Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity

Abstract

Introduction Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication.

Isolation of exosomes from whole blood by integrating acoustics and microfluidics

“…To examine whether our exosomeisolation module could separate EV subgroups, namely microvesicles from exosomes, we input a mixture of purified exosomes and microvesicles derived from primary human trophoblasts (PHTs) to a standalone exosome-isolation module. The isolation and culture of PHT cells from human placentas and the purification of PHT-derived microvesicles and exosomes from PHT-conditioned medium were described elsewhere (16). We identified an optimized driving frequency of 39.4 MHz based on pilot experiments using a nanoparticle mixture of 110 and 340 nm (Fig.…”

“…Exosomes were isolated from whole-blood specimens using an OptiPrep gradient ultracentrifugation as previously described (16). The collection of placentas used for cell isolation and culture was reviewed and approved by the Institutional Review Board (IRB) at the University of Pittsburgh.…”

“…While differential centrifugation achieves high purity, it is time-consuming (several hours to days), expensive, and inefficient (in that the exosome isolation yields from whole blood are typically low, 5-40% of preseparation exosome population) (12,(14)(15)(16)(17). It also requires trained personnel to operate.…”

Microbial Vertical Transmission during Human Pregnancy

“…Primary syncytiotrophoblasts isolated from full-term placentas potently resist infection by diverse viruses and confer broad antiviral resistance to non-trophoblast cells through effectors that operate in a paracrine manner (Bayer et al, 2015; Bayer et al, 2016; Delorme-Axford et al, 2013; Ouyang et al, 2016). Accordingly, the syncytiotrophoblast layer of first-trimester chorionic villi is largely resistant to HCMV infection, whereas CTBs and other cells of the villous core are susceptible (Fisher et al, 2000).…”

“…In most cases, the mechanistic basis for this restriction is unclear, largely given the difficulties in working with trophoblasts at various stages of gestation and modeling the human placenta using small animals. In experiments with primary syncytiotrophoblasts from full term placentas, we discovered that these cells resist viral infections and transfer this resistance to non-placental cells in a paracrine manner through pathways involving placental-specific miRNAs packaged in exosomes as well as type III interferons (IFNs) (Bayer et al, 2015; Bayer et al, 2016; Delorme-Axford et al, 2013; Ouyang et al, 2016). Others have shown that the physical properties of the syncytium function in the restriction of microbial infections.…”