Research Article
Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

https://doi.org/10.1016/j.yexcr.2011.01.014Get rights and content

Abstract

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm ± 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of “normal” early pregnancy.

Introduction

During the past decade, significant attention has been focused on nano-sized vesicular components released by a variety of cells types, which exhibit immunoregulatory activities [1], [2], [3], [4]. These membranous vesicles, termed “exosomes” [5], have been implicated in a variety of physiological functions and pathological disease states where they present promising potential as diagnostic markers [6], [7]. While many cells are capable of secreting a variety of vesicles with different sizes and compositions, the definition of “exosomes” is based on specific criteria, including size (30–100 nm), buoyant density ranging from 1.13 to 1.19 g/ml, cup-shaped morphology observed by electron microscopy, and protein markers [8]. Exosomes are generally considered to be endocytic vesicules, originating from the limiting membrane of multivesicular bodies (MVB) via a mechanism of inward budding [9], [10]. As a result, many of the protein markers are derived from the endosome sorting pathway.

Ultrastructural studies, western blot and mass spectrometry analysis of exosomes from different sources have allowed the distinction between commonly expressed proteins, as well as cell type-specific proteins [11], [12], [13]. Based on their endosomal origin, exosomes, independent of the parental cell of origin, exhibit multiple proteins involved in MVB formation (annexins, Rab family GTPases, and ESCRT complex proteins (TSG101, Alix)). Additional protein markers linked with exosomes include tetraspanins (CD81, CD63, and CD9) and heat shock proteins (HSP60, HSP70, and HSP90) [13], [14]. Cell type-specific markers include classes I and II MHC, co-stimulatory proteins (CD80 and CD86) on antigen presenting cell-derived exosomes [14], [15], integrin CD41a and Von Willebrand factor on platelet-derived exosomes [16], and perforin and granzyme on cytotoxic T lymphocyte-derived exosomes [17].

While elevated levels of exosomes/microvesicles have been demonstrated in the peripheral circulation of pregnant women [18], the role of exosomes in pregnancy is still under investigation [20], [19]. Reports have described syncytiotrophoblastic (ST) cells as being a primary source of exosomes, which have been postulated to play an important role in fetal tolerance [21], [22], [23]. The expression of FasL, during the first trimester of pregnancy by ST, was reported to be localized in intracellular microvesicles and the ST also lacked plasma membrane associated FasL [22]. Mincheva-Nilsson et al. confirmed these findings by electron microscopy, showing cytosolic FasL concentrated in MVB intraluminal nanosized-exosomes like-vesicles that ultimately fused with ST apical membrane and were released in the extracellular space [24]. They demonstrated that ST derived exosomes expressed NKG2D receptor ligands, MICA/B (major histocompatibility class I chain-related antigens A and B) and the human retinoic acid early transcripts 1 (RAET1) [24]. The expressions of FasL and RAET1 molecules were absent on the ST cell surface, but localized in the intraluminal vesicles (ILVs, exosomes). In contrast, MICA/B proteins were both found at the apical cell surface and in the ILVs. Investigating the function of these ST-derived exosomes, Nielson et al. demonstrated that ST-isolated exosomes downregulated the NKG2D receptor on NK, CD8 and γδ T cells in a dose dependant manner [24].

In addition to direct modulation of signaling molecules on T and NK cells, early pregnancy has been characterized as a pro-inflammatory environment, associated with the production of pro-inflammatory cytokines, including interleukin 1β (IL-1β). IL-1β is a potent multifunctional pro-inflammatory cytokine produced by monocytes and tissue macrophages. Our previous work has demonstrated that microvesicles derived from a first trimester extravillous trophoblast (EVT) were capable of inducing the migration of monocytes and “educating” these cells to produce a pro-inflammatory cytokine profile, including IL-1β [25]. The nature of the membranous material derived from these extravillous trophoblast cells has not previously been evaluated, to define whether this material represents exosomes or a different form of shed vesicle. Additionally, the protein composition of these vesicles has not been investigated to define potential biologic activities associated with them and the molecular pathways modulated by these vesicles. In the current study, we performed morphological, biochemical, and proteomic characterizations of Sw71-derived microvesicles to define the nature of the release vesicles and to understand their functions related to pregnancy.

Section snippets

Sw71 cells and culture conditions

For isolation of exosomes from trophoblast cells, Swan71 (Sw71) cells were utilized. The human first trimester cell line, Sw71, was a kind gift from Dr G. Mor (Yale University, New Haven, CT) that was isolated from a 7-week placenta and immortalized by ectopic expression of the catalytic subunit of the human telomerase [26]. Sw71 cells were maintained in DMEM/F12 (Gibco Invitrogen, Carlsbad, CA). Culture media were supplemented with 2 mM L-glutamine, 10% exosome-depleted fetal bovine serum

Density analysis of the exosome-like vesicles

Exosomes possess biochemical properties that allow them to float on 30% sucrose density gradients. To characterize and verify the exosomal nature of SDE, conditioned media from Sw71 cells were ultra-filtrated and layered on a continuous sucrose gradient and ultracentrifuged. This process allowed the separation of floating vesicles from other contaminating aggregates, such as non-membranous serum proteins and fractions were collected from the top of the gradient. ICAM-1, fibronectin, PAI-1, and

Discussion

During pregnancy, the semi-allogeneic fetus implants and survives in a genetically foreign mother in spite of the laws of transplantation. Studies have highlighted the existence of a pro-inflammatory bias in early gestation, which appears to be a pre-requisite for successful pregnancy. A key immunoregulatory component derived from the embryo is microvesicles derived from the trophoblast. Our previous work has demonstrated that membranous vesicles derived from the early trophoblast cells can

Conclusions

In summary, this study provides the first evidence that trophoblasts secrete microvesicles, which based on morphology, size (50–200 nm), density (1.134–1.173 g/ml), and protein composition (CD81, Alix, cytoskeleton related proteins, and Rab family GTPases) are consistent with exosomes. We also provide the first extensive proteome of these trophoblast-derived exosomes, which will be useful in furthering our understanding of “normal” early pregnancy and in the identification of components that may

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