Human Trophoblast Cultures Research Articles

Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring β-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 μm observed at 5 days. Functionality, assessed through β-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.

Zika virus co-opts microRNA networks to persist in placental niches detected by spatial transcriptomics

Zika virus congenital infection evades double-stranded RNA detection and may persist in the placenta for the duration of pregnancy without accompanying overt histopathologic inflammation. Understanding how viruses can persist and replicate in the placenta without causing overt cellular or tissue damage is fundamental to deciphering mechanisms of maternal-fetal vertical transmission. Placenta-specific microRNAs are believed to be a tenet of viral resistance at the maternal-fetal interface. We aimed to test the hypothesis that the Zika virus functionally disrupts placental microRNAs, enabling viral persistence and fetal pathogenesis. To test this hypothesis, we used orthogonal approaches in human and murine experimental models. In primary human trophoblast cultures (n=5 donor placentae), we performed Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation to identify any significant alterations in the functional loading ofmicroRNAs and their targets onto the RNA-induced silencing complex.Trophoblasts from same-donors were split and infected with a contemporary first-passage Zika virus strain HN16 (multiplicity of infection=1 plaque forming unit per cell) or mock infected. To functionally cross-validate microRNA-messenger RNA interactions, we compared our Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation results with an independent analysis of published bulk RNA-sequencing data from human placental disk specimens (n=3 subjects; Zika virus positive in first, second, or third trimester, CD45- cells sorted by flow cytometry) and compared it with uninfected controls (n=2 subjects). To investigate the importance of these microRNA and RNA interference networks in Zika virus pathogenesis, we used a gnotobiotic mouse model uniquely susceptible to the Zika virus. We evaluated if small-molecule enhancement of microRNA and RNA interference pathways with enoxacin influenced Zika viruspathogenesis (n=20 dams total yielding 187 fetal specimens). Lastly, placentae (n=14 total) from this mouse model were analyzed with Visium spatial transcriptomics (9743 spatial transcriptomes) to identify potential Zika virus-associated alterations in immune microenvironments. We found that Zika virus infection of primary human trophoblast cells led to an unexpected disruption of placental microRNA regulation networks. When compared with uninfected controls, Zika virus-infected placentae had significantly altered SLC12A8, SDK1, andVLDLR RNA-induced silencing complex loading and transcript levels (-2<log2(fold-change)>2; adjusted P value <.05; Wald-test with false discovery rate correction q<0.05). In silico microRNA target analyses revealed that 26 of 119 transcripts (22%) in the transforming growth factor-β signaling pathway were targeted by microRNAs that were found to be dysregulated following Zika virus infection in trophoblasts. In gnotobiotic mice, relative to mock controls, Zika virus-associated fetal pathogenesis included fetal growth restriction (P=.036) and viral persistence in placental tissue (P=.011). Moreover, spatial transcriptomics of murine placentae revealed that Zika virus-specific placental niches were defined by significant up-regulation of complement cascade components and coordinated changes in transforming growth factor-β gene expression. Finally, treatment of Zika virus-infected mice with enoxacin abolished placental Zika virus persistence, rescued the associated fetal growth restriction, and the Zika virus-associated transcriptional changes in placental immune microenvironments were no longer observed. These results collectively suggest that (1) Zika virus infection and persistence is associated with functionally perturbed microRNA and RNA interference pathways specifically related to immune regulation in placental microenvironments and (2) enhancement of placental microRNA and RNA interference pathways in mice rescued Zika virus-associated pathogenesis, specifically persistence of viral transcripts in placental microenvironments and fetal growth restriction.

H3K27me3-modulated Hofbauer cell BMP2 signalling enhancement compensates for shallow trophoblast invasion in preeclampsia.

Preeclampsia (PE) is a common hypertensive pregnancy disorder associated with shallow trophoblast invasion. Although bone morphogenetic protein 2 (BMP2) has been shown to promote trophoblast invasion invitro, its cellular origin and molecular regulation in placenta, as well as its potential role in PE, has yet to be established. Additionally, whether BMP2 and/or its downstream molecules could serve as potential diagnostic or therapeutic targets for PE has not been explored. Placentas and sera from PE and healthy pregnant women were subjected to multi-omics analyses, immunoblots, qPCR, and ELISA assays. Immortalized trophoblast cells, primary cultures of human trophoblasts, and first-trimester villous explants were used for invitro experiments. Adenovirus expressing sFlt-1 (Ad Flt1)-induced PE rat model was used for invivo studies. We find globally decreased H3K27me3 modifications and increased BMP2 signalling in preeclamptic placentas, which is negatively correlated with clinical manifestations. BMP2 is derived from Hofbauer cells and epigenetically regulated by H3K27me3 modification. BMP2 promotes trophoblast invasion and vascular mimicry by upregulating BMP6 via BMPR1A-SMAD2/3-SMAD4 signalling. BMP2 supplementation alleviates high blood pressure and fetal growth restriction phenotypes in Ad Flt1-induced rat PE model. Our findings demonstrate that epigenetically regulated Hofbauer cell-derived BMP2 signalling enhancement in late gestation could serve as a compensatory response for shallow trophoblast invasion in PE, suggesting opportunities for diagnostic marker and therapeutic target applications in PE clinical management. National Key Research and Development Program of China (2022YFC2702400), National Natural Science Foundation of China (82101784, 82171648, 31988101), and Natural Science Foundation of Shandong Province (ZR2020QH051, ZR2020MH039).

Physiologically relevant culture medium Plasmax improves human placental trophoblast stem cell function

Human trophoblast cultures provide powerful tools to model key processes of placental development. In vitro trophoblast studies to date have relied on commercial media that contains nonphysiological levels of nutrients, and the impact of these conditions on trophoblast metabolism and function is unknown. Here, we show that the physiological medium (Plasmax) with nutrient and metabolite concentrations recapitulating human plasma improves human trophoblast stem cell (hTSC) proliferation and differentiation compared with standard medium (DMEM-F12). hTSCs cultured in Plasmax-based medium also show altered glycolytic and mitochondrial metabolism, as well as reduced S-adenosylmethionine/S-adenosyl-homocysteine ratio compared with DMEM-F12-based medium. These findings demonstrate the importance of the nutritional environment for phenotyping cultured human trophoblasts.

Testosterone Decreases Placental Mitochondrial Content and Cellular Bioenergetics.

Placental mitochondrial dysfunction plays a central role in the pathogenesis of preeclampsia. Since preeclampsia is a hyperandrogenic state, we hypothesized that elevated maternal testosterone levels induce damage to placental mitochondria and decrease bioenergetic profiles. To test this hypothesis, pregnant Sprague–Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg/day) from gestation day (GD) 15 to 19. On GD20, the placentas were isolated to assess mitochondrial structure, copy number, ATP/ADP ratio, and biogenesis (Pgc-1α and Nrf1). In addition, in vitro cultures of human trophoblasts (HTR-8/SVneo) were treated with dihydrotestosterone (0.3, 1.0, and 3.0 nM), and bioenergetic profiles using seahorse analyzer were assessed. Testosterone exposure in pregnant rats led to a 2-fold increase in plasma testosterone levels with an associated decrease in placental and fetal weights compared with controls. Elevated maternal testosterone levels induced structural damage to the placental mitochondria and decreased mitochondrial copy number. The ATP/ADP ratio was reduced with a parallel decrease in the mRNA and protein expression of Pgc-1α and Nrf1 in the placenta of testosterone-treated rats compared with controls. In cultured trophoblasts, dihydrotestosterone decreased the mitochondrial copy number and reduced PGC-1α, NRF1 mRNA, and protein levels without altering the expression of mitochondrial fission/fusion genes. Dihydrotestosterone exposure induced significant mitochondrial energy deficits with a dose-dependent decrease in basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. In summary, our study suggests that the placental mitochondrial dysfunction induced by elevated maternal testosterone might be a potential mechanism linking preeclampsia to feto-placental growth restriction.

IFITM proteins inhibit placental syncytiotrophoblast formation and promote fetal demise.

Elevated levels of type I interferon (IFN) during pregnancy are associated with intrauterine growth retardation, preterm birth, and fetal demise through mechanisms that are not well understood. A critical step of placental development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast (ST) layer. Fusion is mediated by syncytins, proteins deriving from ancestral endogenous retroviral envelopes. Using cultures of human trophoblasts or mouse cells, we show that IFN-induced transmembrane proteins (IFITMs), a family of restriction factors blocking the entry step of many viruses, impair ST formation and inhibit syncytin-mediated fusion. Moreover, the IFN inducer polyinosinic:polycytidylic acid promotes fetal resorption and placental abnormalities in wild-type but not in Ifitm-deleted mice. Thus, excessive levels of IFITMs may mediate the pregnancy complications observed during congenital infections and other IFN-induced pathologies.

Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

Preeclampsia (PE) is a common cause of maternal morbidity, characterized by impaired trophoblast invasion and spiral artery transformation resulting in progressive uteroplacental hypoxia. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation, and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to PE. Here we show that LIN28B is expressed ∼1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in primary human trophoblast cultures. The expression of LIN28B mRNA and protein levels are significantly reduced in gestational age-matched preeclamptic vs. normal placentas, whereas LIN28A expression is not different. First trimester human placental sections displayed stronger LIN28B immunoreactivity in extravillous (invasive) cytotrophoblasts and syncytial sprouts vs. villous trophoblasts. LIN28B overexpression increased HTR8 cell proliferation, migration, and invasion, whereas LIN28B knockdown in JEG3 cells reduced cell proliferation. Moreover, LIN28B knockdown in JEG3 cells suppressed syncytin 1 (SYN-1), apelin receptor early endogenous ligand (ELABELA), and the chromosome 19 microRNA cluster, and increased mRNA expression of ITGβ4 and TNF-α. Incubation of BeWo and JEG3 cells under hypoxia significantly decreased expression of LIN28B and LIN28A, SYN-1, and ELABELA, whereas TNF-α is increased. These results provide the first evidence that LIN28B is the predominant paralog in human placenta and that decreased LIN28B may play a role in PE by reducing trophoblast invasion and syncytialization, and by promoting inflammation.-Canfield, J., Arlier, S., Mong, E. F., Lockhart, J., VanWye, J., Guzeloglu-Kayisli, O., Schatz, F., Magness, R. R., Lockwood, C. J., Tsibris, J. C. M., Kayisli, U. A., Totary-Jain, H. Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

Activation of Protein C in Human Trophoblasts in Culture and Downregulation of Trophoblast Endothelial Protein C Receptor by TNF-α.

In mice, trophoblasts are equipped with a potent anticoagulant mechanism, the protein C pathway. In human placenta, no functional studies of the protein C pathway are available. Human first-trimester trophoblasts (CK(++) HLA-G(+/-) Vim(-)) were isolated and kept in culture for a maximum of 48 hours. Activation of protein C on trophoblasts was at least as efficient as in endothelial cells (4.43 × 10 (-) (7) nmol/L/min/cell). Endothelial protein C receptor (EPCR) was expressed in syncytiotrophoblasts and extravillous trophoblasts. Downregulation of the messenger RNA of trophoblast EPCR occurred when trophoblasts were challenged with tumor necrosis factor α, and it could be prevented by unfractionated heparin but not by low-molecular-weight heparin at therapeutic doses. In conclusion, there is a functional protein C pathway on human first-trimester trophoblasts which can be modulated by inflammation. This finding has implications for the pathogenesis and prevention of placenta-mediated obstetric complications.

Benzo(a)pyrene exposure during pregnancy: Accumulation and effects on primary cultures of human trophoblasts

Benzo(a)pyrene exposure during pregnancy: Accumulation and effects on primary cultures of human trophoblasts

Estrogen-related receptor γ serves a role in blood pressure homeostasis during pregnancy.

Persistent hypoxia caused by shallow trophoblast invasion and poor placental perfusion may underlie the pathophysiology of preeclampsia, a leading cause of maternal and neonatal morbidity and mortality. Previously, we found that estrogen-related receptor γ (ERRγ) serves a critical and O2-dependent role in differentiation of human trophoblasts in culture and expression of tissue kallikrein and voltage-gated K(+) channels. In this study, we surprisingly observed that ERRγ expression was significantly increased in placentas from preeclamptic women compared with that in gestation-matched normotensive women. To further investigate a functional role for ERRγ during pregnancy, we analyzed ERRγ-deficient mice. Maternal systolic blood pressure was significantly reduced in pregnant ERRγ(+/-) females bred to ERRγ(+/-) males compared with that in wild-type (WT) mice and was markedly up-regulated by treatment of WT pregnant mice with the ERRγ agonist DY131. Placentas of ERRγ(+/-) mice manifested increased vascular endothelial growth factor A expression compared with that in WT mice. Notably, circulating levels of the antiangiogenic factor, soluble fms-like tyrosine kinase-1, were significantly reduced in ERRγ(+/-) pregnant mice as was serum aldosterone. These effects were associated with a decrease in maternal adrenal Cyp11b1 (steroid 11β-hydroxylase) and Cyp11b2 (aldosterone synthase) expression. In contrast, adrenal Cyp11b1 and Cyp11b2 mRNA were increased in pregnant WT mice treated with DY131. Moreover, chromatin immunoprecipitation and luciferase reporter assays identified Cyp11b2 as a transcriptional target of ERRγ. Collectively, these findings reveal a potential role of ERRγ in maternal blood pressure homeostasis during pregnancy and suggest that aberrant ERRγ expression may contribute to the pathogenesis of preeclampsia.

Estrogen-Related Receptor γ (ERRγ) Regulates Oxygen-Dependent Expression of Voltage-gated Potassium (K+) Channels and Tissue Kallikrein during Human Trophoblast Differentiation

Estrogen-related receptor γ (ERRγ) serves a critical O2-dependent regulatory role in the differentiation of human cytotrophoblasts to syncytiotrophoblast. In this study, we investigated expression of genes encoding tissue kallikrein (KLK1) and voltage-gated K(+) channels (KV7) during differentiation of human trophoblasts in culture and the roles of ERRγ and O2 tension in their regulation. Expression of KLK1 and the KV7 channel subunits, KCNQ1, KCNE1, KCNE3, and KCNE5, increased during differentiation of cultured human trophoblast cells in a 20% O2 environment. Notably, together with ERRγ, expression of KLK1, KCNQ1, KCNE1, KCNE3, and KCNE5 was markedly reduced when cells were cultured in a hypoxic environment (2% O2). Moreover, upon transduction of trophoblast cells with short hairpin RNAs for endogenous ERRγ, KLK1, KCNQ1, KCNE1, and KCNE3 expression was significantly decreased. Promoter and site-directed mutagenesis studies in transfected cells identified putative ERRγ response elements within the KLK1 and KCNE1 5'-flanking regions required for ERRγ-stimulated transcriptional activity. Binding of endogenous ERRγ to these ERRγ response elements increased during trophoblast differentiation in culture and was inhibited by hypoxia. The KV7 blocker linopirdine reduced human chorionic gonadotropin secretion and aggregation of cultured human trophoblasts, suggesting a possible role of KV7 channels in cell fusion and differentiation. Illumina gene expression arrays of cultured human trophoblast cells revealed several genes upregulated during syncytiotrophoblast differentiation and downregulated upon ERRγ knockdown involved in cell differentiation, adhesion, and synthesis of steroid and peptide hormones required for placental development and function. Collectively, these findings suggest that ERRγ mediates O2-dependent expression of genes involved in human trophoblast differentiation, function, and vascular homeostasis.

Live-cell imaging shows apoptosis initiates locally and propagates as a wave throughout syncytiotrophoblasts in primary cultures of human placental villous trophoblasts

Human placental villi are surfaced by the syncytiotrophoblast, a multinucleated, epithelial-cell layer that functions in maternal–fetal exchange. Mononucleated cytotrophoblasts are subjacent to the syncytiotrophoblast. Using confocal fluorescence microscopy of third-trimester villi, we previously found that cytotrophoblasts are often interdigitated into the syncytiotrophoblast, that cytotrophoblasts undergo caspase-mediated apoptosis, and that apoptosis is much lower, and perhaps completely inhibited, in intact syncytiotrophoblast lacking fibrin-type fibrinoid. Previous analysis of primary cultures of human trophoblasts also indicated lower levels of apoptosis in syncytiotrophoblast compared to cytotrophoblasts. Here, using confocal microscopy we find that cultured cytotrophoblasts and syncytiotrophoblasts display complex structural relationships, as in vivo, and that apoptosis of a cytotrophoblast or syncytiotrophoblast does not induce apoptosis of adjacent trophoblasts. Using live-cell imaging of mitochondrial depolarization and nuclear condensation in cultured syncytiotrophoblasts, we show apoptosis initiates in a localized region and propagates radially at ∼5 μm/min with no loss of velocity until the entire syncytium has undergone apoptosis. The rate of propagation is similar in cases of spontaneous apoptosis and in apoptosis that occurs in the presence of cobalt chloride or rotenone, two inducers of apoptosis. We suggest that inhibition of syncytiotrophoblast apoptosis in vivo is important to prevent widespread syncytiotrophoblast death, which would result in placental dysfunction and contribute to poor pregnancy outcomes.

Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts

The human placenta is key to pregnancy outcome, and the elevated oxidative stress present in many complicated pregnancies contributes to placental dysfunction and suboptimal pregnancy outcomes. We tested the hypothesis that pomegranate juice, which is rich in polyphenolic antioxidants, limits placental trophoblast injury in vivo and in vitro. Pregnant women with singleton pregnancies were randomized at 35∼38 wk gestation to 8 oz/day of pomegranate juice or apple juice (placebo) until the time of delivery. Placental tissues from 12 patients (4 in the pomegranate group and 8 in the control group) were collected for analysis of oxidative stress. The preliminary in vivo results were extended to oxidative stress and cell death assays in vitro. Placental explants and cultured primary human trophoblasts were exposed to pomegranate juice or glucose (control) under defined oxygen tensions and chemical stimuli. We found decreased oxidative stress in term human placentas from women who labored after prenatal ingestion of pomegranate juice compared with apple juice as control. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in term villous explants and primary trophoblast cultures exposed to hypoxia, the hypoxia mimetic cobalt chloride, and the kinase inhibitor staurosporine. Punicalagin, but not ellagic acid, both prominent polyphenols in pomegranate juice, reduced oxidative stress and stimulus-induced apoptosis in cultured syncytiotrophoblasts. We conclude that pomegranate juice reduces placental oxidative stress in vivo and in vitro while limiting stimulus-induced death of human trophoblasts in culture. The polyphenol punicalagin mimics this protective effect. We speculate that antenatal intake of pomegranate may limit placental injury and thereby may confer protection to the exposed fetus.

Nitric oxide generation affects pro- and anti-angiogenic growth factor expression in primary human trophoblast

ObjectivesPreeclampsia is associated with reduced trophoblast placenta growth factor (PGF) expression, elevated soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased bioactivity of nitric oxide (NO). Elevated sFlt-1 reduces bio-availability of PGF and vascular endothelial growth factor (VEGF) leading to maternal endothelial dysfunction. Although NO can regulate gene expression, its ability to regulate trophoblast expression of angiogenic growth factors is not known. Study designHuman primary term trophoblast and JEG-3 choriocarcinoma cells were cultured under 21%O2 or 1%O2 conditions in the presence or absence of NO donor (SNP) or inhibitor (L-NAME). Effects on PGF, VEGF and Flt-1 isoform mRNA expression were determined by quantitative real-time PCR. Changes in expression of soluble protein isoforms of FLT-1 was monitored by ELISA. ResultsHypoxia decreased PGF mRNA but increased VEGF, sFlt-1 and Flt-1 mRNA expression in trophoblast. Generation of NO in trophoblast under 1%O2 culture conditions significantly reversed sFlt-1 mRNA and protein expression, independent of mFlt-1. Conversely NO generation in hypoxic trophoblast increased VEGF and PGF mRNA expression. ConclusionsNO production in primary human trophoblast cultures had divergent effects on pro-angiogenic (PGF, VEGF) versus anti-angiogenic (sFlt-1) mRNA expression, resulting in an enhanced pro-angiogenic gene expression environment in vitro.

AHR and ARNT expression in the human and rat placentas and their transcription activity in human trophoblast cultures in transactivation AHR battery genes

AHR and ARNT expression in the human and rat placentas and their transcription activity in human trophoblast cultures in transactivation AHR battery genes

Do Antiphospholipid Antibodies Have Direct Pathologic Effects Upon Endometrial and Trophoblast Cells?

The presence of antiphospholipid antibodies (aPL) in association with vascular thromboses (VT) and/or pregnancy morbidity (PM) is the hallmark of the Antiphospholipid Syndrome (APS). Increasingly, evidence indicates that PM in the APS involves direct targeting of endometrium and invading fetal trophoblast by aPL. Intravascular or intervillous blood clots are rarely found on histological examination of miscarriage samples from APS patients. In primary human trophoblast culture, aPL inhibit the expression of human chorionic gonadotrophin, induce apoptosis, decrease trophoblast fusion, alter expression of cell adhesion molecules, and limit trophoblast invasiveness. Maternal tissues may also be targeted by aPL since aPL have been shown to inhibit decidualization of cultured endometrial stromal cells and target the decidua when injected in pregnant mice, resulting in recruitment of neutrophils, complement activation, and a rapid increase in TNF-α levels. Thus the mechanism of pregnancy failure in APS is complex and involves local inflammation, impaired decidualisation, disruption of normal trophoblast function and cell death. In this article we review the evidence that different subsets of aPL directly target trophoblast and endometrial cells and the mechanisms that may be involved. The potential diagnostic and therapeutic implications of these findings will then be discussed.

Soluble Factors From LPS- and PHA-Activated PBMC Induce MAPK, Stat1 and Stat3 Phosphorylation in Primary Cultures of Human Term Placental Trophoblasts: Implications for Infection and Prematurity

Infection of the maternal vaginal tract is one of the single most important antecedents of premature labor. We have hypothesized that the abundant local synthesis of pro-inflammatory cytokines that occurs during infection may disrupt the delicate “immunological cross-talk” that must occur between maternal and fetal tissues in order to carry pregnancy to term. These experiments were undertaken as part of a larger study directed at testing that hypothesis. We prepared primary cultures of human trophoblasts from term placentas. Cell cultures were stimulated with conditioned medium from resting, PHA or LPS-activated peripheral blood mononuclear cells (PBMC). Medium with LPS or PHA at the same concentration as that used to stimulate the PBMC was used as an additional control. Lysates were subjected to western blotting for activated forms of the mitogen-activated protein kinases (MAPK), Stat1, and Stat3. Western blotting showed phosphorylation of the Jun kinase (JNK), p38, and Erk1/Erk2 MAPK in trophoblasts incubated with conditioned medium from LPS or PHA-activated PBMC but not from medium from resting PBMC, or with PHA or LPS alone. Phosphorylation could be detected as early as 5 min and was still observable by 10 min, the latest time point tested. Similarly, Stat1 and Stat3 phosphorylation was observed within 10 min of exposure to conditioned medium and was still observable 10 min after exposure. Immunohistochemistry also demonstrated nuclear translocation of both Stat1 and Stat3 after stimulation of trophoblasts with medium from activated PBMC. These findings are compatible with the hypothesis that immunologic balance at the maternal-fetal interface is maintained by ongoing “cross-talk” between the fetus (and fetally-derived tissues) and the maternal immune system. Infection of the maternal vaginal tract may disrupt this delicate immunologic balance, initiating inflammatory events that ultimately result in preterm labor.

Pregnancy-associated plasma protein A proteolytic activity is associated with the human placental trophoblast cell membrane.

Pregnancy-associated plasma protein-A (PAPP-A) is a product of the placenta and decidua and is secreted into the maternal circulation during human pregnancy. It recently has been identified as an IGF binding protein (IGFBP)-4 protease. Presumed functions at the maternal-fetal interface are to proteolyze IGFBP-4 and thus increase IGF bioavailability locally in the placenta, to promote IGF-II-mediated trophoblast invasion into the maternal decidua, and to modulate IGF regulation of steroidogenesis and glucose and amino acid transport in the villous. Herein, we have investigated the possibility that IGFBP-4 proteolysis may occur on the trophoblast cell membrane, presumably to increase local bioavailable IGF for interactions with cognate IGF membrane receptors. Human trophoblasts were cultured, trophoblast plasma membranes were isolated and solubilized, and IGFBP-4 protease activity and PAPP-A immunoreactivity in the solubilized plasma membrane fraction were investigated. IGFBP-4 protease activity was detected in solubilized human trophoblast membranes, resulting in cleavage of recombinant human IGFBP-4 into 18- and 14-kDa fragments, detected by Western immunoblot analysis. This protease activity was dependent on the presence of IGF-II, and its metal ion dependence was demonstrated by inhibition of the protease by the metal chelators, EDTA and EGTA. The presence of PAPP-A in solubilized human trophoblast membranes was demonstrated by Western immunoblotting. Trophoblast membrane PAPP-A had a relative molecular weight of approximately 200 kDa and comigrated on (reducing) SDS-PAGE with recombinant human PAPP-A and PAPP-A secreted into media conditioned by cultured human trophoblasts. IGFBP-4 protease activity was not detected after immunodepletion of PAPP-A from the trophoblast membrane fraction with PAPP-A polyclonal antibodies, suggesting the identity of the membrane-derived IGFBP-4 protease as PAPP-A. Immunocytochemistry revealed PAPP-A on the cell membrane and in the cytoplasm of human trophoblasts in culture. Together, these data demonstrate the presence of an IGF-II- and metal-dependent IGFBP-4 protease activity in human trophoblast plasma membranes, identified as PAPP-A, which is well situated to proteolyze IGFBP-4 at the maternal-placental interface to facilitate IGF action at the villous surface and/or the invading extravillous cytotrophoblast.

Development of an in vitro human placenta model by the cultivation of human trophoblasts in a fiber-based bioreactor system.

The in vitro human trophoblast culture system is of significant importance in the study of human placenta development and its role as the transport organ between maternal and fetal circulations in normal physiology and pathology pregnancy. But conventional in vitro model systems fail to reproduce many important features of human placenta in vivo. In our study, a perfusion bioreactor system was developed with a chemically modified poly(ethylene terephthalate) (PET) fibrous matrix as the cell culture scaffold. The dual compartment design of the bioreactor simulates maternal and fetal circulation systems in vivo. First trimester human trophoblast cells readily attached on a chemically modified PET fiber surface. The detection of human fibronectin showed that cells were able to form three-dimensional structures by aggregation and bridging between fibers. Moreover, metabolic and hormone secretion data showed that cells in this perfusion culture system maintained their normal functional activities. The results of this study demonstrate the feasibility of tissue engineering human trophoblast cells in a perfusion bioreactor system for the development of an in vitro drug testing model system.

Dexamethasone stimulates gap junctional communication, peptide hormones production and differentiation in human term trophoblast

During trophoblast differentiation, a gap junctional intercellular communication (GJIC) precedes the trophoblastic cell fusion and the formation of the syncytiotrophoblast. The end of GJIC could thus be considered as an objective criterion of trophoblast differentiation. Maternal cortisol level increases during gestation and glucocorticoid receptors have been identified in trophoblastic cells. Therefore, the effects of dexamethasone on intercellular dye diffusion have been investigated by means of the Fluorescence Recovery After Photobleaching (gap-FRAP) technique in human trophoblast in culture. In parallel, trophoblast differentiation, hCG and hCS production were assessed. Dexamethasone stimulated the syncytium formation and significantly increased the percentage of coupled cells (2.5 fold at 0.5 μM). Simultaneously, hCG release was increased. After 2 days, hCS expression, was also increased by 35% compared to control. The glucocorticoid action was specific, since the stimulatory effects were blocked by RU486 (1 μM). Dexamethasone could act by the stimulation of hCG production or by the stimulation of GJIC and trophoblast differentiation. An excess of hCG antibody with dexamethasone did not significantly affect the stimulatory effect of the glucocorticoid, suggesting a self-sufficient effect of the steroid. In conclusion, dexamethasone stimulated intercellular communication, subsequent differentiation and concomitant endocrine functions of the trophoblast.