The Role of Placental Exosomes in Reproduction

The Role of Placental Exosomes in ReproductionLucia Mincheva-Nilsson, Vladimir Baranov

Department of Clinical Immunology, Umea University, Umea, Sweden

Introduction

The mammalian pregnancy, in particular the human

hemochorial pregnancy, is the highest and most

complicated mode of reproduction that puts a great

strain to the maternal organism and comprises a

biological challenge to create an immunologic toler-

ance to the fetal allograft and support its develop-

ment and growth. The key for successful

mammalian reproduction, the placenta, is a transient

organ that mediates not only hormonal, nutritional

and oxygen support to the fetus but also actively

secretes signal substances and immunoregulatory

factors that modulate ⁄ alter the maternal immune

response during pregnancy. Recent reports1–6 have

shown that the main and unique cell type in the

human placenta, the syncytiotrophoblast, releases

biogenetically and morphologically defined, nanome-

ter-sized microvesicles called exosomes, which are

used in the fetal–maternal cross-talk for adaptation

of the maternal organism to the ongoing pregnancy.

Cell-to-cell communication is of crucial impor-

tance for all living organisms. Cells may communi-

cate and exchange information by different

mechanisms: (i) via soluble mediators such as

hormones, cytokines, chemokines, bioactive ions

and lipids that are released from cells and act in an

autocrine or paracrine manner; (ii) via direct adhe-

sion contacts between signaling and target cells,

including trogocytosis; (iii) exchanging information

through intercellular nanotubules.7,8 There is, how-

ever, emerging evidence that a fourth mechanism

built on release and uptake of membrane-bound

microvesicles (MV) exists. Cell–cell communication by

MV comprises a combination of secretion of molecules

on one hand and preservation of their membrane-

association on the other hand. Secreted MV execute

cell-to-cell contact ‘by proxy’ delivering signals and

transferring packages of information from a donor

cell to a target cell locally, and ⁄ or at a distance. The

smallest members of the MV family, the exosomes,

are nanometer-sized and actively secreted by most

Keywords

Exosomes, FasL, MICA ⁄ B, microvesicles,

placenta, ULBP

Correspondence

Lucia Mincheva-Nilsson, Department of Clinical

Immunology, Umea University, S-90185 Umea,

Sweden.

E-mail: [email protected]

Submitted January 21, 2010;

accepted January 21, 2010.

Citation

Mincheva-Nilsson L, Baranov V. The Role of

Placental Exosomes in Reproduction. Am J

Reprod Immunol 2010; 63: 520–533

doi:10.1111/j.1600-0897.2010.00822.x

Cell communication comprises cell–cell contact, soluble mediators and

intercellular nanotubes. There is, however, another cell–cell communi-

cation by released membrane-bound microvesicles that convey cell–cell

contact ‘by proxy’ transporting signals ⁄ packages of information from

donor to recipient cells locally and ⁄ or at a distance. The nanosized

exosomes comprise a specialized type of microvesicles generated within

multivesicular bodies (MVB) and released upon MVB fusion with the

plasma membrane. Exosomes are produced by a variety of immune,

epithelial and tumor cells. Upon contact, exosomes transfer molecules

that can render new properties and ⁄ or reprogram their recipient cells.

Recently, it was discovered that the syncytiotrophoblast constitutively

and throughout the pregnancy secretes exosomes. The placenta-derived

exosomes are immunosuppressive and carry proteins and RNA mole-

cules that in a redundant way influence a number of mechanisms and

promote the fetal allograft survival. In this review, we summarize the

current knowledge on the nature of placenta-derived exosomes and

discuss their role in pregnancy.

REVIEW ARTICLE

American Journal of Reproductive Immunology 63 (2010) 520–533

520 ª 2010 John Wiley & Sons A/S

cell types throughout the body. Their functions are

diverse and related to the physiological functions of

the donor cells.9 Most published reports demonstrate

their immunomodulatory features (reviewed in ref.

10,11).

Microvesicles’ secretion in the placenta occurs

both by blebbing ⁄ shedding of syncytiotrophoblast

microparticles (STBM) from the plasma mem-

brane12–14 and by exosome release through the

endosomal pathway.1–6 STBM are 0.2–2 lm in size

and are present in a low plasma concentration in

normal pregnancy. They are distinct from exosomes

and comprise a heterogeneous population, involving

shed microvilli and apoptotic fragments. Enhanced

shedding of STBM promotes endothelial and

immune cell dysfunction, inflammation and necro-

sis ⁄ apoptosis and is somehow associated with the

placental pathophysiology in preeclampsia.12–14

Distinguishing between STBM and exosomes as

separate bioactive entities is required to determine

precisely their role in reproduction. In this review,

we will focus on the placenta-derived exosomes and

discuss their role in normal pregnancy. In the

following text, a brief characterization of various

microvesicles and a description of the biogenesis and

nature of exosomes are given as a background.

Microvesicles are everywhere

Although various membrane-bound MV have been

observed for years in the intercellular space by elec-

tron microscopy, the existing general dogma consid-

ered them as an inert cellular debris ⁄ dust that

should be more or less ignored. Only recently has it

become clear that the MV are not a uniform entity

but differ in size, morphology and nature and can be

either shed from the plasma membrane or secreted

through the endosomal membrane compartment.

The realization that various MV are found in health

and disease, and that they might have different roles

depending on their type and the cells from which

they are derived, has opened new perspectives in

understanding biological processes and their regula-

tion. To state that MV are everywhere, in the blood

and all bodily effusions, is not an overstatement. In

fact, they are an integral part of the intercellular

environment. MV are a heterogeneous population

that vary in size (0.03–2 lm), shape and composition

depending on the cellular sources and how they are

generated. The MV can be divided by size into large

(0.1–2 lm) and small (30–100 nm). Two main types

comprise the large MV: (i) those produced by blebbing

of the plasma membrane upon programmed cell death

called apoptotic bodies ⁄ apoptotic blebs ⁄ apoptotic

vesicles that are a product of dying cells;15,16 and (ii)

those produced by direct budding from the plasma

membrane of living cells called microvesicles (some-

times named microparticles or ectosomes). Examples

of the latter are MV shed by plasma membrane of

various epithelia17–19 including syncytiotrophoblast,

such as shed microvilli from cell surface membranes;

prostasomes,20 budded from the apical plasma mem-

brane of prostate epithelium; and prominosomes,21

prominin-1-expressing plasma membrane microvesi-

cles from brain stem cells. The small-sized MV

(<100 nm) called exosomes, which represent an

apparently distinct class of MV, arise from the endo-

somal membrane compartment, have a definite

morphology and are more homogeneous in their size

and biological composition. MV, including exosomes,

are produced by many hematopoietic and non-

hematopoietic cells, such as reticulocytes, mast cells,

T and B cells, platelets, dendritic cells, neurons and

microglia, various epithelial cells like enterocytes

from small and large intestine, uroepithelia, bron-

chial epithelia, syncytiotrophoblast, hepatocytes as

well as tumor cells.10,11,19 MV have been isolated

from blood and various bodily fluids such as saliva,

urine, amniotic fluid, malignant effusions, bronchial

lavage fluid, synovial fluid, and breast milk

(reviewed in ref. 22). The major part (about 80%) of

MV, constitutively present at low levels in the

peripheral blood of healthy subjects, are platelet

derived.23 The rate of clearance of circulating MV in

humans is presently not known. In rat experiments,

80% of labeled MV were cleared from the circula-

tion within 5 min.24 Various populations of micro-

vesicles and some of their characteristics regarding

morphology, size and genesis are presented in

Table I.

General description of the biogenesis, structural

organization and biological properties of exosomes

History of exosome discovery

The first reports of exosome-like microvesicles

produced by ovarian cancer and neoplasic cell lines

appeared about 30 years ago.25,26 Independently,

Johnston et al. discovered that normal reticulocytes

used microvesicles to eliminate the transferrin receptor

and made the important discovery that they were

secreted from the endosomal compartment.9,27 Since

PLACENTAL EXOSOMES IN REPRODUCTION


ª 2010 John Wiley & Sons A/S 521

then, for many years, exosomes remained in obliv-

ion and only in the past decade were they ‘rediscov-

ered’ as a new and exciting ‘fourth mechanism of

cellular communication’ with a powerful influence

and a biological role.

Definition and biogenesis

Exosomes are defined as secreted membrane-bound

nanovesicles that are identified by the following

characteristics: (i) cup-shaped form; (ii) 30- to 100-nm

size; (iii) buoyant density of 1,13-1,19 g ⁄ mL on

sucrose gradient; (iv) endosomal origin; (v) tetraspanins

present in their lipid raft–rich membrane.10 The sug-

gested biogenesis of exosomes, illustrated schemati-

cally in Fig. 1, separates them from all other MV so

far known. They are formed in the late endosomal

membrane compartment by inward budding of the

limiting membrane of late multivesicular endo-

somes ⁄ multivesicular bodies (MVB) and contain cell

surface–expressed proteins and cytosolic compo-

nents. They are actively secreted into the extracellular

space by fusion of the MVB with the plasma

membrane.

The mechanism(s) of protein sorting during

exosome formation is not fully understood. It is

Table I Summary of some of the main characteristics of different types of microvesicles

Characteristics Exosomes Microvesicles ⁄ microparticles Shed microvilli

Apoptotic

bodies ⁄ vesicles

Size 30–100 nm 0.1–2 lm >400 nm 100–600

to700 nm

Density in sucrose 1.13–1.19 g ⁄ mL Undetermined Undetermined 1.16–1.28 g ⁄ mL

Sedimentation (g) 100,000–110,000 10,000–100,000 10,000 1500–100,000

Morphological

shape

Cup-shaped, electron

translucent

Various shapes,

electron-dense and ⁄ or

electron translucent

Various shapes, round,

elongated and cylinder-like

Irregular and

heterogeneous in

shape

Membrane

composition

Cholesterol-, sphingomyelin-,

and ceramide-rich lipid rafts,

expose phosphatidylserine

Expose phosphatidylserine,

some enriched in

cholesterol and

diacylglycerol, some

undetermined

Undetermined Undetermined

Specific marker(s)

for identification

Tetraspanins (CD63, CD9,

CD83), ESCRT complex

members (Alix, TSG101)

Integrins, selectins, CD40 and

others, depending on the

cell type

Various, depending on the

cell type

Histones, DNA

Origin in the cell Multivesicular bodies (MVB) Plasma membrane Plasma membrane Fragments of

dying cells,

undetermined

Mode of

release ⁄ secretion

Fusion of MVB with the

plasma membrane

Plasma membrane blebbing Plasma membrane blebbing Plasma membrane

blebbing and

cellular

fragmentation

Fig. 1 Biogenesis of exosomes – schematic presentation of the

endosomal pathway by which exosomes are generated and secreted.

CG, Complex Golgi; RER, rough endoplasmic reticulum; MV, micro-

vesicle; MVB, multivesicular body.

MINCHEVA-NILSSON AND BARANOV



suggested that proteins, carried by exosomes, are

sorted to the MVB in two ways: (i) by endocytosis

and transport of plasma membrane proteins to the

early recycling- and further to the late endosomal

compartment; and (ii) by direct transportation from

the Golgi complex to MVB and insertion in the MVB

membrane to bud as exosomes. For many years,

MVB were considered to be the late stage in the

maturation of endosomes to lysosomes – ‘garbage

stations’ for proteins destined for destruction.

However, approximately two decades ago, an alter-

native function for the MVB was described, namely

their ability to move to the plasma membrane and

fuse with it. After fusion, the MVB secrete their

intraluminal vesicle cargo in the extracellular space

as exosomes.10 Thus, the MVB are situated at a

‘cross-road’ in the endosomal pathway, where the

fate of the proteins sorted to the MVB is decided –

either secretion to the extracellular space as exo-

somes or degradation in the lysosomal ‘dustbin’.28

Accordingly, two classes of MVB are proposed – deg-

radative MVB that evolve to lysosomes and exocy-

totic MVB that fuse with the plasma membrane.

Recently, it was shown that different biochemical

properties may govern the alternative fates of these

two types of MVB.29 The mechanisms governing

degradative MVB involve the well-characterized

multiprotein network called endosomal-sorting

complexes required for transport (ESCRT complexes)

and an ubiquitinylation process resulting in

ubiquitin tagging of both cell surface proteins and

intracellular proteins targeted for lysosomal degrada-

tion.28,30–32 The mechanism underlying exocytic

MVB trafficking is less clear. The transmembrane

protein TSAP6 has been suggested to regulate

exosome production,33 and Rab11, a member of the

small GTPase family, together with calcium was

shown to be important for the docking and fusion of

MVB with the plasma membrane.34 It is, further-

more, suggested that sorting of proteins for exosome

release might be ubiquitin independent and instead

involve sphingomyelin metabolites, such as ceramide

as well as the ESCRT multiprotein complex.29

Although considerable progress is made to reveal the

key links between ubiquitin, phospholipids and the

ESCRT proteins, more studies are needed to eluci-

date the biogenesis of exosomes.

Morphology and general biochemical composition

Electron microscopy (EM) is a superior methodologi-

cal approach for studies of exosome morphology and

biogenesis. Exosomes, visualized in situ by EM, are

uniform spherical vesicles 50–90 nm in size situated

within the lumen of MVB. The EM image of isolated

exosomes is different – they are typically uniformly

cup-shaped but heterogeneous in size, varying

a b

c

d

Fig. 2 Micrographs illustrating exosome biogenesis in human first-trimester syncytiotrophoblast visualized by IEM staining for FasL, MICA ⁄ B, ULBP

and TSG101. a. Perinuclear-located multivesicular bodies, stained with monoclonal antibodies against FasL. Note the staining of the limiting

membrane and the numerous intraluminal vesicles. b. Multivesicular bodies stained with antibodies against MICA ⁄ B proteins localized near the api-

cal microvillous surface of the syncytiotrophoblast membrane. One of them (star) is fused with the plasma membrane, and the content of exo-

somes is released in the intervillous space. c. Complex Golgi (star), stained with monoclonal antibodies against FasL illustrating protein synthesis.

d. Two MVB stained with monoclonal antibody against the endosomal-sorting complexes required for transport (ESCRT) protein TSG101. Note the

staining of the limiting membrane and intraluminal microvesicles. MVB, multivesicular body; N, nucleus; CG, complex Golgi. Magnification:

a: ·15,000; b: ·18,000; c and d: ·30,000.




between 30–100 nm. The reason for this appearance

(Fig. 3a), currently used as the morphological

hallmark for isolated exosomes, is not known but

might be a consequence of the isolation procedure.

The exosomal membrane is detergent resistant with

a lipid raft–rich bilayer, built up of cholesterol,

sphingolipids and tetraspanins, where proteins with

transmembrane or GPI linkage are inserted.35 As

other MV, they expose phosphatidylserine, but at

low level, on their surface and can be captured by

phospatidylserine receptors expressed on surface of

activated T lymphocytes and phagocytes.36 The

molecular content of exosomes includes proteins and

the ribonucleic acids mRNA and micro RNA

(miRNA) and is dependent on the tissue ⁄ cell type

from which the exosomes originate.37 Nearly all exo-

somes, regardless of their origin, carry a conserved

set of proteins. Examples of commonly found exo-

somal proteins are cytoplasmic proteins such as

tubulin, actin, actin-binding proteins, annexins and

Rab proteins; the heat shock proteins hsp70 and

hsp90; signal transduction kinases, heterotrimeric

G-proteins; members of the ESCRT complex Alix

and TSG101. Common surface–expressed proteins

are MHC class I molecules; adhesion molecules such

as b integrins, and ICAM-1, and the class of proteins

Fig. 3 Electron micrographs of placental

exosomes illustrating cup-shaped morphology

and immunogold staining showing CD63-,

PLAP-, MIC- and ULBP1-3 expression.

Bar = 100 nm.




that have become exosomal markers – the tetraspanins

CD9, CD63, CD81 and CD82 (reviewed in ref. 37).

Advantages of exosome-mediated protein secretion and

suggested functions of secreted exosomes

Exosome release seems to be a powerful way of

intercellular communication. It has emerged as a

‘non-classical’ form of secretion compared to the

‘classical’ excretory pathway. The advantages of

exosome-mediated protein secretion are many: (i)

preservation of the three-dimensional structure of

the transported proteins and thus their biological

activity; (ii) independence of cell-to-cell contact for

signal delivery; (iii) packages of carried molecules

give lower mobility and higher concentration of the

carried molecules; (iv) independence of de novo

protein synthesis; (v) biological effect at a distance.

A variety of important biological functions like

intercellular signaling, antigen presentation, immune

regulation, pro- or antiapoptotic effects, delivery of

protein molecules via binding to the target’s plasma

membrane or internalization by endocytosis and

transport of bioactive RNA between cells19,38–40 have

been ascribed to exosomes. A major role of secreted

exosomes is their powerful effect on the immune

system that divides them into two groups –

exosomes with immunoactivating properties and

those with tolerogenic or even immunosuppressive

effect. In general, exosomes produced by antigen-

presenting cells such as dendritic cells (DC),

macrophages and B cells are immune activating.

They activate directly or indirectly, via DC or cyto-

kine production of T helper cells, immune effector

mechanisms such as cytotoxicity, antibody and

cytokine production, and priming of T cells.19 Impor-

tantly, in activated T and NK cells, the cytotoxic

molecules perforin, granzyme, the proapoptotic

molecules FasL and TRAIL are carried by endosomal

vesicles of specialized MVB, also named secretory

lysosomes.2,41,42 On the other side, exosomes pro-

duced by epithelial cells and the great majority of

tumors are immune inhibitory.43,44 Normal epithelial

cell-derived exosomes exert an immunosuppressive

effect promoting homeostasis and immune toler-

ance.43 Remarkably, many tumors ‘hijack’ features

of the immune cells by expression and release of

ligands to important immune receptors and ⁄ or key

immune molecules and signal substances on exo-

somes that are used as decoys, disturbing and down

regulating normal immune effector mechanisms or

enhancing the suppressive function of T regulatory

cells.44,45 Thus, the net effect of the function of

tumor exosomes originating from mammary, lung,

colon, prostate and ovarian cancer46 is a powerful

inhibition of the host immune system promoting the

establishment of the primary tumor and its metasta-

ses. Placenta-derived exosomes, like tumor-derived

ones, are immunosuppressive.

Secretion of placental exosomes – a constitutive

feature of human normal pregnancy

Biogenesis of placental exosomes

Taylor et al. did some pioneering work on describing

circulating ‘shed placental fragments’47,48 that later

they characterized as exosomes and studied their

role in the down regulation of T-cell responses of

pregnant women.3,4 Mor et al.1 reported that

isolated trophoblast cells from first trimester preg-

nancy lacked plasma membrane–associated FasL but

expressed cytoplasmic FasL that was secreted via

microvesicles. We confirmed these findings at the

ultrastructural level, and using immunoelectron

microscopy (IEM), we provided the first data of the

biogenesis of placental exosomes demonstrating that

the intracellular FasL in human placenta was

concentrated to MVB in the syncytiotrophoblast and

expressed and secreted by microvesicles ⁄ exosomes of

60–100 nm size.2 Since then, we have studied the

biogenesis, composition and role of syncytiotropho-

blast-derived exosomes in situ and in vitro from

placental explant cultures. We studied exosome

biogenesis in the syncytiotrophoblast of human

placenta by IEM expression analyses of FasL and the

two families of the NKG2D receptor ligands, i.e. the

MHC class I chain–related antigens A and B (MICA

⁄ B) and the human retinoic acid early transcript 1

(RAET1) proteins also called ULBP 1-5, as marker

molecules of endosomal compartments.2,5,6 We were

surprised to find that in case of FasL and ULBP, the

protein expression was absent on the surface of

the syncytiotrophoblast and instead found only in

the late endosomal compartment, where the proteins

were located on the MVB’s limiting membrane and

on numerous intraluminal vesicles with the size of

exosomes (Fig. 2a). MVB, filled with exosomes

carrying a particular protein, could be found at dif-

ferent levels in the syncytioplasm and frequent

fusion of MVB with the apical microvillous surface

and release of their vesicles into the intervillous

extracellular space could be observed as illustrated

in Fig 2b. In contrast to FasL2 and ULBP ⁄ RAET1




proteins (Fig. 2a, and ref. 6), which were typically

retained in late endosomes ⁄ MVB (Fig. 2a) and

released as exosomes, the MICA and B molecules

were expressed both on the apical cell surface and

inside MVB (Fig 2b, ref. 5). At present, the mecha-

nisms regulating the expression, distribution and

turnover of proteins in the plasma membrane or

MVB (exosomes) are not clear. It has been suggested

that in some cell types, post-transcriptional mecha-

nisms exist that regulate the levels of NKG2D ligands

at the cell surface by retaining them in intracellular

compartments, particularly in MVB.49 Indeed, it has

very recently been shown in several cellular systems

that MICB molecules have a very short half-life at

the plasma membrane but are accumulated in late

endosomal compartment.50 It is possible that sorting

of MICB, FasL, ULBP4 and 5 to MVB is controlled

by ubiquitination as they possess lysine residues

within their cytoplasmic tail.51,52 In Fig. 2d, syncy-

tiotrophoblastic MVB, stained with the ESCRT mem-

ber TSG101, is shown, supporting this suggestion. In

contrast, GPI-linked ULBP1-3 and the transmem-

brane MICA molecule from 008 locus, which is the

most frequent allele in human populations and has

truncated cytoplasmic tail, are preferentially

expressed in lipid rafts at the cell surface.53,54 It has

been suggested that these raft domains may support

the sorting of proteins to MVB and formation of exo-

somes.54,55 From these studies can be concluded that

both plasma membranal and exosomal protein

expressions are used in the syncytiotrophoblast. The

conspicuous endosomal membrane compartment,

represented primarily by MVB, and the frequent

observation of MVB fused with the apical plasma

membrane suggested that exosome secretion might

be a constitutive feature of the syncytiotrophoblast

and seemed to be preferentially used. It is obvious

that the choice ⁄ preference of exosomal release of

FasL, ULBP and, partially, of MICA ⁄ B over plasma

membranal expression is of crucial importance for

the protection of the fetal allograft. Surface expres-

sion of FasL in the placenta would be easily involved

in induction and promotion of inflammatory

response at the fetal–maternal interface,56 while

exosomal FasL is proapoptotic and promotes

immunotolerance.1–4 Similarly, a strategy of releas-

ing NKG2D ligand-carrying exosomes from placenta

would be a decoy mechanism to downregulate the

cognate cytotoxic receptor; in contrast to a plasma

membranal ligand expression that would make the

syncytiotrophoblast a target for attack by NKG2D

receptor-bearing maternal lymphocytes. In conclu-

sion, our IEM data show that the exosome biogene-

sis in the syncytiotrophoblast follow that of other

cells and occurs within MVB. As a vigorous protein

producer, the syncytiotrophoblast is an excellent

model for studies of the biogenesis of exosomes.

Composition, structure and functional properties of

secreted placental exosomes

Methodological consideration

Phenotypic and functional studies of isolated

exosomes demand stringent purification procedures.

The choice of isolation method is of crucial impor-

tance for obtaining ‘pure’ exosome populations, i.e.

free from contaminating proteins, non-exosomal

microvesicles ⁄ microparticles and apoptotic bodies.

This is even more important when studies of exo-

somes in pregnancy are performed, as it is well

known that the syncytiotrophoblast in vivo constitu-

tively generates considerable amounts of microvesi-

cles ⁄ microparticles.13 The most used method,

consisting of a series of centrifugations to remove

dead cells ⁄ debris followed by ultracentrifugation,

will pellet the exosomes in a mixture with other

MV. For enrichment and purification of the exo-

somes, additional ultrafiltration and continuous

sucrose gradient (floating density 1,13-1,19) or

ultracentrifugation with sucrose cushions57,58

should be performed. Another method is using

adherence to magnetic beads (MACS, Miltenyi

Biotec, or Dynabeads, Dynal A ⁄ S) coated with

specific markers expressed on the exosome surface,

followed by elution and ultracentrifugation.59,60

New methods improving the yield and purity of

exosome isolation are under constant develop-

ment.61 Only then can phenotypic and functional

studies produce reliable results.

Another consideration is how fluorescence-acti-

vated cell sorting (FACS) is used for exosome pheno-

typing. In some reports, a direct FACS analysis of

ultracentrifuged MV pellets is used for phenotypic

characterization of exosomes. The discriminative

capacity of FACS is down to 300 nm,62 i.e. a size

three times the upper limit for the size of exosomes.

Thus, a FACS analysis of directly stained ultracentri-

fuged pellets63,64 will exclude the exosome fraction.

The use of FACS for exosome phenotyping must

be proceeded by loading of exosomes, directly or

via antibody capture on artificial beads.6,59 In con-

clusion, it is essential to elaborate on and choose




appropriate methods in all work with exosomes.

Most studies today focus on quantity, cell origin and

biological activity of MV without specific analysis of

the inherent organization and the purity of the micr-

ovesicle ⁄ microparticle populations studied. We

strongly advise against work63–67 where an isolated

crude mixture of all kind of MV, apoptotic bodies,

exosomes and other particles is included in pheno-

typic or functional analyses.

Morphology and protein composition of placental exosomes

Taking the methodological consideration described

earlier, only studies with reliable exosome-derived

data will be reviewed here. There are few investiga-

tions into isolated pregnancy-specific exosomes so

far. They comprise studies of exosomes isolated from

the peripheral blood of pregnant women performed

by the group of Taylor et al.3,4 and studies of

exosomes isolated from ex vivo placental explant

cultures from first trimester normal pregnancy

performed by our group.2,5,6 An illustration of the

morphology, size and some proteins present on the

membrane of placental exosomes isolated from

explant cultures is given in Fig. 3. As can be seen,

they have the typical exosomal cup shape and a size

of 40–90 nm. They express the exosome-specific

marker tetraspanin CD63 and the placenta-specific

enzyme placental-type alkaline phosphatase indicat-

ing their origin. In contrast to other exosomes, such

as those derived from immune cells, placental exo-

somes lack MHC molecule expression. Instead, the

MHC-related molecules MICA ⁄ B and RAE-

T1 ⁄ ULBP1-5, ligands of the activating NK cell recep-

tor NKG2D, were expressed on their surface (Fig. 3).

Furthermore, the placental exosomes express the

proapoptotic molecules FasL1–4 and TRAIL. Interest-

ingly, Western blot analyses revealed that these mol-

ecules were aggregated on the exosomal membrane

in a functional trimeric form which is required for

Fas signaling (LM-N, manuscript in preparation). In

addition, we found that the membranal form of the

regulatory cytokine TGFb is also expressed on the

placental exosome membrane (unpublished result).

Based on these studies, a schematic drawing of a

‘typical’ placental exosome with some of its protein

composition is presented in Fig. 4.

The entire protein composition of different

exosomes is not yet known and that is also valid for

placental exosomes. We have recently completed a

preliminary proteomic analysis of exosomes isolated

from supernatants of placental explant cultures, and

in Table II are listed some of the proteins, commonly

identified in exosomes, which were present in pla-

cental exosomes as well. The detailed presentation of

the data is currently under processing (LM-N, manu-

script in preparation). Largely, there were two

groups of proteins (i) those present on the exosomal

membrane and (ii) those entrapped in the lumen.

The proteomic analysis revealed some membrane-

associated proteins like the tetraspanins CD9 and

CD63 and proteins involved in adhesion and target-

ing (integrins, CD47, transferrin receptor, epidermal

growth factor receptor), but the great majority were

cytosolic such as cytoskeleton and cytoskeleton-

binding proteins (tubulin, actin, cofilin1, profilin1);

proteins involved in protein biosynthesis and degra-

dation such as ribosomal proteins, elongation factors

and proteasomes; proteins involved in intracellular

transport, fusion and signal transduction such as

annexins, Rab- and Ras-related proteins, vesicle

transport proteins, small GTP-as family members,

clatrin, dysferlin, syntaxin; heat shock proteins and

chaperons (HSP27, 70 and 90); ESCRT-associated

proteins such as TSG101, ALIX, vascular sorting

protein 29 and charged MVB protein 1B and 4B;

and enzymes such as a-enolase, 5¢nucleotidase and

dipeptidyl peptidases.

Identification of RNA in trophoblast-derived exosomes

One of the most intriguing and powerful role of

exosomes is their transport of selected mRNAs and

microRNAs (miRNA) and thus their ability to

Fig. 4 A schematic presentation of a putative

human placental exosome and a list of some

of the placenta exosome–associated proteins.




transfer genetic information to target cells and regu-

late their cellular metabolic pathways.7,39,40 These

findings suggest that exosomes may represent a new

mechanism of exchange of genetic information

between cells. Moreover, the possibility to analyze

mRNA and miRNA patterns in exosomes generated

in health and diseases opens a great opportunity to

refine disease diagnostics.40,68,69

Very recently, the first detailed miRNA profile of

human placenta was reported.70 In this study,

performed on human early and term placental

tissue, was demonstrated that most placenta-specific

miRNAs were linked to a miRNA cluster on chromo-

some 19. These miRNA cluster genes were upregu-

lated in placental development. Six novel miRNAs

were identified, and of those only four were

expressed in placenta. Simultaneously, miRNAs were

identified in maternal plasma, a finding supporting a

previous report by Chim et al.71 on placental miRNA

presence in plasma. The authors suggested that

syncytiotrophoblast secretes miRNA in the maternal

circulation by exosomes. To prove this notion, the

trophoblast cell line BeWo was used as a model of

villous trophoblast and exosomes were isolated from

culture supernatant. Two placenta-specific miRNAs

MIR517A and MIR21 were demonstrated in the exo-

some-enriched fraction. Proteome analysis suggested

that MIR17A might be involved in the regulation of

TNF signal transduction. It will be of interest to char-

acterize the placental miRNAs enriched in placenta-

derived exosomes in connection with normal and

pathologic pregnancies, different fetal developmental

stages or pregnancies with fetal abnormalities.

Retrieval of such exosomal profiles from the maternal

plasma will open new revenues for infertility and

prenatal diagnostics. In summary, these results are

promising first steps toward establishing the

exosomal miRNA profiles in pregnancy. More

Table II List of proteins, generally identified in exosomes and

revealed in placental exosomes by proteomic analysis

Protein family Members

Cytoskeleton

proteins

Actin

Myosin

Tubulin

Ezrin

Profilin 1

Cofilin 1

Heat shock

proteins

chaperons

HSP27

HSP70

HSP90

Tetraspanins CD9

CD63

MVB biogenesis Ubiquitin

TSG101

Alix

Vacuolar sorting protein 29 (ESCRT)

Charged MVB proteins 1B and 4B

(ESCRT)

Apoptosis

regulation

Programmed cell death proteins 6

and 10

Protein

biosynthesis

and

degradation

60S ribosomal proteins

40S ribosomal proteins

Elongation factors 1-a1, a2, a3 and c

Proteasome a4 subunit

Proteasome a5 subunit

Proteasome 26S non-ATPase subunit

Adhesion,

targeting

Integrins a5, aV, b1, b3

CD47

Transferrin receptor

Epidermal growth factor receptor

Liprin b-2

Signal

transduction

14-3-3 proteins

Rab 1A, 1B, 35

Ras-related proteins 1B and R

Guanine nucleotide binding protein

Ras GTPase-activating protein

Transforming protein RhoA

Sorcin

Enzymes a-enolase

5¢ nucleotidase

Dipeptidyl peptidases

Membrane

transport

and fusion

Annexins

Rab proteins: 2A, 5A, 5B, 5C, 6, 7, 10, 14

Clatrin heavy chain

Copine-3

Dysferlin

Testilin

Myoferlin

Syntaxin

Vesicle transport through interaction

protein 1B

Table II Continued

Protein family Members

Others Histones

LAMP2 (CD107B)

Multidrug resistance protein 1

S-100 proteins

Lysosomal membrane protein 2

Protein DI-1

ESCRT, endosomal-sorting complexes required for transport;

MVB, multivesicular bodies.




investigations are needed to elucidate the role of the

placenta-specific mRNA and miRNA and their capac-

ity to enter and reprogram maternal cells in favor of

fetal survival.

Functions associated with placenta-derived exosomes

Information about the function of placenta-derived

exosomes is presented in few reports1–6 and is based

on functional studies of placental exosomes isolated

from (i) peripheral blood of pregnant women3,4 and

(ii) supernatants of cultured trophoblast cells1 and

placental explants.5,6 Taylor et al. has characterized

placenta-derived exosomes in peripheral blood of

pregnant women that completed normal-term preg-

nancies or had a preterm delivery. The exosomal

concentration in plasma was elevated in women

with normal pregnancies delivering at term in

comparison with preterm delivery and non-pregnant

women. Incubation of T cells (Jurkat cell line) with

placental exosomes resulted in downregulation of

the expression of CD3-f and Janus kinase 3 (JAK3)

and activation of caspase 3. These responses affected

the clonal T-cell selection and lead to impaired

T-cell-mediated responses supporting the existing

hypothesis of Th2 deviation of immune responses

during pregnancy. The observed downregulation of

the CD3-f chain correlated to FasL- and PD-L1

expression on the exosomes and could be explained

by induction of apoptosis in target cells.4 These

results are in line with the report of Mor et al.1 that

trophoblast-derived FasL-expressing microvesicles

have proapoptotic function. Furthermore, it was

shown that exosomes from term-delivering mothers

could inhibit IL-2 production by activated T cells,

while the exosomes from preterm-delivering moth-

ers could not.3,4

Our studies of exosomes from placental explant

cultures included analyses of FasL2, TRAIL and the

NKG2D ligands MICA ⁄ B and ULBP1-5.5,6 Initially,

we suggested that exosome-associated MIC mole-

cules were present in the serum of pregnant women

and could suppress NKG2D receptor-mediated cyto-

toxicity.5 Thereafter, we demonstrated that isolated

exosomes carried all NKG2D ligands and were able

to downregulate the NKG2D receptor on NK-, CD8+-

and cdT cells in a dose-dependent manner. The

exosome-induced cellular internalization of NKG2D

resulted in impairment of the receptor-mediated

cytotoxicity without affecting the lytic potential of

the cells measured by perforin mRNA expression

and perforin protein content. Our results were indi-

rectly confirmed in a very recent publication by

Ashiru et al. who reported that cancer-derived

exosomes expressed MICA and suppressed NK cell

cytotoxicity in a similar way as placental exosomes,

i.e. by downregulation of the NKG2D receptor.71 In

our recent studies, we have found that placental

exosomes display bioactive trimeric FasL and TRAIL

and are proapoptotic. Using IEM, we have further

proven that placental exosomes are indeed formed

in the MVB ⁄ endosomal compartment by the syncy-

tiotrophoblast of the explant cultures.2,5,6 Summariz-

ing ours and others results, several functions for the

placenta-derived exosomes can be proposed: (i)

impairment of T-cell signaling; (ii) impairment of

cytotoxicity by downregulation of the major activat-

ing NK cell receptor NKG2D and (iii) apoptotic activ-

ity through FasL-, TRAIL- and PD-L1-mediated

pathways. Thus, it is clear that placental exosomes

are involved in the control of critical immune mech-

anisms such as cytotoxicity, T-cell response and

apoptosis in the local vicinity and ⁄ or at a distance

from the fetal–maternal interface. These functions

define the placental exosomes as inhibitory ⁄ immune

suppressive, using in a redundant way a number of

mechanisms that promote maternal immune toler-

ance of the fetal allograft.

Exosomes in amniotic fluid

Finally, we would like to briefly mention and

comment on the fact that exosomes are present in

and can be isolated from the amniotic fluid in

human72–74 and murine74 pregnancies. Here again,

one can find studies with insufficiently characterized

amniotic microvesicles and speculative suggestions

about their origin and functions.73 In the first report

of amniotic exosomes,72 their connection with the

fetal kidney was made, complying with the fact that

the renal system of the fetus is the main contributor

to the production of amniotic fluid. Moreover, the

group of Keller et al.74 proved that these exosomes

were released by the fetus, not the mother, and

investigated their phenotype in human and murine

pregnancies. The amniotic exosomes expressed CD24

as their specific address marker, annexin-1, and

kidney markers such as aquaporin-2, and have a

similar composition to exosomes from urine of new-

born infants. At this stage, more studies are needed

to evaluate if amniotic exosomes have a pregnancy-

promoting role. We are inclined to think that their

presence in the amniotic fluid is because of the fetal




urine production rather than that they have a

special role in the immunomodulation of the mater-

nal immune system. However, we are open for

convincingly proven suggestions otherwise. On the

other side, the amniotic exosome discovery72,74

opens fantastic possibilities to monitor prenatal kid-

ney development and develop prenatal diagnosis of

kidney diseases and genetic malformations.

Final remarks and future directions

Intercellular communication by MV has opened new

perspectives in understanding cross-talk mediated

between donor ⁄ signaling and target cells. From this

point of view, we can say that shedding MV from

the plasma membrane or releasing exosomes from

the endosomal compartment of the syncytiotropho-

blast expands the boundaries of the maternal–fetal

communication and affirms the role of the placenta

as a powerful regulatory organ that uses MV-medi-

ated signaling to target and reprogram maternal cells

for the benefit of reproduction.

Among the developments we expect in the near

future is the detailed differential characterization of

the STBM and exosomes. New optimized and refined

techniques for their separation are urgently needed.

Applying stringency and accuracy in isolation proce-

dures is the only way to ensure the possibility to

reveal the typical protein and RNA pattern profiles

of STBM and placental exosomes. Using isolated

pure fractions of MV in functional experiments will

clarify their functional differences and roles in

normal and pathological pregnancies.

How do we envisage the placental exosomes and

their role in pregnancy? From the accumulated

knowledge so far is clear that the main cell type of

the placenta, the syncytiotrophoblast, continuously

and constitutively produces and releases exosomes

that are used in the intercellular communication

between the mother and the child throughout the

pregnancy. Evidence summarized in this review

shows that the placental exosomes are packages

transporting important signaling molecules and

genetic information to be delivered to specific targets

locally or at a systemic level. They seem to be immu-

nosuppressive in nature, and have the ability to

shape the maternal immune system through different

mechanisms, which makes them pluripotent. The

exosomes directly secreted in the maternal blood

are, probably, at the highest abundance in the inter-

villous space of the chorionic villi and decrease in

concentration with increasing distance away from

the placenta. Thus, the continuous release of

exosomes by the syncytiotrophoblast creates an

exosomal concentration gradient where the protec-

tion against maternal immune attack is strongest at

the fetal–maternal interface, i.e. in the immediate

vicinity of the chorionic villi. One can imagine that

the fetus, together with the placenta, is surrounded

by a ‘cloud of exosomes’ that modulate the maternal

defense and create a protective and beneficial milieu

for the fetus.

The exciting field of placental exosome research is

still just at its beginning. An important progress will

be the exact identification of the mechanisms that

govern the exosomal biogenesis – molecular sorting,

inward MVB membrane budding and mechanisms of

placental exosome release. How is the fate of MVB

decided: why and how do some MVB become degra-

dative and others exocytotic? New molecules and

additional exosomal functions will be elucidated and

added. More such analyses will be performed to

come to a consensus regarding the mRNAs’ and

miRNAs’ role and contribution to the control of

successful pregnancy.

Finally, the role of exosomes in the pathogenesis

of pregnancy disturbances and related diseases,

recurrent abortions and infertility waits to be

evaluated.

Pregnancy is not the sole condition gaining from

studies of placental exosomes. Understanding how

the well-being of the fetal allograft is created can

benefit transplantation. Moreover, many prolifera-

tive, invasive and immune tolerance mechanisms

that support normal human pregnancy are also

exploited by malignancies to establish a nutrient

supply and evade the host immune response.

Placenta-derived and tumor-derived exosomes share

similar composition and properties. Thus, release of

exosomes that can edit immune responses to pro-

mote survival and well-being of the fetus or the

tumor, respectively, might be one common link

between the physiological state of pregnancy and

the pathological state of cancer. Knowledge, gained

from exosome research in reproduction, could lead

to identification of novel diagnostic and therapeutic

approaches in both conditions.

Acknowledgments

This work was supported by grants from the Swedish

National Cancer Research Foundation Cancerfonden




(CAN 2008 ⁄ 627, # 08 0360), Cancerforskningsfon-

den i Norrland, (AMP 08-587) and Insamlingsstiftel-

sen Umea University (223-438-07).

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The Role of Placental Exosomes in Reproduction

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