Human Trophoblast Research Articles

Parabens promotes invasive properties of multiple human cells: A potential cancer-associated adverse outcome pathway

Parabens are esters of p-hydroxybenzoic acid, which have been used as preservatives and considered safe for nearly a century, until the last two decades when concerns began to be raised about their association with cancers. Knowledge of the mode of action of parabens on the metastatic properties of different cancer cells is still very limited. In the present study, we investigated the effects of methylparaben (MP) and propylparaben (PP) on cell invasion and/or migration in multiple human cancerous and noncancerous cells, including hepatocellular carcinoma cells (HepG2), cervical carcinoma cells (HeLa), breast carcinoma cells (MCF-7), and human placental trophoblasts (HTR-8/SVneo). MP and PP at concentrations in a range of 5–500 μg/L significantly promoted the invasion of four cell lines, with a minimum effective concentration of 5 μg/L. MP and PP up-regulated the expression levels and enzymatic activities of matrix metalloproteinase 2 and 9 (MMP2 and MMP9), as well as altered the expression of the tissue inhibitors of metalloproteinase 1 and 2 (TIMP1 and TIMP2) in four cell lines, suggesting MMPs/TIMPs as potential key events (KEs) for paraben-induced cell invasion. Activation of the p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal protein kinases 1/2 (JNK1/2) signaling pathways was required for MP- and PP-promoted invasion of four cell lines, suggesting MAPK signaling pathways as candidates for KEs in cancer or noncancerous cells response to paraben exposure. This study showed for the first time that the two widely used parabens, MP and PP, promoted invasive capacity of multiple human cells through a common mode of action. This study provides evidence for the establishment of a potential cancer-associated AOP for parabens based on pathway-specific mechanism(s), which contributes towards assessing the health risks of these environmental chemicals.

In vitro blastocyst implantation and trophoblast migration are disrupted by the UV filter benzophenone-3 (BP3)

Benzophenone-3 (BP3) is a common ingredient in personal care products (PCPs) due to its well-established effectiveness in absorbing UV radiation. Sunscreen products are among the most widely used PCPs-containing BP3 applied to the skin, resulting in significant human exposure to BP3 primarily through a dermal application. In the present work, we have tested the action of three environmentally relevant concentrations of BP3 (2, 20 and 200 μg/L) on an in vitro model of implantation of murine blastocysts and on migration ability of the human trophoblast cell line Swan 71. We showed that BP3 caused a significant reduction of blastocyst expansion and a delayed hatching in a non-monotonic way. Besides, embryos displayed a delayed attachment in the three BP3 groups, resulting in a smaller implantation area on the 6th day of culture: BP3(2) (0.32 ± 0.07 mm2); BP3(20) (0.30 ± 0.08 mm2) and BP3(200) (0.25 ± 0.06 mm2) in comparison to the control (0.42 ± 0.07 mm2). We also found a reduced migration capacity of the human first-trimester trophoblast cell line Swan 71 in a scratch assay when exposed to BP3: the lowest dose displayed a higher uncovered area (UA) at 6h when compared to the control, whereas a higher UA of the wound was observed for the three BP3 concentrations at 18 and 24 h of exposure. The changes in UA provoked by BP3 restored to normal values in the presence of flutamide, an androgen receptor (AR) inhibitor. These results indicate that a direct impairment on early embryo implantation and a defective migration of extravillous trophoblast cells through the androgen receptor pathway can be postulated as mechanisms of BP3-action on early gestation with potential impact on fetal growth.

Abstract 5804: Preclinical evaluation of LCB84, a novel next-generation TROP2 directed ADC utilizing a cancer selective linker and an MMAE payload

Abstract LCB84 is an antibody-drug conjugate (ADC) that consists of an antibody targeting human trophoblast cell-surface antigen-2 (TROP2) linked to monomethyl auristatin E (MMAE) via a beta-glucuronidase cleavable linker with a drug-to-antibody ratio (DAR) of 4. LCB84 demonstrates tumor selective killing by multiple mechanisms, increasing both safety and efficacy in parallel. Firstly, the over-expression of TROP2 in many solid tumors allows for targeted delivery of MMAE by LCB84 to Trop2-positive tumor tissues. Secondly, the overexpression of beta-glucuronidase in the lysosome of most tumors facilitates the selective release of active payload within tumor cells compared to normal healthy tissues. Thirdly, the intrinsic selectivity of tubulin inhibitor payloads, such as MMAE, for proliferating cells provides additional protection for non-proliferating normal cells. MMAE is also cell-permeable in its free drug form and can lead to increased efficacy of LCB84 via a bystander effect, enabling activity in heterogenous tumors. LCB84 demonstrates superior efficacy in mouse tumor xenograft studies when compared to benchmark TROP2-targeting ADCs, including sacituzumab govitecan and datopotamab deruxtecan. LCB84 also shows an improved safety profile compared to these clinical benchmarks, as well as compared to traditional MMAE-bearing ADCs, in both in vitro and in vivo toxicity assessments. Additionally, LCB84 shows a stable preclinical pharmacokinetic profile with minimal loss of payload over time. The utilization of an ADC for target-mediated delivery, in combination with a stable beta-glucuronide trigger for selective payload release in tumors, allows for a superior preclinical therapeutic index which is expected to translate to a best-in-class ADC in the clinic. Citation Format: Stephen Slocum, Hyejung Kim, Chul-Woong Chung, Changsik Park. Preclinical evaluation of LCB84, a novel next-generation TROP2 directed ADC utilizing a cancer selective linker and an MMAE payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5804.

Metformin ameliorates PM2.5-induced functional impairment of placental trophoblasts by inhibiting ferroptosis

To investigate the protective effect of metformin against PM2.5-induced functional impairment of placental trophoblasts and explore the underlying mechanism. Sixteen pregnant Kunming mice were randomly assigned into two groups (n=8) for intratracheal instillation of PBS or PM2.5 suspension at 1.5, 7.5, and 12.5 days of gestation. The pregnancy outcome of the mice was observed, and placental zonal structure and vascular density of the labyrinth area were examined with HE staining, followed by detection of ferroptosis-related indexes in the placenta. In cultured human trophoblasts (HTR8/SVneo cells), the effects of PM2.5 exposure and treatment with metformin on cell viability, proliferation, migration, invasion, and tube formation ability were evaluated using CCK8 assay, EDU staining, wound healing assay, Transwell experiment, and tube formation experiment; the cellular expressions of ferroptosis-related proteins were analyzed using ELISA and Western blotting. M2.5 exposure of the mice during pregnancy resulted in significantly decreased weight and number of the fetuses and increased fetal mortality with a reduced placental weight (all P<0.001). PM2.5 exposure also caused obvious impairment of the placental structure and trophoblast ferroptosis. In cultured HTR8/SVneo cells, PM2.5 significantly inhibited proliferation, migration, invasion, and angiogenesis of the cells by causing ferroptosis. Metformin treatment obviously attenuated PM2.5-induced inhibition of proliferation, migration, invasion, and angiogenesis of the cells, and effectively reversed PM2.5-induced ferroptosis in the trophoblasts as shown by significantly increased intracellular GSH level and SOD activity, reduced MDA and Fe2+ levels, and upregulated GPX4 and SLC7A11 protein expression (P<0.05 or 0.01). PM2.5 exposure during pregnancy causes adverse pregnancy outcomes and ferroptosis and functional impairment of placental trophoblasts in mice, and metformin can effectively alleviate PM2.5-induced trophoblast impairment.

An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells.

Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.

Endogenous retrovirus HERVH-derived lncRNA UCA1 controls human trophoblast development

Endogenous retroviruses (ERVs) are frequently reactivated in mammalian placenta. It has been proposed that ERVs contribute to shaping the gene regulatory network of mammalian trophoblasts, dominantly acting as species- and placental-specific enhancers. However, whether and how ERVs control human trophoblast development through alternative pathways remains poorly understood. Besides the well-recognized function of human endogenous retrovirus-H (HERVH) in maintaining pluripotency of early human epiblast, here we present a unique role of HERVH on trophoblast lineage development. We found that the LTR7C/HERVH subfamily exhibits an accessible chromatin state in the human trophoblast lineage. Particularly, the LTR7C/HERVH-derived Urothelial Cancer Associated 1 (UCA1), a primate-specific long non-coding RNA (lncRNA), is transcribed in human trophoblasts and promotes the proliferation of human trophoblast stem cells (hTSCs), whereas its ectopic expression compromises human trophoblast syncytialization coinciding with increased interferon signaling pathway. Importantly, UCA1 upregulation is detectable in placental samples from early-onset preeclampsia (EO-PE) patients and the transcriptome of EO-PE placenta exhibits considerable similarities to that of the syncytiotrophoblasts differentiated from UCA1-overexpressing hTSCs, supporting up-regulated UCA1 as a potential biomarker of this disease. Altogether, our data shed light on the versatile regulatory role of HERVH in early human development and provide a unique mechanism whereby ERVs exert a function in human placentation and placental syndromes.

Triphenyl phosphate interferes with the synthesis of steroid hormones through the PPARγ/CD36 pathway in human trophoblast JEG-3 cells.

Triphenyl phosphate (TPhP), a chemical commonly found in human placenta and breast milk, has been shown to disturb the endocrine system. Our previous study confirmed that TPhP could accumulate in the placenta and interference with placental lipid metabolism and steroid hormone synthesis, as well as induce endoplasmic reticulum (ER) stress through PPARγ in human placental trophoblast JEG-3 cells. However, the molecular mechanism underlying this disruption remains unknown. Our study aimed to identify the role of the PPARγ/CD36 pathway in TPhP-induced steroid hormone disruption. We found that TPhP increased lipid accumulation, total cholesterol, low- and high-density protein cholesterol, progesterone, estradiol, glucocorticoid, and aldosterone levels, and genes related to steroid hormones synthesis, including 3βHSD1, 17βHSD1, CYP11A, CYP19, and CYP21. These effects were largely blocked by co-exposure with either a PPARγ antagonist GW9662 or knockdown of CD36 using siRNA (siCD36). Furthermore, an ER stress inhibitor 4-PBA attenuated the effect of TPhP on progesterone and glucocorticoid levels, and siCD36 reduced ER stress-related protein levels induced by TPhP, including BiP, PERK, and CHOP. These findings suggest that ER stress may also play a role in the disruption of steroid hormone synthesis by TPhP. As our study has shed light on the PPARγ/CD36 pathway's involvement in the disturbance of steroid hormone biosynthesis by TPhP in the JEG-3 cells, further investigations of the potential impacts on the placental function and following birth outcome are warranted.

Exposure to high dose of polystyrene nanoplastics causes trophoblast cell apoptosis and induces miscarriage

BackgroundWith rapid increase in the global use of various plastics, microplastics (MPs) and nanoplastics (NPs) pollution and their adverse health effects have attracted global attention. MPs have been detected out in human body and both MPs and NPs showed female reproductive toxicological effects in animal models. Miscarriage (abnormal early embryo loss), accounting for 15-25% pregnant women worldwide, greatly harms human reproduction. However, the adverse effects of NPs on miscarriage have never been explored.ResultsIn this study, we identified that polystyrene (PS) plastics particles were present in women villous tissues. Their levels were higher in villous tissues of unexplained recurrent miscarriage (RM) patients vs. healthy control (HC) group. Furthermore, mouse assays further confirmed that exposure to polystyrene nanoplastics (PS-NPs, 50 nm in diameter, 50 or 100 mg/kg) indeed induced miscarriage. In mechanism, PS-NPs exposure (50, 100, 150, or 200 µg/mL) increased oxidative stress, decreased mitochondrial membrane potential, and increased apoptosis in human trophoblast cells by activating Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3 signaling through mitochondrial pathway. The alteration in this signaling was consistent in placental tissues of PS-NPs-exposed mouse model and in villous tissues of unexplained RM patients. Supplement with Bcl-2 could efficiently suppress apoptosis in PS-NPs-exposed trophoblast cells and reduce apoptosis and alleviate miscarriage in PS-NPs-exposed pregnant mouse model.ConclusionsExposure to PS-NPs activated Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3, leading to excessive apoptosis in human trophoblast cells and in mice placental tissues, further inducing miscarriage.Graphical

Human trophoblast stem cells restrict human cytomegalovirus replication.

Placental infection plays a central role in human cytomegalovirus (HCMV) pathogenesis during pregnancy, but the species specificity of HCMV and the limited availability and lifespan of primary trophoblasts have been persistent barriers to understanding how infection impacts this vital organ. Human trophoblast stem cells (TSCs) represent a new approach to modeling viral infection early in placental development. This study reveals that TSCs, like other stem cell types, restrict HCMV replication. However, infection perturbs the expression of genes involved in differentiation and cell fate determination, pointing to a mechanism by which HCMV could cause placental injury.

Low levels of nicotine and cotinine but not benzo[a]pyrene induce human trophoblast cell proliferation

E-cigarettes use constitutes a source of thirdhand nicotine exposure. The increasing use of electronic cigarettes in homes and public places increases the risk of exposure of pregnant women to thirdhand nicotine. The effects of exposure of pregnant women to very low levels of nicotine have not been studied in humans but detrimental in experimental animals. The objective of this study is to investigate the effect of nanomolar concentrations of nicotine and its metabolite cotinine on the proliferation of JEG-3, a human trophoblast cell line. We also studied the proliferative effect of nanomolar concentrations of benzo[a]pyrene (B[a]P), a polycyclic hydrocarbon in tobacco smoke, for comparison.We treated JEG-3 cells in culture with nanomolar concentrations of nicotine, cotinine, and B[a]P. Their effect on cell proliferation was determined, relative to untreated cells, by MTT assay. Western blotting was used to assess the mitogenic signaling pathways affected by nicotine and cotinine.In contrast to the inhibitory effects reported with higher concentrations, we showed that nanomolar concentrations of nicotine and cotinine resulted in significant JEG-3 cell proliferation and a rapid but transient increase in levels of phosphorylated ERK and AKT, but not STAT3. Biphasic, non-monotonic effect on cell growth is characteristic of endocrine disruptive chemicals like nicotine. The mitogenic effects of nicotine and cotinine potentially contribute to increased villous epithelial thickness, seen in placentas of some smoking mothers. This increases the diffusion distance for oxygen and nutrients between mother and fetus, contributing to intrauterine growth restriction in infants of smoking mothers.

Inhibition of the endoplasmic reticulum stress-associated IRE-1 pathway alleviates preterm birth.

Premature birth (PTB) remains a major global health concern due to its association with neonatal morbidity and mortality. The unfolded protein response (UPR) within the endoplasmic reticulum (ER) is tightly regulated by Inositol-requiring enzyme type 1(IRE-1), a pivotal cellular modulator. This study seeks to elucidate the role of the ER stress (ERS)-related IRE-1 pathway in PTB. Human placental trophoblast cells HTR8/Svneo were exposed to the ER-stress inducer tunicamycin (TM). The expression of IRE-1 and ERS-associated proteins ATF6, GRP78, and XBP-1 was assessed in placental tissues and TM-treated cells. Cellular viability, migration, invasion, and apoptosis were evaluated through a series of experimental assays. Additionally, various methods were employed to assess and verify the activation of autophagy, using the autophagy marker, microtubule-associated protein 1A/1B-light chain 3 (LC3). Additionally, TUDCA (an ERS inhibitor) was used to assess its potential to counteract the TM-induced cell effects. Elevated levels of ATF6, GRP78, and XBP-1 were observed in PTB tissues and cells. TM treatment substantially reduced cell viability, migration, and invasion while promoting apoptosis. Treatment with TUDCA (an ERS inhibitor) counteracted the effects of TM on the cells. Furthermore, we identified an overexpression of IRE-1 in PTB tissues and cells and its knockdown enhanced cell viability, migration, and invasion while suppressed apoptosis and autophagy under TM stimulation. Notably, IRE-1 was found to modulate the activity of the IRE-1/XBP1/CHOP signaling pathway in TM-treated cells. The upregulation of IRE-1 in PTB placental tissues is implicated in the pathogenesis of PTB. Importantly, inhibiting the ERS-associated IRE-1/XBP1/CHOP pathway may be a good strategy in mitigating PTB.

Trophoblast organoid systems to study human placentation

Human trophoblast organoids provide a valuable invitro system to investigate human placental development and function. In this issue of Developmental Cell, Shannon etal. benchmark two organoid models against primary trophoblast at single-cell resolution, identifying their strengths and limitations.

Pregnancy-Specific Beta-1-Glycoprotein 1 Increases HTR-8/SVneo Cell Migration through the Orai1/Akt Signaling Pathway.

The impaired invasion ability of trophoblast cells is related to the occurrence of preeclampsia (PE). We previously found that pregnancy-specific beta-1-glycoprotein 1 (PSG1) levels were decreased in the serum of individuals with early-onset preeclampsia (EOPE). This study investigated the effect of PSG1 on Orai1-mediated store-operated calcium entry (SOCE) and the Akt signaling pathway in human trophoblast cell migration. An enzyme-linked immunosorbent assay (ELISA) was used to determine the level of PSG1 in the serum of pregnant women with EOPE. The effects of PSG1 on trophoblast proliferation and migration were examined using cell counting kit-8 (CCK8) and wound healing experiments, respectively. The expression levels of Orai1, Akt, and phosphorylated Akt (p-Akt) were determined through Western blotting. The results confirmed that the serum PSG1 levels were lower in EOPE women than in healthy pregnant women. The PSG1 treatment upregulated the protein expression of Orai1 and p-Akt. The selective inhibitor of Orai1 (MRS1845) weakened the migration-promoting effect mediated by PSG1 via suppressing the Akt signaling pathway. Our findings revealed one of the mechanisms possibly involved in EOPE pathophysiology, which was that downregulated PSG1 may reduce the Orai1/Akt signaling pathway, thereby inhibiting trophoblast migration. PSG1 may serve as a potential target for the treatment and diagnosis of EOPE.

Sh- Ambra1 inhibits IRS-1/PI3K/Akt signalling pathway to reduce autophagy in gestational diabetes.

Gestational diabetes mellitus (GDM) is the most common metabolic disease in pregnancy. However, studies of activating molecule of Beclin1-regulated autophagy (Ambra1) affecting the insulin substrate receptor 1/phosphatidylinositol 3 kinase/protein kinase B (IRS-1/PI3K/Akt) signalling pathway in GDM have not been reported. The aim of the study was to detect the difference of Ambra1 expression in the placenta of normal pregnant women and GDM patients. An in vitro model of gestational diabetes mellitus was established by inducing HTR8/Svneo cells from human chorionic trophoblast layer with high glucose. The changes of cell morphology were observed by inverted microscope, and the expression levels of Ambra1 gene and protein in model cells were detected. After this, Ambra1 gene was silenced by shRNA transfection, and PI3K inhibitor was added to detect changes in Ambra1, autophagy, and insulin (INS) signalling pathways. The protein expression levels of Ambra1, Bcl-2 interacting protein (Beclin-1), and microtubule-associated proteins 1A/1B light chain 3B (LC3-II) in the placentas of GDM pregnant women were higher than those of normal pregnant women. High glucose induces morphological changes in HTR8/Svneo cells and increases Ambra1 transcription and translation levels. sh-Ambra1 increased survival of HTR8/SvNEO-HG cells and inhibited Ambra1, Beclin1, and LC3-II transcription and translation levels. Also, sh-Ambra1 increased IRS-1/PI3K/Akt protein phosphorylation levels and inhibited the IRS-1/PI3K/Akt signalling pathway and its resulting autophagy. sh-Ambra1 increased IRS-1/PI3K/Akt protein phosphorylation levels to reduce autophagy in gestational diabetes.

A Potential Role of CD82/KAI1 during Uterine Decidualization in Mice.

The tumor metastasis suppressor gene CD82/KAI1 has been demonstrated to impact human trophoblast invasion and migration. Communication between trophoblasts and decidual stromal cells plays a crucial role in controlling the normal invasiveness of trophoblasts. However, whether CD82/KAI1 is involved in decidualization and what role it plays remain unclear. CD82/KAI1 demonstrates specific spatiotemporal expression patterns in stromal cells undergoing decidualization during pregnancy. This is observed in both naturally pregnant females post-implantation and pseudopregnant mice undergoing induced decidualization, as detected through in situ hybridization and immunofluorescence. CD82/KAI1 expression showed a significant time-dependent increase in cultured stromal cells after 24 and 48 h of progesterone (P4) and estrogen (E2) treatment. This was accompanied by a notable upregulation of decidualization markers, including cyclin D3 and PR. After transducing stromal cells with the adenovirus-overexpressing CD82/KAI1 for 48 h, the expression of cyclin D3 protein increased. Meanwhile, there was an attenuated expression of CD82/KAI1 due to an adenovirus siRNA knockdown, whereas cyclin D3 and PR expressions were not affected. Our findings suggest a potential role of CD82/KAI1 in regulating the process of decidualization, providing insights into stromal cell differentiation.

The Impact of SLC2A8 RNA Interference on Glucose Uptake and the Transcriptome of Human Trophoblast Cells.

While glucose is the primary fuel for fetal growth, the placenta utilizes the majority of glucose taken up from the maternal circulation. Of the facilitative glucose transporters in the placenta, SLC2A8 (GLUT8) is thought to primarily function as an intracellular glucose transporter; however, its function in trophoblast cells has not been determined. To gain insight into the function of SLC2A8 in the placenta, lentiviral-mediated RNA interference (RNAi) was performed in the human first-trimester trophoblast cell line ACH-3P. Non-targeting sequence controls (NTS RNAi; n = 4) and SLC2A8 RNAi (n = 4) infected ACH-3P cells were compared. A 79% reduction in SLC2A8 mRNA concentration was associated with an 11% reduction (p ≤ 0.05) in ACH-3P glucose uptake. NTS RNAi and SLC2A8 RNAi ACH-3P mRNA were subjected to RNAseq, identifying 1525 transcripts that were differentially expressed (|log2FC| > 1 and adjusted p-value < 0.05), with 273 transcripts derived from protein-coding genes, and the change in 10 of these mRNAs was validated by real-time qPCR. Additionally, there were 147 differentially expressed long non-coding RNAs. Functional analyses revealed differentially expressed genes involved in various metabolic pathways associated with cellular respiration, oxidative phosphorylation, and ATP synthesis. Collectively, these data indicate that SLC2A8 deficiency may impact placental uptake of glucose, but that its likely primary function in trophoblast cells is to support cellular respiration. Since the placenta oxidizes the majority of the glucose it takes up to support its own metabolic needs, impairment of SLC2A8 function could set the stage for functional placental insufficiency.

Dynamic and distinct histone modifications facilitate human trophoblast lineage differentiation

The placenta serves as an essential organ for fetal growth throughout pregnancy. Histone modification is a crucial regulatory mechanism involved in numerous biological processes and development. Nevertheless, there remains a significant gap in our understanding regarding the epigenetic regulations that influence trophoblast lineage differentiation, a fundamental aspect of placental development. Here, through comprehensive mapping of H3K4me3, H3K27me3, H3K9me3, and H3K27ac loci during the differentiation of trophoblast stem cells (TSCs) into syncytiotrophoblasts (STs) and extravillous trophoblasts (EVTs), we reveal dynamic reconfiguration in H3K4me3 and H3K27ac patterns that establish an epigenetic landscape conducive to proper trophoblast lineage differentiation. We observe that broad H3K4me3 domains are associated with trophoblast lineage-specific gene expression. Unlike embryonic stem cells, TSCs lack robust bivalent domains. Notably, the repression of ST- and EVT-active genes in TSCs is primarily attributed to the weak H3K4me3 signal rather than bivalent domains. We also unveil the inactivation of TSC enhancers precedes the activation of ST enhancers during ST formation. Our results provide a comprehensive global map of diverse histone modifications, elucidating the dynamic histone modifications during trophoblast lineage differentiation.

Growth differentiation factor-11 upregulates matrix metalloproteinase 2 expression by inducing Snail in human extravillous trophoblast cells

The human extravillous trophoblast (EVT) cell invasion is an important process during placentation. Although the placenta is normal tissue, the EVT cells exhibit some features common to cancer cells, including high migratory and invasive properties. Snail and Slug are transcription factors that mediate the epithelial-mesenchymal transition (EMT), a crucial event for cancer cell migration and invasion. It has been shown that GDF-11-induced matrix metalloproteinase 2 (MMP2) expression is required for EVT cell invasion. Whether GDF-11 can regulate Snail and Slug expression in human EVT cells remains unknown. If it does, the involvement of Snail and Slug in GDF-11-induced MMP2 expression and EVT cell invasion must also be defined. In the present study, using the immortalized human EVT cell line, HTR-8/SVneo, and primary cultures of human EVT cells as experimental models, our results show that GDF-11 upregulates Snail and Slug expression. ALK4 and ALK5 mediate the stimulatory effects of GDF-11 on Snail and Slug expression. In addition, we demonstrate that SMAD2 and SMAD3 are required for the GDF-11-upregulated Snail expression, while only SMAD3 is involved in GDF-11-induced Slug expression. Moreover, our results reveal that Snail mediates GDF-11-induced MMP2 expression and cell invasion but not Slug. This study increases our understanding of the biological function of GDF-11 in human EVT cells and provides a novel mechanism for regulating MMP2 and EVT cell invasion.

TBX3 reciprocally controls key trophoblast lineage decisions in villi during human placenta development in the first trimester

Human trophoblastic lineage development is intertwined with placental development and pregnancy outcomes, but the regulatory mechanisms underpinning this process remain inadequately understood. In this study, based on single-nuclei RNA sequencing (snRNA-seq) analysis of the human early maternal-fetal interface, we compared the gene expression pattern of trophoblast at different developmental stages. Our findings reveal a predominant upregulation of TBX3 during the transition from villous cytotrophoblast (VCT) to syncytiotrophoblast (SCT), but downregulation of TBX3 as VCT progresses into extravillous trophoblast cells (EVT). Immunofluorescence analysis verified the primary expression of TBX3 in SCT, partial expression in MKi67-positive VCT, and absence in HLA-G-positive EVT, consistent with our snRNA-seq results. Using immortalized trophoblastic cell lines (BeWo and HTR8/SVneo) and human primary trophoblast stem cells (hTSCs), we observed that TBX3 knockdown impedes SCT formation through RAS-MAPK signaling, while TBX3 overexpression disrupts the cytoskeleton structure of EVT and hinders EVT differentiation by suppressing FAK signaling.In conclusion, our study suggests that the spatiotemporal expression of TBX3 plays a critical role in regulating trophoblastic lineage development via distinct signaling pathways. This underscores TBX3 as a key determinant during hemochorial placental development.

Single-cell assessment of primary and stem cell-derived human trophoblast organoids as placenta-modeling platforms

Human trophoblast stem cells (hTSCs) and related trophoblast organoids are state-of-the-art culture systems that facilitate the study of trophoblast development and human placentation. Using single-cell transcriptomics, we evaluate how organoids derived from freshly isolated first-trimester trophoblasts or from established hTSC cell lines reproduce developmental cell trajectories and transcriptional regulatory processes defined invivo. Although organoids from primary trophoblasts and hTSCs overall model trophoblast differentiation with accuracy, specific features related to trophoblast composition, trophoblast differentiation, and transcriptional drivers of trophoblast development show levels of misalignment. This is best illustrated by the identification of an expanded progenitor state in stem cell-derived organoids that is nearly absent invivo and transcriptionally shares both villous cytotrophoblast and extravillous trophoblast characteristics. Together, this work provides a comprehensive resource that identifies strengths and limitations of current trophoblast organoid platforms.