High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping: FLOAT.

Extracellular vesicles (EVs) have been identified as promising biomarkers for the noninvasive diagnosis of various diseases. However, challenges in separating EVs from soluble proteins have resulted in variable EV recovery rates and low purities. Here, we report a high-yield ( > 90%) and rapid ( < 10 min) EV isolation method called FLocculation via Orbital Acoustic Trapping (FLOAT). The FLOAT approach utilizes an acoustofluidic droplet centrifuge to rotate and controllably heat liquid droplets. By adding a thermoresponsive polymer flocculant, nanoparticles as small as 20 nm can be rapidly and selectively concentrated at the center of the droplet. We demonstrate the ability of FLOAT to separate urinary EVs from the highly abundant Tamm-Horsfall protein, addressing a significant obstacle in the development of EV-based liquid biopsies. Due to its high-yield nature, FLOAT reduces biofluid starting volume requirements by a factor of 100 (from 20 mL to 200 µL), demonstrating its promising potential in point-of-care diagnostics.

Defining trophoblast injury patterns in the transcriptomes of dysfunctional placentas.

Trophoblast injury is central to clinically relevant placenta dysfunction. We hypothesized that the mRNA of primary human trophoblasts, exposed to distinct injuries in vitro, capture transcriptome patterns of placental biopsies obtained from common obstetrical syndromes. We deployed a CIBERSORTx deconvolution method to correlate trophoblastic RNAseq-based expression matrices with the transcriptome of omics-defined placental dysfunction patterns in vivo. We found distinct trophoblast injury patterns in placental biopsies from women with fetal growth restriction and a hypertensive disorder, or in biopsies clustered by their omics analysis. Our RNAseq data are useful for defining the contribution of trophoblast injuries to placental dysfunction syndromes.

The impact of opioids on the transcriptional landscape of human villous trophoblasts.

INTRODUCTION: Opioid use disorder (OUD) is implicated in major obstetrical diseases such as fetal growth restriction. Whether or not opioids directly impact placental trophoblast development and function remains unclear. We sought to examine the expression of opioid receptors (OPRs) in villous trophoblasts and the effect of opioids on placental transcriptomics. METHODS: Trophoblast stem (TS) cells and primary human trophoblast (PHT) cells from healthy term placentas were used to assess OPR expression in conditions that enhance trophoblast stemness vs differentiation. Placental RNAseq was conducted using our retrospective cohorts of pregnant people with OUD vs controls, both without major obstetrical complications. RT-qPCR was used to determine the effect of fentanyl on the expression of putative opioid targets and stemness or differentiation-associated genes in TS and PHT cells. RESULTS: Three main OPRs, including OPRM1, OPRD1, and OPRK1 were expressed in term PHT cells cultured in the stemness medium, whereas only OPRD1 and OPRK1 were expressed in TS cells. Interestingly, upon induction of differentiation, the expressed OPR mRNAs in TS or in PHT cells were downregulated. We found 286 differentially expressed long RNAs in placentas from the OUD participants vs controls. While three putative opioid targets differed their expression in stemness vs differentiation states of trophoblasts, fentanyl had no effect on their expression or the expression of major stemness or differentiation-relevant genes in TS and PHT cells. DISCUSSION: Trophoblastic expression of OPRs and opioid RNA targets is impacted by cell differentiation, suggesting differential susceptibility of villous trophoblasts to the effect of opioids.

Integrated unbiased multiomics defines disease-independent placental clusters in common obstetrical syndromes.

BACKGROUND: Placental dysfunction, a root cause of common syndromes affecting human pregnancy, such as preeclampsia (PE), fetal growth restriction (FGR), and spontaneous preterm delivery (sPTD), remains poorly defined. These common, yet clinically disparate obstetrical syndromes share similar placental histopathologic patterns, while individuals within each syndrome present distinct molecular changes, challenging our understanding and hindering our ability to prevent and treat these syndromes. METHODS: Using our extensive biobank, we identified women with severe PE (n = 75), FGR (n = 40), FGR with a hypertensive disorder (FGR + HDP; n = 33), sPTD (n = 72), and two uncomplicated control groups, term (n = 113), and preterm without PE, FGR, or sPTD (n = 16). We used placental biopsies for transcriptomics, proteomics, metabolomics data, and histological evaluation. After conventional pairwise comparison, we deployed an unbiased, AI-based similarity network fusion (SNF) to integrate the datatypes and identify omics-defined placental clusters. We used Bayesian model selection to compare the association between the histopathological features and disease conditions vs SNF clusters. RESULTS: Pairwise, disease-based comparisons exhibited relatively few differences, likely reflecting the heterogeneity of the clinical syndromes. Therefore, we deployed the unbiased, omics-based SNF method. Our analysis resulted in four distinct clusters, which were mostly dominated by a specific syndrome. Notably, the cluster dominated by early-onset PE exhibited strong placental dysfunction patterns, with weaker injury patterns in the cluster dominated by sPTD. The SNF-defined clusters exhibited better correlation with the histopathology than the predefined disease groups. CONCLUSIONS: Our results demonstrate that integrated omics-based SNF distinctively reclassifies placental dysfunction patterns underlying the common obstetrical syndromes, improves our understanding of the pathological processes, and could promote a search for more personalized interventions.

The lipase cofactor CGI58 controls placental lipolysis.

In eutherians, the placenta plays a critical role in the uptake, storage, and metabolism of lipids. These processes govern the availability of fatty acids to the developing fetus, where inadequate supply has been associated with substandard fetal growth. Whereas lipid droplets are essential for the storage of neutral lipids in the placenta and many other tissues, the processes that regulate placental lipid droplet lipolysis remain largely unknown. To assess the role of triglyceride lipases and their cofactors in determining placental lipid droplet and lipid accumulation, we assessed the role of patatin like phospholipase domain containing 2 (PNPLA2) and comparative gene identification-58 (CGI58) in lipid droplet dynamics in the human and mouse placenta. While both proteins are expressed in the placenta, the absence of CGI58, not PNPLA2, markedly increased placental lipid and lipid droplet accumulation. These changes were reversed upon restoration of CGI58 levels selectively in the CGI58-deficient mouse placenta. Using co-immunoprecipitation, we found that, in addition to PNPLA2, PNPLA9 interacts with CGI58. PNPLA9 was dispensable for lipolysis in the mouse placenta yet contributed to lipolysis in human placental trophoblasts. Our findings establish a crucial role for CGI58 in placental lipid droplet dynamics and, by extension, in nutrient supply to the developing fetus.

Assessing hypoxic damage to placental trophoblasts by measuring membrane viscosity of extracellular vesicles.

INTRODUCTION: As highly sophisticated intercellular communication vehicles in biological systems, extracellular vesicles (EVs) have been investigated as both promising liquid biopsy-based disease biomarkers and drug delivery carriers. Despite tremendous progress in understanding their biological and physiological functions, mechanical characterization of these nanoscale entities remains challenging due to the limited availability of proper techniques. Especially, whether damage to parental cells can be reflected by the mechanical properties of their EVs remains unknown. METHODS: In this study, we characterized membrane viscosities of different types of EVs collected from primary human trophoblasts (PHTs), including apoptotic bodies, microvesicles and small extracellular vesicles, using fluorescence lifetime imaging microscopy (FLIM). The biochemical origin of EV membrane viscosity was examined by analyzing their phospholipid composition, using mass spectrometry. RESULTS: We found that different EV types derived from the same cell type exhibit different membrane viscosities. The measured membrane viscosity values are well supported by the lipidomic analysis of the phospholipid compositions. We further demonstrate that the membrane viscosity of microvesicles can faithfully reveal hypoxic injury of the human trophoblasts. More specifically, the membrane of PHT microvesicles released under hypoxic condition is less viscous than its counterpart under standard culture condition, which is supported by the reduction in the phosphatidylethanolamine-to-phosphatidylcholine ratio in PHT microvesicles. DISCUSSION: Our study suggests that biophysical properties of released trophoblastic microvesicles can reflect cell health. Characterizing EV's membrane viscosity may pave the way for the development of new EV-based clinical applications.

Small extracellular vesicles from plasma of women with preeclampsia increase myogenic tone and decrease endothelium-dependent relaxation of mouse mesenteric arteries.

Preeclampsia (PE) is a common syndrome of pregnancy, characterized by new-onset hypertension and proteinuria after gestational week 20, or new onset of hypertension and significant end-organ dysfunction. In the worst cases, it can threaten the survival of both mother and baby. Extracellular vesicles (EVs) are lipid-bilayer nanoparticles released from cells. They are involved in cell-cell communication and transport of diverse cargo molecules. Small extracellular vesicles (sEVs, exosomes) are defined by their size and biogenesis within the endocytic compartment of the cell or reverse budding of the plasma membrane. The function of circulating gestational EVs, released from maternal organs or the placenta, remains to be explored. Here, we focused on sEVs that circulate in the maternal blood in the third trimester of human pregnancy and hypothesized that sEVs from pregnant women with PE play a role in regulation of vessel tone. When compared to sEVs from women with uncomplicated pregnancies, ex vivo exposure of isolated mouse mesenteric arteries to sEVs purified from the plasma of pregnant women with PE led to constriction in response to intraluminal pressure. This effect was not observed using microvesicles from the plasma of women with PE or using PE plasma that was depleted of EVs. Blood vessels exposed to sEVs from women with PE were also more resistant to methacholine-stimulated relaxation. Immunofluorescence microscopy confirmed the presence of sEVs within the vessel wall. Together, these data support the notion that circulating sEVs from pregnant women play a role in the regulation of arterial tone.

Ferroptosis induces membrane blebbing in placental trophoblasts.

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp) forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in the trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture of wild-type target cells with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp-phosphatidylethanolamine in the cell membrane promoted its cell ability to be stretched. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.

Placental response to maternal SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic affected people at all ages. Whereas pregnant women seemed to have a worse course of disease than age-matched non-pregnant women, the risk of feto-placental infection is low. Using a cohort of 66 COVID-19-positive women in late pregnancy, we correlated clinical parameters with disease severity, placental histopathology, and the expression of viral entry and Interferon-induced transmembrane (IFITM) antiviral transcripts. All newborns were negative for SARS-CoV-2. None of the demographic parameters or placental histopathological characteristics were associated with disease severity. The fetal-maternal transfer ratio for IgG against the N or S viral proteins was commonly less than one, as recently reported. We found that the expression level of placental ACE2, but not TMPRSS2 or Furin, was higher in women with severe COVID-19. Placental expression of IFITM1 and IFITM3, which have been implicated in antiviral response, was higher in participants with severe disease. We also showed that IFITM3 protein expression, which localized to early and late endosomes, was enhanced in severe COVID-19. Our data suggest an association between disease severity and placental SARS-CoV-2 processing and antiviral pathways, implying a role for these proteins in placental response to SARS-CoV-2.

RNA Network Interactions During Differentiation of Human Trophoblasts.

In the human placenta, two trophoblast cell layers separate the maternal blood from the villous basement membrane and fetal capillary endothelial cells. The inner layer, which is complete early in pregnancy and later becomes discontinuous, comprises the proliferative mononuclear cytotrophoblasts, which fuse together and differentiate to form the outer layer of multinucleated syncytiotrophoblasts. Because the syncytiotrophoblasts are responsible for key maternal-fetal exchange functions, tight regulation of this differentiation process is critical for the proper development and the functional role of the placenta. The molecular mechanisms regulating the fusion and differentiation of trophoblasts during human pregnancy remain poorly understood. To decipher the interactions of non-coding RNAs (ncRNAs) in this process, we exposed cultured primary human trophoblasts to standard in vitro differentiation conditions or to conditions known to hinder this differentiation process, namely exposure to hypoxia (O2 < 1%) or to the addition of dimethyl sulfoxide (DMSO, 1.5%) to the culture medium. Using next generation sequencing technology, we analyzed the differential expression of trophoblastic lncRNAs, miRNAs, and mRNAs that are concordantly modulated by both hypoxia and DMSO. Additionally, we developed a model to construct a lncRNA-miRNA-mRNA co-expression network and inferred the functions of lncRNAs and miRNAs via indirect gene ontology analysis. This study improves our knowledge of the interactions between ncRNAs and mRNAs during trophoblast differentiation and identifies key biological processes that may be impaired in common gestational diseases, such as fetal growth restriction or preeclampsia.

Term Human Placental Trophoblasts Express SARS-CoV-2 Entry Factors ACE2, TMPRSS2, and Furin.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had a massive impact on human lives worldwide. While the airborne SARS-CoV-2 primarily affects the lungs, viremia is not uncommon. As placental trophoblasts are directly bathed in maternal blood, they are vulnerable to SARS-CoV-2. Intriguingly, the human fetus is largely spared from SARS-CoV-2 infection. We tested whether the human placenta expresses the main SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin and showed that ACE2 and TMPRSS2 are expressed in the trophoblast rather than in other placental villous cells. While furin is expressed in the main placental villous cell types, we surveyed, trophoblasts exhibit the highest expression. In line with the expression of these entry factors, we demonstrated that a SARS-CoV-2 pseudovirus could enter primary human trophoblasts. Mechanisms underlying placental defense against SARS-CoV-2 infection likely involve postentry processing, which may be germane for mitigating interventions against SARS-CoV-2.IMPORTANCE Pregnant women worldwide have been affected by COVID-19. As the virus is commonly spread to various organs via the bloodstream and because human placental trophoblasts are directly bathed in maternal blood, feto-placental infection by SARS-CoV-2 seems likely. However, despite the heightened risk to pregnant women, thus far the transmission risk of COVID-19 to the feto-placental unit seems extremely low. This has been recently attributed to a negligible expression of SARS-CoV-2 entry factors in the human placenta. We therefore sought to explore the expression of the entry factors ACE2 and TMPRSS2 in the different cell types of human placental villi. Using a combination of transcriptome sequencing (RNA-seq), real-time quantitative PCR (RT-qPCR), in situ hybridization, and immunofluorescence, we found that trophoblasts, but not the other main villous cell types, express ACE2 and TMPRSS2, with a broad expression of furin. Correspondingly, we also showed that primary human trophoblasts are permissive to entry of SARS-CoV-2 pseudovirus particles.

Acoustofluidic centrifuge for nanoparticle enrichment and separation.

Liquid droplets have been studied for decades and have recently experienced renewed attention as a simplified model for numerous fascinating physical phenomena occurring on size scales from the cell nucleus to stellar black holes. Here, we present an acoustofluidic centrifugation technique that leverages an entanglement of acoustic wave actuation and the spin of a fluidic droplet to enable nanoparticle enrichment and separation. By combining acoustic streaming and droplet spinning, rapid (<1 min) nanoparticle concentration and size-based separation are achieved with a resolution sufficient to identify and isolate exosome subpopulations. The underlying physical mechanisms have been characterized both numerically and experimentally, and the ability to process biological samples (including DNA segments and exosome subpopulations) has been successfully demonstrated. Together, this acoustofluidic centrifuge overcomes existing limitations in the manipulation of nanoscale (<100 nm) bioparticles and can be valuable for various applications in the fields of biology, chemistry, engineering, material science, and medicine.

Extracellular vesicles promote transkingdom nutrient transfer during viral-bacterial co-infection.

Extracellular vesicles (EVs) are increasingly appreciated as a mechanism of communication among cells that contribute to many physiological processes. Although EVs can promote either antiviral or proviral effects during viral infections, the role of EVs in virus-associated polymicrobial infections remains poorly defined. We report that EVs secreted from airway epithelial cells during respiratory viral infection promote secondary bacterial growth, including biofilm biogenesis, by Pseudomonas aeruginosa. Respiratory syncytial virus (RSV) increases the release of the host iron-binding protein transferrin on the extravesicular face of EVs, which interact with P. aeruginosa biofilms to transfer the nutrient iron and promote bacterial biofilm growth. Vesicular delivery of iron by transferrin more efficiently promotes P. aeruginosa biofilm growth than soluble holo-transferrin delivered alone. Our findings indicate that EVs are a nutrient source for secondary bacterial infections in the airways during viral infection and offer evidence of transkingdom communication in the setting of polymicrobial infections.

Trophoblastic extracellular vesicles and viruses: Friends or foes?

Cells produce cytoplasmic vesicles to facilitate the processing and transport of RNAs, proteins, and other signaling molecules among intracellular organelles. Moreover, most cells release a range of extracellular vesicles (EVs) that mediate intercellular communication in both physiological and pathological settings. In addition to a better understanding of their biological functions, the diagnostic and therapeutic prospects of EVs, particularly the nano-sized small EVs (sEVs, exosomes), are currently being rigorously pursued. While EVs and viruses such as retroviruses might have evolved independently, they share a number of similar characteristics, including biogenesis pathways, size distribution, cargo, and cell-targeting mechanisms. The interplay of EVs with viruses has profound effects on viral replication and infectivity. Our research indicates that sEVs, produced by primary human trophoblasts, can endow other non-placental cell types with antiviral response. Better insights into the interaction of EVs with viruses may illuminate new ways to attenuate viral infections during pregnancy, and perhaps develop new antiviral therapeutics to protect the feto-placental unit during critical times of human development.

Placental small extracellular vesicles: Current questions and investigative opportunities.

The discovery of regulated trafficking of extracellular vesicles (EVs) has added a new dimension to our understanding of local and distant communication among cells and tissues. Notwithstanding the expanded landscape of EV subtypes, the majority of research in the field centers on small and large EVs that are commonly termed exosomes, microvesicles and apoptotic cell-derived vesicles. In the context of pregnancy, EV-based communication has a special role in the crosstalk among the placenta, maternal and fetal compartments, with most studies focusing on trophoblastic EVs and their effect on other placental cell types, endothelial cells, and distant tissues. Many unanswered questions in the field of EV biology center on the mechanisms of vesicle biogenesis, loading of cargo molecules, EV release and trafficking, the interaction of EVs with target cells and the endocytic pathways underlying their uptake, and the intracellular processing of EVs inside target cells. These questions are directly relevant to EV-based placental-maternal-fetal communication and have unique implications in the context of interaction between two organisms. Despite rapid progress in the field, the number of speculative, unsubstantiated assumptions about placental EVs is concerning. Here we attempt to delineate existing knowledge in the field, focusing primarily on placental small EVs (exosomes). We define central questions that require investigative attention in order to advance the field.

Unc-13 homolog D mediates an antiviral effect of the chromosome 19 microRNA cluster miR-517a.

The function of microRNAs (miRNAs) can be cell autonomous or communicated to other cell types and has been implicated in diverse biological processes. We previously demonstrated that miR-517a-3p (miR-517a), a highly expressed member of the chromosome 19 miRNA cluster (C19MC) that is transcribed almost exclusively in human trophoblasts, attenuates viral replication via induction of autophagy in non-trophoblastic recipient cells. However, the molecular mechanisms underlying these effects remain unknown. Here, we identified unc-13 homolog D (UNC13D) as a direct, autophagy-related gene target of miR-517a, leading to repression of UNC13D. In line with the antiviral activity of miR-517a, silencing UNC13D suppressed replication of vesicular stomatitis virus (VSV), whereas overexpression of UNC13D increased VSV levels, suggesting a role for UNC13D silencing in the antiviral activity of miR-517a. We also found that miR-517a activated NF-κB signaling in HEK-293XL cells expressing TLR8, but the effect was not specific to C19MC miRNA. Taken together, our results define mechanistic pathways that link C19MC miRNA with inhibition of viral replication.

Internalization of trophoblastic small extracellular vesicles and detection of their miRNA cargo in P-bodies.

Pregnancy is a unique situation, in which placenta-derived small extracellular vesicles (sEVs) may communicate with maternal and foetal tissues. While relevant to homoeostatic and pathological functions, the mechanisms underlying sEV entry and cargo handling in target cells remain largely unknown. Using fluorescently or luminescently labelled sEVs, derived from primary human placental trophoblasts or from a placental cell line, we interrogated the endocytic pathways used by these sEVs to enter relevant target cells, including the neighbouring primary placental fibroblasts and human uterine microvascular endothelial cells. We found that trophoblastic sEVs can enter target cells, where they retain biological activity. Importantly, using a broad series of pharmacological inhibitors and siRNA-dependent silencing approaches, we showed that trophoblastic sEVs enter target cells using macropinocytosis and clathrin-mediated endocytosis pathways, but not caveolin-dependent endocytosis. Tracking their intracellular course, we localized the sEVs to early endosomes, late endosomes, and lysosomes. Finally, we used coimmunoprecipitation to demonstrate the association of the sEV microRNA (miRNA) with the P-body proteins AGO2 and GW182. Together, our data systematically detail endocytic pathways used by placental sEVs to enter relevant fibroblastic and endothelial target cells, and provide support for "endocytic escape" of sEV miRNA to P-bodies, a key site for cytoplasmic RNA regulation.

Correction: Separating extracellular vesicles and lipoproteins via acoustofluidics.

Correction for 'Separating extracellular vesicles and lipoproteins via acoustofluidics' by Mengxi Wu et al., Lab Chip, 2019, 19, 1174-1182, DOI: .

Unique microRNA Signals in Plasma Exosomes from Pregnancies Complicated by Preeclampsia.

Although preeclampsia is a common and serious complication of pregnancy, insight into its pathobiology and diagnosis is lacking. Circulating plasma exosomes, which contain RNA and other molecules and have recently become accessible for diagnostics, may be informative in this regard. We tested the hypothesis that preeclampsia may affect the miRNA cargo within circulating maternal blood exosomes. We collected plasma from 60 pregnant women at term, including 20 women with pregnancy complicated by preeclampsia, and 20 women with fetal growth restriction and 20 with healthy pregnancy, serving as controls. We isolated exosomes from the maternal plasma by continuous density gradient ultracentrifugation. Our main outcome variable was exosomal miRNA cargo, analyzed by quantitative polymerase chain reaction-based TaqMan advanced miRNA assay in a card format and the expression of differentially expressed exosomal miRNA in whole plasma from the same participants. We found that 7 miRNA species were differentially expressed in exosomes from women with preeclampsia and those from controls. In contrast, there was no significant difference in exosomal miRNA expression between women with fetal growth restriction and controls. The results were not affected by fetal sex. Only one of the preeclampsia-related, differentially expressed exosomal miRNAs was significantly different in whole plasma miRNA analysis. We concluded that unlike whole plasma miRNA, exosomes extracted from the plasma of women with preeclampsia exhibit a unique miRNA profile, suggesting that plasma exosomal miRNA could provide insight into the pathophysiology of preeclampsia, and may play a role in disease diagnostics.

Separating extracellular vesicles and lipoproteins via acoustofluidics.

Extracellular vesicles (EVs) and lipoproteins are abundant and co-exist in blood. Both have been proven to be valuable as diagnostic biomarkers and for therapeutics. However, EVs and lipoproteins are both on the submicron scale and overlap in size distributions. Conventional methods to separate EVs and lipoproteins are inefficient and time-consuming. Here we present an acoustofluidic-based separation technique that is based on the acoustic property differences of EVs and lipoproteins. By using the acoustofluidic technology, EVs and subgroups of lipoproteins are separated in a label-free, contact-free, and continuous manner. With its ability for simple, rapid, efficient, continuous-flow isolation, our acoustofluidic technology could be a valuable tool for health monitoring, disease diagnosis, and personalized medicine.