Regulation Of Trophoblast Differentiation Research Articles

Charting the Dynamic Trophoblast Plasma Membrane Identifies LYN As a Functional Regulator of Syncytialization.

The differentiation of placental cytotrophoblasts (CTBs) into the syncytiotrophoblast (STB) layer results in a significant remodeling of the plasma membrane proteome. Here, we use a peroxidase-catalyzed proximity labeling strategy to map the dynamic plasma membrane proteomes of CTBs and STBs. Coupled with mass-spectrometry-based proteomics, we identify hundreds of plasma membrane proteins and observe relative changes in protein abundance throughout differentiation, including the upregulation of the plasma-membrane-localized nonreceptor tyrosine kinase LYN. We show that both siRNA-mediated knockdown and small molecule inhibition of LYN kinase function impairs CTB fusion and reduces the expression of syncytialization markers, presenting a function for LYN outside of its canonical role in immunological signaling. Our results demonstrate the use of the proximity labeling platform to discover functional regulators within the plasma membrane and provide new avenues to regulate trophoblast differentiation.

Mitochondrial pyruvate flux regulates trophoblast differentiation

Mitochondrial pyruvate flux regulates trophoblast differentiation

Spatially resolved transcriptomic profiling of placental development in dairy cow.

The placenta plays a crucial role in successful mammalian reproduction. Ruminant animals possess a semi-invasive placenta characterized by a highly vascularized structure formed by maternal endometrial caruncles and fetal placental cotyledons, essential for full-term fetal development. The cow placenta harbors at least two trophoblast cell populations: uninucleate (UNC) and binucleate (BNC) cells. However, the limited capacity to elucidate the transcriptomic dynamics of the placental natural environment has resulted in a poor understanding of both the molecular and cellular interactions between trophoblast cells and niches, and the molecular mechanisms governing trophoblast differentiation and functionalization. To fill this knowledge gap, we employed Stereo-seq to map spatial gene expression patterns at near single-cell resolution in the cow placenta at 90 and 130 days of gestation, attaining high-resolution, spatially resolved gene expression profiles. Based on clustering and cell marker gene expression analyses, key transcription factors, including YBX1 and NPAS2, were shown to regulate the heterogeneity of trophoblast cell subpopulations. Cell communication and trajectory analysis provided a framework for understanding cell-cell interactions and the differentiation of trophoblasts into BNCs in the placental microenvironment. Differential analysis of cell trajectories identified a set of genes involved in regulation of trophoblast differentiation. Additionally, spatial modules and co-variant genes that help shape specific tissue structures were identified. Together, these findings provide foundational insights into important biological pathways critical to the placental development and function in cows.

Single-nuclei RNA sequencing (snRNA-seq) uncovers trophoblast cell types and lineages in the mature bovine placenta

Ruminants have a semi-invasive placenta, which possess highly vascularized placentomes formed by maternal endometrial caruncles and fetal placental cotyledons and required for fetal development to term. The synepitheliochorial placenta of cattle contains at least two trophoblast cell populations, including uninucleate (UNC) and binucleate (BNC) cells that are most abundant in the cotyledonary chorion of the placentomes. The interplacentomal placenta is more epitheliochorial in nature with the chorion developing specialized areolae over the openings of uterine glands. Of note, the cell types in the placenta and cellular and molecular mechanisms governing trophoblast differentiation and function are little understood in ruminants. To fill this knowledge gap, the cotyledonary and intercotyledonary areas of the mature day 195 bovine placenta were analyzed by single nuclei analysis. Single-nuclei RNA-seq analysis found substantial differences in cell type composition and transcriptional profiles between the two distinct regions of the placenta. Based on clustering and cell marker gene expression, five different trophoblast cell types were identified in the chorion, including proliferating and differentiating UNC and two different types of BNC in the cotyledon. Cell trajectory analyses provided a framework for understanding the differentiation of trophoblast UNC into BNC. The upstream transcription factor binding analysis of differentially expressed genes identified a candidate set of regulator factors and genes regulating trophoblast differentiation. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta.

SLC20a1/PiT-1 is required for chorioallantoic placental morphogenesis.

The placenta mediates the transport of nutrients, such as inorganic phosphate (Pi), between the maternal and fetal circulatory systems. The placenta itself also requires high levels of nutrient uptake as it develops to provide critical support for fetal development. This study aimed to determine placental Pi transport mechanisms using in vitro and in vivo models. We observed that Pi (P33) uptake in BeWo cells is sodium dependent and that SLC20A1/Slc20a1 is the most highly expressed placental sodium-dependent transporter in mouse (microarray), human cell line (RT-PCR) and term placenta (RNA-seq), supporting that normal growth and maintenance of the mouse and human placenta requires SLC20A1/Slc20a1. Slc20a1 wild-type (Slc20a1+/+) and knockout (Slc20a1-/-) mice were produced through timed intercrosses and displayed yolk sac angiogenesis failure as expected at E10.5. E9.5 tissues were analyzed to test whether placental morphogenesis requires Slc20a1. At E9.5, the developing placenta was reduced in size in Slc20a1-/-. Multiple structural abnormalities were also observed in the Slc20a1-/-chorioallantois. We determined that monocarboxylate transporter 1 protein (MCT1+) cells were reduced in developing Slc20a1-/-placenta, confirming that Slc20a1 loss reduced trophoblast syncytiotrophoblast 1 (SynT-I) coverage. Next, we examined the cell type-specific Slc20a1 expression and SynT molecular pathways in silico and identified Notch/Wnt as a pathway of interest that regulates trophoblast differentiation. We further observed that specific trophoblast lineages express Notch/Wnt genes that associate with endothelial cell tip-and-stalk cell markers. In conclusion, our findings support that Slc20a1 mediates the symport of Pi into SynT cells, providing critical support for their differentiation and angiogenic mimicry function at the developing maternal-fetal interface.

N-Oleoyldopamine promotes the differentiation of mouse trophoblast stem cells into parietal trophoblast giant cells

The placenta plays various roles in a healthy pregnancy, and abnormalities in the placenta result in adverse outcomes. Adequate differentiation of trophoblast subtypes is necessary for placental function, but the molecular mechanisms that determine trophoblast cell fate remain unclear. Here, we screened small molecular compound (SMC) libraries (1904 SMCs) to identify particular SMCs which regulate trophoblast differentiation in mouse trophoblast stem cells (mTSCs) to understand the molecular mechanisms underlying cell fate decision in trophoblast cells. The two-step screening revealed a novel effect of N-oleoyldopamine (OLDA), an endogenic vanilloid, to promote differentiation into parietal trophoblast giant cells (P-TGCs) and repress them into spongiotrophoblast cells in mTSCs. Analyses by gene deletion and inhibitor treatments indicated that transient receptor potential cation channel subfamily V member 3 (Trpv3), one of the candidates for targeting by OLDA, was involved in maintaining stem status and P-TGC differentiation in mTSCs. Finally, transcriptome analysis revealed that Fosl1, a key regulatory factor in differentiation into P-TGCs, was upregulated by OLDA treatment, suggesting that OLDA promoted the differentiation of mTSCs into P-TGCs via regulation of Fosl1 expression.

Hyperosmolarity Impairs Human Extravillous Trophoblast Differentiation by Caveolae Internalization.

We recently reported that an intact caveolar structure is necessary for adequate cell migration and tubulogenesis of the human extravillous trophoblast (EVT) cells. Emerging evidence supports that hyperosmolarity induces the internalization of caveolae into the cytoplasm and accelerates their turnover. Furthermore, signaling pathways associated with the regulation of trophoblast differentiation are localized in caveolae. We hypothesized that hyperosmolarity impairs EVT differentiation and caveolae/caveolin−1 (Cav-1) participates in this process. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 and exposed to hyperosmolar condition (generated by the addition of 100 mM sucrose). Hyperosmolarity altered the EVT cell migration and the formation of tube-like structures. In addition, cell invasion was decreased along with a reduction in the latent and active forms of matrix metalloproteinase-2 (MMP−2) secreted by these cells. With respect to Cav-1 protein abundance, we found that hyperosmolarity enhanced its degradation by the lysosomal pathway. Accordingly, in the hyperosmolar condition, we also observed a significant increase in the number of vacuoles and the internalization of the caveolae into the cytoplasm. Taken together, our findings suggest that hyperosmolarity may induce caveolae internalization and increase their turnover, compromising the normal differentiation of EVT cells.

BAP1/ASXL complex modulation regulates epithelial-mesenchymal transition during trophoblast differentiation and invasion.

Normal function of the placenta depends on the earliest developmental stages when trophoblast cells differentiate and invade into the endometrium to establish the definitive maternal-fetal interface. Previously, we identified the ubiquitously expressed tumour suppressor BRCA1-associated protein 1 (BAP1) as a central factor of a novel molecular node controlling early mouse placentation. However, functional insights into how BAP1 regulates trophoblast biology are still missing. Using CRISPR/Cas9 knockout and overexpression technology in mouse trophoblast stem cells, here we demonstrate that the downregulation of BAP1 protein is essential to trigger epithelial-mesenchymal transition (EMT) during trophoblast differentiation associated with a gain of invasiveness. Moreover, we show that the function of BAP1 in suppressing EMT progression is dependent on the binding of BAP1 to additional sex comb-like (ASXL1/2) proteins to form the polycomb repressive deubiquitinase (PR-DUB) complex. Finally, both endogenous expression patterns and BAP1 overexpression experiments in human trophoblast stem cells suggest that the molecular function of BAP1 in regulating trophoblast differentiation and EMT progression is conserved in mice and humans. Our results reveal that the physiological modulation of BAP1 determines the invasive properties of the trophoblast, delineating a new role of the BAP1 PR-DUB complex in regulating early placentation.

The role of the tumour suppressor BRCA1-associated protein 1 (Bap1) in regulating trophoblast differentiation and invasiveness

The role of the tumour suppressor BRCA1-associated protein 1 (Bap1) in regulating trophoblast differentiation and invasiveness

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.

MicroRNAs: crucial regulators of placental development.

MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that are integral to a wide range of cellular processes mainly through the regulation of translation and mRNA stability of their target genes. The placenta is a transient organ that exists throughout gestation in mammals, facilitating nutrient and gas exchange and waste removal between the mother and the fetus. miRNAs are expressed in the placenta, and many studies have shown that miRNAs play an important role in regulating trophoblast differentiation, migration, invasion, proliferation, apoptosis, vasculogenesis/angiogenesis and cellular metabolism. In this review, we provide a brief overview of canonical and non-canonical pathways of miRNA biogenesis and mechanisms of miRNA actions. We highlight the current knowledge of the role of miRNAs in placental development. Finally, we point out several limitations of the current research and suggest future directions.

Mapping the chromatin landscape and Blimp1 transcriptional targets that regulate trophoblast differentiation

Trophoblast stem cells (TSCs) give rise to specialized cell types within the placenta. However, the regulatory mechanisms that guide trophoblast cell fate decisions during placenta development remain ill defined. Here we exploited ATAC-seq and transcriptional profiling strategies to describe dynamic changes in gene expression and chromatin accessibility during TSC differentiation. We detect significantly increased chromatin accessibility at key genes upregulated as TSCs exit from the stem cell state. However, downregulated gene expression is not simply due to the loss of chromatin accessibility in proximal regions. Additionally, transcriptional targets recognized by the zinc finger transcriptional repressor Prdm1/Blimp1, an essential regulator of placenta development, were identified in ChIP-seq experiments. Comparisons with previously reported ChIP-seq datasets for primordial germ cell-like cells and E18.5 small intestine, combined with functional annotation analysis revealed that Blimp1 has broadly shared as well as cell type-specific functional activities unique to the trophoblast lineage. Importantly, Blimp1 not only silences TSC gene expression but also prevents aberrant activation of divergent developmental programmes. Overall the present study provides new insights into the chromatin landscape and Blimp1-dependent regulatory networks governing trophoblast gene expression.

Alterted SLIT2/ROBO1 signalling is linked to impaired placentation of missed and threatened miscarriage in early pregnancy.

Two-thirds of early pregnancy failures present with reduced trophoblast invasion, and SLIT2/ROBO1 signalling is considered to play an important role in trophoblast function during pregnancy. We investigated SLIT2/ROBO1 signalling associated with missed and threatened miscarriage during early gestation. Human placenta samples were collected from women with missed miscarriage (n = 25), threatened miscarriage (n = 22) and termination of pregnancy controls (n = 32). Corresponding decreases in beta human chorionic gonadotrophin (β-hCG) levels and shallow trophoblast invasion were observed in patients with missed and threatened miscarriage, immunohistological staining revealed abnormal Slit2 and Robo1, as well as E-cadherin and activating protein-2 alpha (AP-2α) expression in villi and extravillous trophoblasts, and the expression of these proteins were confirmed in villi and decidua of miscarriage material by Western blotting. Using HTR8/SVneo cells, blocking SLIT2/ROBO1 signalling promoted cell migration, proliferation and suppressed differentiation. Moreover, blocking SLIT2/ROBO1 signalling in HTR8/SVneo cells altered trophoblast differentiation-related and angiogenesis-related gene mRNA expression, which also occurred in the tissues of missed and threatened miscarriage. SLIT2/ROBO1 signalling may regulate trophoblast differentiation and invasion causing restricting β-hCG production, shallow trophoblast invasion and inhibiting placental angiogenesis in missed and threatened miscarriage during the first trimester.

P45 NF-E2 regulates syncytiotrophoblast differentiation by post-translational GCM1 modifications in human intrauterine growth restriction

Placental insufficiency jeopardizes prenatal development, potentially leading to intrauterine growth restriction (IUGR) and stillbirth. Surviving fetuses are at an increased risk for chronic diseases later in life. IUGR is closely linked with altered trophoblast and placental differentiation. However, due to a paucity of mechanistic insights, suitable biomarkers and specific therapies for IUGR are lacking. The transcription factor p45 NF-E2 (nuclear factor erythroid derived 2) has been recently found to regulate trophoblast differentiation in mice. The absence of p45 NF-E2 in trophoblast cells causes IUGR and placental insufficiency in mice, but mechanistic insights are incomplete and the relevance of p45 NF-E2 for human syncytiotrophoblast differentiation remains unknown. Here we show that p45 NF-E2 negatively regulates human syncytiotrophoblast differentiation and is associated with IUGR in humans. Expression of p45 NF-E2 is reduced in human placentae complicated with IUGR compared with healthy controls. Reduced p45 NF-E2 expression is associated with increased syncytiotrophoblast differentiation, enhanced glial cells missing-1 (GCM1) acetylation and GCM1 desumoylation in IUGR placentae. Induction of syncytiotrophoblast differentiation in BeWo and primary villous trophoblast cells with 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) reduces p45 NF-E2 expression. Of note, p45 NF-E2 knockdown is sufficient to increase syncytiotrophoblast differentiation and GCM1 expression. Loss of p45 NF-E2 using either approach resulted in CBP-mediated GCM1 acetylation and SENP-mediated GCM1 desumoylation, demonstrating that p45 NF-E2 regulates post-translational modifications of GCM1. Functionally, reduced p45 NF-E2 expression is associated with increased cell death and caspase-3 activation in vitro and in placental tissues samples. Overexpression of p45 NF-E2 is sufficient to repress GCM1 expression, acetylation and desumoylation, even in 8-Br-cAMP exposed BeWo cells. These results suggest that p45 NF-E2 negatively regulates differentiation and apoptosis activation of human syncytiotrophoblast by modulating GCM1 acetylation and sumoylation. These studies identify a new pathomechanism related to IUGR in humans and thus provide new impetus for future studies aiming to identify new biomarkers and/or therapies of IUGR.

Association of dysfunctional synapse defective 1 (SYDE1) with restricted fetal growth - SYDE1 regulates placental cell migration and invasion.

The transcription factor glial cells missing 1 (GCM1) regulates trophoblast differentiation and function during placentation. Decreased GCM1 expression is associated with pre-eclampsia, suggesting that abnormal expression of GCM1 target genes may contribute to the pathogenesis of pregnancy complications. Here we identified a novel GCM1 target gene, synapse defective 1 (SYDE1), which encodes a RhoGAP that is highly expressed in human placenta, and demonstrated that SYDE1 promotes cytoskeletal remodelling and cell migration and invasion. Importantly, genetic ablation of murine Syde1 results in small fetuses and placentas with aberrant phenotypes in the placental-yolk sac barrier, maternal-trophoblast interface, and placental vascularization. Microarray analysis revealed altered expression of renin-1, angiotensin I converting enzyme 2, angiotensin II type 1a receptor, and membrane metalloendopeptidase of the renin-angiotensin system in Syde1-knockout placenta, which may compensate for the vascular defects to maintain normal blood pressure. As pregnancy proceeds, growth restriction of the Syde1-/- fetuses and placentas continues, with elevated expression of the Syde1 homologue Syde2 in placenta. Syde2 may compensate for the loss of Syde1 function because SYDE2, but not the GAP-dead SYDE2 mutant, reverses migration and invasion activities of SYDE1-knockdown JAR trophoblast cells. Clinically, we further detected decreased SYDE1 expression in preterm and term IUGR placentas compared with gestational age-matched controls. Our study suggests a novel mechanism for GCM1 and SYDE1 in regulation of trophoblast cell migration and invasion during placental development and that decreased SYDE1 expression is associated with IUGR. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells

Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface.

HLA-G expression is regulated by miR-365 in trophoblasts under hypoxic conditions

IntroductionHypoxia occurs in the first trimester of placental development and is implicated in the regulation of trophoblast differentiation. Prolonged hypoxic conditions in the placenta are related to the development of preeclampsia. MicroRNAs (miRNAs) are noncoding, single-stranded RNAs that modulate gene expression by targeting messenger RNA. We hypothesized that, under hypoxic conditions, trophoblasts may have a unique miRNA profile that may play a critical role in placental development. MethodsTotal RNA was extracted from human trophoblast, HChEpC1b, exposed to normoxia (20% O2) or hypoxia (2% O2) for 24 h, and the miRNA expression profiles were investigated using a microRNA array. Several differential miRNAs were selected and validated using real-time reverse transcription PCR. We identified potential targets of these miRNAs using in silico analysis. We confirmed a potential target protein by western blot analysis and luciferase assays. ResultsThe expression of miR-365 was significantly upregulated under hypoxic conditions. In silico analysis showed that miR-365 targeted human leukocyte antigen (HLA)-G. Both hypoxic conditions and overexpression of miR-365 inhibited the expression of HLA-G proteins. The overexpression of miR-365 also decreased the activity of the luciferase reporter containing the 3′-untranslated region (UTR) of HLA-G with the predicted miR-365-binding site. DiscussionHLA-G is a non-classical HLA class-Ib molecule that is expressed mainly in extravillous trophoblasts and which plays a key role in maintaining immune tolerance at the maternal–fetal interface. Our results indicate that miR-365 targets the HLA-G 3′ UTR to repress its expression. The expression of miR-365 may play an important role in human placental development and in immunoprotection of the semiallogenic embryo.

Generation and Use of Trophoblast Stem Cells and Uterine Myocytes to Study the Role of Connexins for Pregnancy and Labor.

Transgenic mouse models have demonstrated critical roles for gap junctions in establishing a successful pregnancy. To study the cellular and molecular mechanisms, the use of cell culture systems is essential to discriminate between the effects of different connexin isoforms expressed in individual cells or tissues of the developing conceptus or in maternal reproductive tissues. The generation and analysis of gene-deficient trophoblast stem cell lines from mice clearly revealed the functions of connexins in regulating placental development. This chapter focuses on the use of connexin gene-deficient trophoblast stem cell cultures to reveal the individual role of gap junctions in regulating trophoblast differentiation and proliferation in vitro under controlled conditions. In addition, cultures of primary uterine myocytes, isolated from mice or rats, allow studying the effects of mechanical stretch or ovarian hormones on regulating connexin expression, and thus, to model the molecular mechanisms of uterine growth and development during pregnancy. Here, we describe the derivation of primary uterine myocyte cultures and their use in in vitro stretch experiments to study the mechanisms of myometrial remodeling essential to accommodate the growing fetus throughout gestation.

Insights into the mechanism of CXCL12-mediated signaling in trophoblast functions and placental angiogenesis.

The chemokine CXCL12 and its receptor CXCR4 are important signaling components required for human blastocyst implantation and the progression of pregnancy. Growing evidence indicates that the CXCL12/CXCR4 axis can regulate trophoblast function and uterine spiral artery remodeling, which plays a fundamental role in placentation and fetal outcome. The orphan receptor CXCR7 is also believed to partly regulate the function of the CXCL12/CXCR4 axis. Additionally, the CXCL12/CXCR4/CXCR7 axis can enhance the cross-talk between trophoblasts and decidual cells such as uterine natural killer cells and decidual stromal cells which are involved in regulation of trophoblast differentiation and invasion and placental angiogenesis. In addition, recent studies proved that CXCL12 expression is elevated in the placenta and mid-trimester amniotic fluid of pregnant women with preeclampsia, implying that dysregulation of CXCL12 plays a role in the pathogenesis of preeclampsia. Further understanding of the regulatory mechanisms of CXCL12-mediated signaling in trophoblast functions and placental angiogenesis may help to design novel therapeutic approaches for pregnancy-associated diseases.

317-POS

Objectives Infection with human cytomegalovirus (HCMV) during pregnancy may cause spontaneous abortions, premature delivery, pre-eclampsia, or permanent neurological disabilities in infants infected in utero. Recent studies demonstrated that HCMV infection inhibits cytotrophoblast (CTB) differentiation into an invasive phenotype resulting in shallow placentation, however the mechanisms involved remain unclear. The lack of an in vitro model that recapitulates the early stages of trophoblast differentiation has hindered efforts to determine the effect of HCMV infection during CTB differentiation. We sought to develop an in vitro model of placental trophoblast differentiation that will allow detailed examination of factors that regulate trophoblast differentiation, as well as, the effect of HCMV infection on trophoblast differentiation and function. Methods Primary trophoblast progenitor cells (TBPCs) isolated from chorions of first-trimester human placentas (generously provided by Dr. M. McMaster) were allowed to attach to collagen beads and cultured in rotating wall vessel (RWV) bioreactors. Differentiation of TBPCs was assessed by immunofluorescence staining, Western blot and qRT-PCR analyses for loss of chorionic villi progenitor markers and gain of invasive cytotrophoblast markers. The invasive capacity of cells was monitored by fibrin clot assays. Results Undifferentiated TBPCs express Cytokeratin 7, HMGA2 and OCT4. TBPCs cultured in RWVs, without added growth factors, initially proliferated and formed aggregates. After 5 days in RWVs, TBPCs downregulated expression of OCT4 and upregulated expression of HLA-G, a marker of invasive cytotrophoblasts. Importantly, TBPCs cultured in RWVs displayed invasive ability in the fibrin clot assay. Multinucleated cells characteristic of syncytiotrophoblasts were not observed. Both undifferentiated and differentiated TBPCs were susceptible to infection with clinical and laboratory-adapted strains of HCMV. Conclusions We have developed an in vitro model of placental trophoblast differentiation that will allow detailed examination of factors that regulate trophoblast differentiation, as well as, pathological placental development that increases risk of pre-eclampsia. Disclosures K.F. Swan: None. M. Ferris: None. G. Pridjian: None. C.A. Morris: None. D.E. Sullivan: None.