miRNAs} of tissue cells. (b) MVs produced by stem
cells recruited from the circulation or from resident stem cells may reprogram tissue-injured cells by delivering mRNA and/or miRNA that induce
the de-differentiation, the production of soluble paracrine mediators, and the cell cycle re-entry of these cells favoring tissue regeneration. BM,
bone marrow; EGF, epiotermal growth factor; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; MSP, macrophage stimulating
protein; VEGF, vascular endothelial growth factor.
842 Kidney lnternatianal (201 O) 78, 838-848
G Camussi et al.: Microvesicles and cell-to-cell communication
cells were also found to release large amount of MVs. The
number of circulating MVs is increased in patients with
cancer and correlate with poor prognosis. 14 It has been
suggested that the release of MVs may protect tumor cells
from apoptosis by extrusion from the cell of apoptosisinducing
proteins. 77 ’78 In addition, cancer cells resistant to
chemotherapy were found to release significant more MVs
than those sensitive to chemotherapy. 79 It has been suggested
that chemotherapeutic agents may be extruded from cells via
MV s. 80 Moreover, it was found that MV s may favor the
escape of tumor cells from immune surveillance. This may
occur either by a mechanism called complement resistance
related to vesicular shedding of terminal components of
complement from the cell plasma membrane, 35•81 or by
shedding of Fas ligand that reduces sensitivity to T-cell Fasmediated
apoptosis. 82 In addition, it was found that tumorderived
MVs can induce apoptosis in activated antitumor
T cells, impairment of monocyte differentiation into
dendritic cells, and induction of myeloid-suppressive
cells.15•83 By carrying active metalloproteinases, MVs may
contribute to stromal remodeling and favor tumor cell
invasion. 84 Moreover, MV s may carry pro-angiogenic signals
that favor the tumor vascularization. 85 ’86 Recently, it has been
shown that tumor-derived MVs may form the pre-metastatic
niche that allow the development of lung metastasis. 87
Finally, it has been suggested that MVs may act by
transferring oncogenes from tumor cells to stromal cells. 88
On the other hand, exosomes derived from mature
dendritic cells have been used as vaccines to stimulate
efficient antitumor cytotoxic T-lymphocyte response. 89
ROLE OF MV-MEDIATED CELL-TO-CELL INTERACTION IN STEM
CELL BIOLOGY
Stem cells are characterized by an unlimited self-renewal and
by high multilineage differentiation potential. Stem cells have
essential roles in organogenesis during the embryonic
development and in many adult tissues are responsible for
the growth, homeostasis, and repair. Depending on the
developmental status and origin, stem cells are classified as
embryonic and adult stem cells. The ESCs are derived from
the inner cell mass of the blastocyst-stage mammalian
embryo few days after fertilization. ESCs are pluripotent as
they generate the germ line during development and virtually
all tissues.
The adult stem cells are undifferentiated cells resident
in tissues, with a more limited self-renewal and differentiation
capabilities. 90-J) 2 When partially committed to
differentiate in a defined cell lineage, they are named
progenitor cells. Adult stem/progenitor cells are present in
most tissues and organs such as bone marrow, liver, pancreas,
heart, kidney, brain, lung, digestive tract, retina, breast,
ovaries, prostate, testis, dental pulp, hair follicles, skin,
skeletal muscle, adipose tissue, and blood. 93 It was assumed
that stem cell self-renewal and differentiation may depend
on an asymmetric division with a regulation that is
hierarchical in nature, leading to a progressive loss of
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proliferative potential when they gain differentiated characteristics.
94 As an alternative to hierarchical model, a
continuum model of stem cell biology has been recently
proposed. 95 •96 According to this theory, the phenotype of
stem cells may vary with cell cycle state and may be reversible.
Therefore, the phenotype of stem cells is reversibly changing
during the cell cycle transit until a terminal-differentiating
stimulus is encountered at a cycle-susceptible time. 95 •96
Recently, Quesenberry and Aliotta97 proposed that the
interaction of stem cells with the microenvironment, also
named niche, have a critical role in defining the stem cell
phenotypes. In this context, MVs may have a regulatory task
by transfer of genetic information between cells. These
researchers proposed that a continuous genetic modulation
through MV transfer between cells is a critical determinant
of stem cell phenotype variation. Indeed, stem cells are an
abundant source of MVs. It has been suggested that MVs
derived from ESCs may represent one of the critical
components supporting self-renewal and expansion of stem
cells.4 •45 In fact, Ratajczak et al.45 demonstrated that MVs
released from ESCs may reprogram hematopoietic progenitors
by a horizontal transfer of mRNA and by delivery of
specific proteins.
MV s, by transferring selected patterns of proteins, mRNAs
and microRNAs, may also act as paracrine mediators of
signaling between stem cells and differentiated cells. We can
envisage a bidirectional exchange of genetic information
from injured cells to bone marrow-derived or resident stem
cells (Figure 3). In the first scenario, MVs released from
injured tissue may reprogram the phenotype of stem cells to
acquire tissue-specific features, whereas in the second, MVs
derived from stem cells may induce cell cycle re-entry of cells
survived to injury allowing tissue regeneration.
MVs derived from injured tissue may reprogram the
phenotype of bone marrow or resident stem cells
It is still debated whether bone marrow-derived stem cells
have the capacity to generate tissue-specific cells after their
engraftment in injured tissues.98 •99 Poulsom et al.100 demonstrated
that bone marrow-derived cells could contribute
to regeneration of the renal tubular epithelium, and in
subsequent studies Fang et al. 101 suggested that the
hematopoietic stem cells rather than the mesenchymal stem
cells (MSCs) contribute to the repair of AKI. However,
transdifferentiation as a mechanism of stem cell plasticity has
never been conclusively proved and several studies challenged
the ability of bone marrow-derived stem cells to differentiate
in tubular epithelial cells.99 •102 •103 Fusion studied with crosssex
transplantation experiments has been suggested as a
mechanism of bone marrow stem cell plasticity in some
reports but not in others.
As an alternative to transdifferentiation and fusion,
Quesenberry and Aliotta97 suggested that stem cell differentiation
depends on epigenetic cell changes mediated by signals
received from injured cells and delivered by MVs. 104 Coculture
of bone marrow cells with injured lung cells induced
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the expression of lung-specific genes and proteins such as
Clara cell-specific protein, surfactant B, and surfactant C in
bone marrow cells.105 It was found that changes in bone
marrow stem cell phenotype depend on MV s released from
injured cells that contain high levels of lung-specific mRNAs
b
MSC TEC TEC+MV
-90 bp
SUM0-1
SUM0-1 Merge
844
G Camussi et al.: Microvesicles and cell-to-cell communication
and deliver these mRNAs to bone marrow cells. This may also
explain the observation that the conditioned medium derived
from renal tubular epithelial cells initiates differentiation of
human MSCs.106 Indeed, in preliminary experiments we found
that MVs derived from injured renal tubular epithelial cells
SUM0-1 Merge
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G Camussi et al.: Microvesicles and cell-to-cell communication
may induce expression of tubular cell markers in human
MSCs. One can speculate that MVs released from injured
tissue may reprogram not only bone marrow-derived stem
cells, but also resident stem cells. Several studies indicate the
presence of resident stem cell populations within the kidney
that may contribute to renal repair.47 •107•108
Taken together, these results suggest that MVs derived
from injured tissues mediated transfer of genetic information
that could explain not only the plasticity and phenotypic
changes of stem cells, but also the functional effects without
the need of their transdifferentiation into tissue cells.
MVs derived from stem cells may reprogram cells survived to
injury and favor tissue regeneration
Experiments based on exogenous MSC administration in
AKI demonstrate a functional and morphological recovery
from acute tubular injury induced by toxic and ischemiareperfusion
injury 99 •109•1 ‘ 0 and a functional improvement in
chronic renal failure. 111 As these beneficial effects are
associated only with a transient recruitment of MSC
within the renal vasculature with a minimal incorporation
within the regenerating tubules, 102•103 it has been suggested
that MSC may provide a paracrine support to the repair
of injured tissue.112 On the other hand, many studies on
tubular repopulation after acute injury indicate a prominent
contribution of renal tubular cells.112′ 113 Strong support of a
paracrine/endocrine mechanism for tissue repair comes from
experiments of Bi et al., 114 showing that the administration
of conditioned medium from MSC is able to mimic the
beneficial effects of the stem cell therapy. They demonstrated
that MSC may favor renal regeneration independently from
engraftment within tubules by producing factors that
limit apoptosis and enhance proliferation of tubular cells.
A growing body of evidence supports the hypothesis of
a paracrine mechanism in bone marrow-derived stem cell
therapy in other organs also, such as infarcted hearts. 115
Indeed, the frequency of stem cell engraftment and
review
transdifferentiation or fusion to generate new cardiomyocytes
and vascular cells appear too low to explain the beneficial
effects observed. Conversely, several studies indicate that
stem cell-released soluble factors may contribute to cardiac
repair and regeneration. 116
The paracrine mediators involved in the beneficial effect of
exogenous stem cell administration may include not only
growth factors, 103•114 but also the MVs released from stem
cells. We envisage the possibility that MVs released from
stem cells recruited at the site of tissue injury may induce
de-differentiation of resident cells survived to injury with
re-entry to cell cycle and activation of tissue regenerative
programs (Figure 3b). Indeed, human MVs released from
MSCs are able to enter in the epithelial cells, delivering their
mRNA cargo (Figure 4). This stimulates in vitro proliferation
and apoptosis resistance of tubular epithelial cells that
acquire a mesenchymal phenotype. In vivo, MVs accelerate
the functional and morphological recovery of glycerolinduced
acute kidney injury in severe combined immunodeficient
mice (Figure 4).47 As the efficacy of MVs is
comparable to that of MSC administration in inducing renal
repair, our own bias is that the beneficial effect of MSCs is
largely due to the release of MVs. RNA inactivation in MVs
abrogated both the in vitro and the in vivo effects of MVs,
suggesting a mechanism dependent on RNA delivery. Indeed,
MVs contain a defined subset of transcripts representative of
the multiple differentiative and functional properties of
MSCs.47 Preliminary results indicate that MSC-derived MVs
also contain defined patterns of microRNAs that may serve as
molecular signature and suggest a specific rather than a
random accumulation in MVs.117 A stimulus-dependent
variation of RNA species packed within MVs suggests
a tightly regulated process in their generation within the
cells. We are currently investigating whether mRNA and
microRNA entry in target cells activates translational control
mechanisms or specific checkpoints for the transcripts.
Whether MVs produced by stem cells may provide a
Figure 41 Effect of mesenchymal stem cell (MSC)-derived microvesicles (MVs) in vitro on cultured mouse tubular epithelial cells
(TECs) and in vivo on glycerol-induced acute kidney injury (AKI) in severe combined immunodeficient (SCID) mice (see Bruno
et al.47 ). (a) Representative confocal micrograph showing the internalization by mouse TECs (30 min at 37 °C) of 30 μg/ml MVs labeled with
PKH26 (red). Nuclei were stained by Hoechst dye (blue; original magnification x 400). The mRNA horizontal transfer and human protein
translation by mouse TECs treated with human MSC-derived MVs was shown by reverse transcriptase-PCR (RT-PCR) for a specific human
mRNA using small ubiquitin-like modifier-1 (SUMO-1) as target mRNA and by immunofluorescence using anti-human SUMO-1 antibodies.
(b) A band of PCR products specific for human SUMO-1 of the expected size (90 bp) was detected in a 4% agarose gel electrophoresis in
TECs cultured in the presence of 30 μg/ml MVs, whereas it was absent in TEC alone. As positive control, the extract of human bone marrowderived
MSC (BM-MSC) was used. (c, d) Representative micrographs showing the expression of human SUMO-1 proteins by mouse TECs
cultured in the absence or in the presence of 30 μg/ml MVs for 24 h. SUMO-1 was detectable in the cytoplasm and nuclei of TECs incubated
with MVs (d) but not in untreated TECs (c). Nuclei were counterstained with Hoechst dye (blue; original magnification x 400). (e-g)
Representative micrographs of semifine sections (e, f) and transmission electron microscopy (g) showing the diffuse tubular injury
characterized by blebbing, loss of brush border, and necrosis of TECs and by the presence of intraluminal tubular casts in mice 5 days after
glycerol-induced AKI. (h-j) Representative micrographs of semifine sections (h, i) and transmission electron microscopy (j) showing the
morphological recovery induced by treatment with 10 μg MSC-derived MVs in mice 5 days after glycerol-induced AKI. The inset in (h) shows
the accumulation of PKH26-labeled MVs within the TECs (original magnification e and h x 150; f, g, and i x 600; and j x 3000). (k-n) The
detection of human protein expression in kidneys of mice treated with human MSC-derived MVs indicated the translation of human
proteins by the horizontally transferred mRNA into TECs in vivo. Representative confocal micrographs showing the presence of staining for
human SUMO-1 protein with cytoplasmic and nuclear expression in kidney sections of AKI mice treated with MVs and killed 48 h later (k, I)
or in control mice untreated with MVs (m, n). Nuclei were counterstained with Hoechst dye (original magnification x 400).
Kidney International (2010) 78, 838-848 845
review
potential therapeutic strategy to avoid the possible maldifferentiation
of stem cells once engrafted in the kidney in
the long term 118 requires further investigations. We recently
showed that MV-mediated transfer of RNA-based information
from human liver stem cells stimulates liver regeneration
in a model of 75% hepatectomy. 48
CONCLUSION
The main function of MVs is signaling through specific
interactions with target cells and transferring gene products.
Therefore, they may partmpate in physiological and
pathological processes. Gaining further insights into the
molecular specificity of MVs may allow the identification of
the cellular source and may provide new diagnostic tools.
Indeed, an increasing body of evidence indicates that
MVs may offer prognostic information in various diseases
such as chronic inflammation, cardiovascular and renal
diseases, pathological pregnancy, and tumors. The presence
ofMVs in body fluid makes them readily accessible, and their
number, cellular origin, composition, and function can be
disease state dependent. Cancer cells, for example, shed
MVs that might not only help tumor and metastasis
development but also represent an important non-invading
diagnostic tool especially with regard to the fact that they
contain genetic material under the form of RNA, which
could be easily screened for cancer genetic markers.
In addition, the recognition of the signals delivered by
MVs may open new therapeutic strategies. The removal
from plasma of harmful MVs may be beneficial in
pathological conditions where MVs deliver thrombogenic
and inflammatory signals or in tumors. On the other
hand, MVs derived from stem cells may reprogram altered
functions in target cells, suggesting that they could be
exploited in regenerative medicine to repair damaged tissues.
Moreover, MV-mediated transfer of genetic information
could explain the observed plasticity and the functional
effects of stem cells without the need of their transdifferentiation
into tissue cells. Many points require further
investigation: (1) the stimuli and the molecular pathways
that regulate the assembly within MVs of the biologically
active molecules that they shuttle; (2) the stimuli that trigger
their release; (3) the surface receptors that may confer
selective specificity; (4) the full diagnostic potential of MVs
in different pathological conditions; (5) the strategy to
inhibit formation or to remove from circulation potentially
harmful MVs; and (6) the therapeutic exploitation in
regenerative medicine of the ability of MVs to modify the
phenotype and function of target cells. The recognition of
the importance of MVs may open new perspectives of
investigation.
DISCLOSURE
All the authors declared no competing interests.
ACKNOWLEDGMENTS
Our research was supported by grants from Regione Piemonte,
Piattaforme Biotecnologiche, progetto PiSTEM, and from Oncoprot.
846
G Camussi et al.: Microvesicles and cell-to-cell communication
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