Human Placental Trophoblast Cells Research Articles

NBR1-dependent autophagy activation protects against environmental cadmium-evoked placental trophoblast senescence

Cadmium (Cd), a well-established developmental toxicant, accumulates in the placentae and disrupts its structure and function. Population study found adverse pregnancy outcomes caused by environmental Cd exposure associated with cell senescence. However, the role of autophagy activation in Cd-induced placental cell senescence and its reciprocal mechanisms are unknown. In this study, we employed animal experiments, cell culture, and case-control study to investigate the above mentioned. We have demonstrated that exposure to Cd during gestation induces placental senescence and activates autophagy. Pharmacological and genetic interventions further exacerbated placental senescence induced by Cd through the suppression of autophagy. Conversely, activation of autophagy ameliorated Cd-induced placental senescence. Knockdown of NBR1 exacerbated senescence in human placental trophoblast cells. Further investigations revealed that NBR1 facilitated the degradation of p21 via LC3B. Our case-control study has demonstrated a positive correlation between placental senescence and autophagy activation in all-cause fetal growth restriction (FGR). These findings offer a novel perspective for mitigating placental aging and placental-origin developmental diseases induced by environmental toxicants.

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.

Placental Trophoblast Cell Isolation from the Term Placenta.

Multiple cell lines have been utilized over time in studying placental biology. Still, most of them rely on choriocarcinoma cells or immortalized trophoblast cells that may not be entirely comparable with actual human placental trophoblast cells. Term placentas can be a source of primary villous trophoblasts. However, challenges remain in isolating them and maintaining them in extended culture. This manuscript describes our three-phase protocol utilizing enzymatic/mechanical digestion, modified Percoll gradient density separation, and immunopurification using magnetic beads. The resulting trophoblast culture remains viable for an extended period and highly pure after initial passaging.

Toxoplasma gondii Induces Pyroptosis in Human Placental Trophoblast and Amniotic Cells by Inducing ROS Production and Activation of Cathepsin B and NLRP1/NLRP3/NLRC4/AIM2 Inflammasome

Toxoplasma gondii infection in pregnant women may cause fetal anomalies; however, the underlying mechanisms remain unclear. The current study investigated whether T. gondii induces pyroptosis in human placental cells and the underlying mechanisms. Human placental trophoblast (BeWo and HTR-8/SVneo) and amniotic (WISH) cells were infected with T. gondii, and then reactive oxygen species (ROS) production, cathepsin B (CatB) release, inflammasome activation, and pyroptosis induction were evaluated. The molecular mechanisms of these effects were investigated by treating the cells with ROS scavengers, a CatB inhibitor, or inflammasome-specific siRNA. T. gondii infection induced ROS generation and CatB release into the cytosol in placental cells but decreased mitochondrial membrane potential. T. gondii-infected human placental cells and villi exhibited NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation and subsequent pyroptosis induction, as evidenced by increased expression of ASC, cleaved caspase-1, and mature IL-1β and gasdermin D cleavage. In addition to inflammasome activation and pyroptosis induction, adverse pregnancy outcome was shown in a T. gondii-infected pregnant mouse model. Administration of ROS scavengers, CatB inhibitor, or inflammasome-specific siRNA into T. gondii-infected cells reversed these effects. Collectively, these findings show that T. gondii induces NLRP1/NLRP3/NLRC4/AIM2 inflammasome-dependent caspase-1-mediated pyroptosis via induction of ROS production and CatB activation in placental cells. This mechanism may play an important role in inducing cell injury in congenital toxoplasmosis.

The mechanisms of regulation between glucose metabolism and syncytialization in human placental trophoblast cells

The mechanisms of regulation between glucose metabolism and syncytialization in human placental trophoblast cells

ZIKV infection differentially affects the transcriptional profiles in HTR8 and U251 cells

The mechanism by which Zika virus (ZIKV) causes severe birth defects in pregnant women remains unclear. Cell tropisms in placenta and brain play a crucial role in ZIKV pathogenesis, leading to congenital Zika syndrome (CZS). To identify the host factors involved in ZIKV infection, we compared the transcriptional profiles of ZIKV-infected human first-trimester placental trophoblast cells HTR8/SVneo and a human glioblastoma astrocytoma cell line U251. Our results demonstrated that ZIKV exhibited lower rates of mRNA replication and protein expression in HTR8 than in U251 cells, while showing a higher release of infectious viral particles. However, a greater number of differentially expressed genes (DEGs) were found in ZIKV-infected U251 cells than in ZIKV-infected HTR8 cells. Several of these DEGs were enriched in distinct biological processes related to the characteristics of each cell type that may contribute to foetal damage. Both cell types exhibited activation of common interferons, inflammatory cytokines, and chemokine production upon ZIKV infection. Moreover, the neutralization of tumour necrosis factor-alpha (TNF-α) promoted ZIKV infection in both trophoblasts and glioblastoma astrocytoma cells. Overall, we identified multiple DEGs associated with ZIKV pathogenesis.

Effects of valproic acid on syncytialization in human placental trophoblast cell lines

The placenta is a critical organ for fetal development and a healthy pregnancy, and has multifaceted functions (e.g., substance exchange and hormone secretion). Syncytialization of trophoblasts is important for maintaining placental functions. Epilepsy is one of the most common neurological conditions worldwide. Therefore, this study aimed to reveal the influence of antiepileptic drugs, including valproic acid (VPA), carbamazepine, lamotrigine, gabapentin, levetiracetam, topiramate, lacosamide, and clobazam, at clinically relevant concentrations on syncytialization using in vitro models of trophoblasts. To induce differentiation into syncytiotrophoblast-like cells, BeWo cells were treated with forskolin. Exposure to VPA was found to dose-dependently influence syncytialization-associated genes (ERVW-1, ERVFRD-1, GJA1, CGB, CSH, SLC1A5, and ABCC4) in differentiated BeWo cells. Herein, the biomarkers between differentiated BeWo cells and the human trophoblast stem model (TSCT) were compared. In particular, MFSD2A levels were low in BeWo cells but abundant in TSCT cells. VPA exposure affected the expression of ERVW-1, ERVFRD-1, GJA1, CSH, MFSD2A, and ABCC4 in differentiated cells (ST-TSCT). Furthermore, VPA exposure attenuated BeWo and TSCT cell fusion. Finally, the relationships between neonatal/placental parameters and the expression of syncytialization markers in human term placentas were analyzed. MFSD2A expression was positively correlated with neonatal body weight, head circumference, chest circumference, and placental weight. Our findings have important implications for better understanding the mechanisms of toxicity of antiepileptic drugs and predicting the risks to placental and fetal development.

Transcriptome-wide profile of 1α,25 dihydroxyvitamin D3 in HTR-8/SVneo cells.

Vitamin D3 has been implicated in multiple reproductive events, whereas the effect of its bioactive metabolite 1α, 25 dihydroxyvitamin D3 (1,25(OH) 2 D3 ) on transcriptome profile of the placenta is unclear. The aim of this article is to determine transcriptome-wide profile caused by 1,25(OH) 2 D3 in human placental trophoblast cells. We performed RNA sequencing after stimulation of HTR-8/SVneo cells with 0.1, 1, 10, and 100 nM 1,25(OH)2 D3 for 24 h, identified differentially expressed genes by edgeR package (version 3.38.4), and analyzed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways by webtool Metascape. Also, common genes and specific genes in different concentrations of 1,25(OH) 2 D3 were identified. There were 180, 158, 161, and 174 differentially expressed genes after 0.1, 1, 10, and 100 nM 1,25(OH) 2 D3 stimulation, respectively. KEGG pathway analysis displayed that "lipid and atherosclerosis" were significantly enriched at 0.1 and 1 nM 1,25(OH)2 D3 , while "cytokine-cytokine receptor interaction," "TGF-beta signaling pathway" and "hippo signaling pathway" were significantly enriched in 1, 10, and 100 nM 1,25(OH)2 D3 . CYP24A1 was a significantly expressed common gene. UCP3 was significantly expressed in low concentrations and might affect energy metabolism. TCF24, EIF3CL, ABCD2, EPHA7, CRLF1, and SECTM1 were specific genes at physiological concentration. Similarly, SPDYE1, IQUB, IL18R1, and ZNF713 were considered as specific genes at supraphysiological concentration. 1,25(OH)2 D3 mainly affected the expression of CYP24A1 gene in HTR-8/SVneo cells. Specific genes accounted for the majority of differentially expressed genes at different concentrations. However, their functions need to be further confirmed.

COLEC12 expression participates in trophoblast insulin resistance, and reverses celecoxib-mediated inhibition of COX2-PGE2 axis in gestational diabetes

Purpose: To evaluate the contribution of collectin subfamily member 12 (COLEC12) in insulin resistance associated with gestational diabetes mellitus (GDM).
 Methods: Insulin resistance was induced in human placental trophoblast (HTR-8/SVneo) cells in vitro by incubating them with 1 μM insulin for 48 h. Expression of COLEC12 was assessed using western blot and quantitative real time-polymerase chain reaction (qRT-PCR). Distribution of glucose transporters GLUT1 and GLUT4 was visualized using immunofluorescent staining. Furthermore, the level of reactive oxygen species (ROS) was evaluated by flow cytometry.
 Results: Insulin-resistant HTR-8/SVneo (IR-HTR-8/SVneo) cells showed significantly elevated COLEC12 expression (p < 0.001). Knocking down of COLEC12 in these cells led to a significant reduction in GLUT1 expression, and increase in both glucose consumption and GLUT4 expression (p < 0.001). Furthermore, COLEC12 knockdown levels of ROS increased SOD expression in IR-HTR8/SVneo, while knockdown of COLEC12 reduced p-insulin receptor substrate (IRS) (Ser307) protein expression, but increased p-IRS-1 (Tyr896), p-insulin receptor β (IRβ) (Tyr1361), and p-AKT levels. COLEC12 knockdown also decreased cyclooxygenase-2 (COX-2) expression, resulting in lower prostaglandin E2 (PGE2) levels in IR-HTR-8/SVneo. COLEC12 over-expression attenuated celecoxibinduced increase in glucose consumption, resulting in decreased COX2 and PGE2 levels.
 Conclusion: COLEC12 in IR-HTR-8/SVneo cells has antioxidant effects that protect against insulin resistance. This protective effect is achieved through the down-regulation of COX-2/PGE2. These findings provide a potential strategy for the treatment of GDM.

Research progress of E3 ubiquitin ligase regulating biological behavior of human placental trophoblast cells.

E3 ubiquitin ligases are important components of the ubiquitin protease system. This family includes many proteins, which can catalyze the ubiquitination of a variety of protein substrates and promote the degradation of them by the proteasome system. Recent studies have shown that E3 ubiquitin ligase plays a key role in the process of fetal development and placental formation. It affects the biological behavior of placental trophoblast cells, leading to a series of pregnancy complications that threaten mothers and babies greatly. This review focuses on the regulation, target and mechanism of E3 ubiquitin ligase on the biological behavior of human placental trophoblast cells.

Activation of Atg5-dependent placental lipophagy ameliorates cadmium-induced fetal growth restriction

Cadmium (Cd), an environmental contaminant, can result in placental non-selective autophagy activation and fetal growth restriction (FGR). However, the role of placental lipophagy, a selective autophagy, in Cd-induced FGR is unclear. This work uses case-control study, animal experiments and cultures of primary human placental trophoblast cells to explore the role of placental lipophagy in Cd-induced FGR. We found association of placental lipophagy and all-cause FGR. Meanwhile, pregnancy Cd exposure induced FGR and placental lipophgay. Inhibition of placental lipophagy by pharmacological and genetic means (Atg5−/− mice) exacerbated Cd-caused FGR. Inversely, activating of placental lipophagy relieved Cd-stimulated FGR. Subsequently, we found that activation of Atg5-dependent lipophagy degrades lipid droplets to produce free cholesterol, and promotes placental progesterone (P4) synthesis. Gestational P4 supplementation significantly reversed Cd-induced FGR. Altogether, activation of Atg5-dependent placental lipophagy ameliorates Cd-induced FGR.

MiR-141-3p promotes hypoxia-induced autophagy in human placental trophoblast cells.

Preeclampsia (PE) is a pregnancy-specific disorder and a significant contributor to maternal, fetal and neonatal morbidity and mortality worldwide. Its pathogenesis is generally accepted as insufficient trophoblast invasion of the maternal endometrium and inadequate remodeling of the maternal spiral arteries. These impairments lead to elevated levels of hypoxia and oxidative stress. Autophagy has become a highly researched field in obstetrics, and this process may be essential for preimplantation development beyond the four- and eight-cell stages, and for blastocyst survival, extra-villous trophoblast functions, invasion and vascular remodeling. Several studies have shown that autophagy activation, shown by an increase in autophagy vacuoles or microtubule-associated protein 1A/1B-light chain 3 (LC3) dots, was more common in PE than in normal pregnancy. Thus, changes in autophagic status are seen in preeclamptic placentas. MicroRNA-141-3p (miR-141-3p), a multifunctional miRNA, is involved in a variety of physiological and pathological processes, including PE and autophagy. However, the influence of miR-141-3p on autophagy regulation in trophoblast cells has yet to be described. Therefore, the objective of our study was to investigate the role of miR-141-3p in autophagy induced by hypoxia in human placental trophoblast cells. Our results found that hypoxia induced autophagy in trophoblast cells and dramatically elevated the expression of miR-141-3p. Overexpression of miR-141-3p improved autophagic activity, whereas low expression of miR-141-3p inhibited autophagic activity. Therefore, our data demonstrated that miR-141-3p promoted hypoxia-induced autophagy in placental trophoblast cells, which may be related to the development of preeclampsia.

Circ_0014736 induces GPR4 to regulate the biological behaviors of human placental trophoblast cells through miR-942-5p in preeclampsia.

Previous studies have indicated that the development of preeclampsia (PE) involves the regulation of circular RNA (circRNA). However, the role of hsa_circ_0014736 (circ_0014736) in PE remains unknown. Thus, the study proposes to reveal the function of circ_0014736 in the pathogenesis of PE and the underlying mechanism. The results showed that circ_0014736 and GPR4 expression were significantly upregulated, while miR-942-5p expression was downregulated in PE placenta tissues when compared with normal placenta tissues. circ_0014736 knockdown promoted the proliferation, migration, and invasion of placenta trophoblast cells (HTR-8/SVneo) and inhibited apoptosis; however, circ_0014736 overexpression had the opposite effects. circ_0014736 functioned as a sponge for miR-942-5p and regulated HTR-8/SVneo cell processes by interacting with miR-942-5p. Additionally, GPR4, a target gene of miR-942-5p, was involved in miR-942-5p-mediated actions in HTR-8/SVneo cells. Moreover, circ_0014736 stimulated GPR4 production through miR-942-5p. Collectively, circ_0014736 inhibited HTR-8/SVneo cell proliferation, migration, and invasion and induced cell apoptosis through the miR-942-5p/GPR4 axis, providing a possible target for the treatment of PE.

WITHDRAWN: Circ_0014736 induces GPR4 to regulate the biological behaviors of a human placental trophoblast cell line through miR-942-5p in preeclampsia

WITHDRAWN: Circ_0014736 induces GPR4 to regulate the biological behaviors of a human placental trophoblast cell line through miR-942-5p in preeclampsia

Workflow for Performing Genetic Manipulation in Human Trophoblast Stem Cells Using CRISPR/Cas9 Technology.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based tool kits greatly facilitate the investigation of gene function, especially gene editing and transcriptional regulation technology. Human trophoblast stem cell (hTSC) is a promising cell model for simulating the development and function of human placental trophoblast cells. Here, we provide an optimized workflow for efficient genome editing and transcriptional activation in hTSCs using a lentivirus-dependent method. We also present methods to evaluate the gene targeted mutations and transcriptional activation.

Maternal obesity: new placental paradigms unfolded.

The prevalence of maternal obesity is increasing at an alarming rate, and is providing a major challenge for obstetric practice. Adverse effects on maternal and fetal health are mediated by complex interactions between metabolic, inflammatory, and oxidative stress signaling in the placenta. Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are common downstream pathways of cell stress, and there is evidence that this conserved homeostatic response may be a key mediator in the pathogenesis of placental dysfunction. We summarize the current literature on the placental cellular and molecular changes that occur in obese women. A special focus is cast onto placental ER stress in obese pregnancy, which may provide a novel link for future investigation.

Watermelon-Derived Extracellular Vesicles Influence Human Ex Vivo Placental Cell Behavior by Altering Intestinal Secretions.

During pregnancy, mother-to-fetus transfer of nutrients is mediated by the placenta; sub-optimal placental development and/or function results in fetal growth restriction (FGR), and the attendant risk of stillbirth, neurodevelopmental delay, and non-communicable diseases in adulthood. A maternal diet high in fruit and vegetables lowers the risk of FGR but the association cannot be explained fully by known macro- and micronutrients. This study investigates if dietary-derived extracellular vesicles (EVs) can regulate placental function. The study characterizes the microRNA and protein cargo of EVs isolated from watermelon, show they are actively internalized by human intestinal epithelial cells in vitro, use mass spectrometry to demonstrate that they alter the intestinal secretome and bioinformatic analyses to predict the likely affected pathways in cells/tissues distal to gut. Application of the watermelon EV-modified intestinal secretome to human placental trophoblast cells and ex vivo tissue explants affects the trophoblast proteome and key aspects of trophoblast behavior, including migration and syncytialization. Dietary-derived plant EVs can modify intestinal communication with distal tissues, including the placenta. Harnessing the beneficial properties of dietary-derived plant EVs and/or exploiting their potential as natural delivery agents may provide new ways to improve placental function and reduce rates of FGR.

Long noncoding RNA PVT1 accelerates the growth of placental trophoblasts in preeclampsia through the microRNA‐24‐3p/HSD11B2 axis

Long noncoding RNA plasmacytoma variant translocation 1 (PVT1) is essential for the maintenance of normal functions of trophoblasts in preeclampsia (PE).This study aims to decipher the concrete mechanism of PVT1 with the microRNA-24-3p/Type-2 11β-hydroxysteroid dehydrogenase (miR-24-3p/HSD11B2) axis in PE. PVT1, miR-24-3p, and HSD11B2 expression levels in normal placental tissues and PE placental tissues were defined. HTR-8/SVneo cells were transfected to determine the effects of PVT1, miR-24-3p, and HSD11B2 on the growth of HTR-8/SVneo cells. The relationships among PVT1/miR-24-3p/HSD11B2 in HTR-8/SVneo cells were identified. PVT1 and HSD11B2 were downregulated, while miR-24-3p was upregulated in the placenta of PE. Upregulated/downregulated PVT1 promoted/impeded the growth of human placental trophoblast (HTR-8/SVneo) cells in PE. Restored/knocked down miR-24-3p impeded/enhanced the growth of HTR-8/SVneo cells in PE. PVT1 inhibited miR-24-3p to mediate HSD11B2. PVT1 sponges miR-24-3p to regulate HSD11B2; thereby, the growth of placental trophoblasts is promoted in PE.

A High-Throughput Toxicity Screen of 42 Per- and Polyfluoroalkyl Substances (PFAS) and Functional Assessment of Migration and Gene Expression in Human Placental Trophoblast Cells.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants that have been associated with adverse pregnancy outcomes in women and experimental research models. Adverse developmental and reproductive outcomes have been investigated for relatively few PFAS, and such studies are not scalable to address the thousands of unique chemical structures. As the placenta has been reported as a PFAS target tissue, the human placental trophoblast JEG-3 cell line was employed in a high-throughput toxicity screen (HTTS) to evaluate the effects of 42 unique PFAS on viability, proliferation, and mitochondrial membrane potential (MMP). HTTS concentration-response curve fitting determined EC50 values for 79% of tested compounds for at least one of the three endpoints. Trophoblast migratory potential was evaluated for a subset of six prioritized PFAS using a scratch wound assay. Migration, measured as the percent of wound closure after 72 h, was most severely inhibited by exposure to 100 µM perfluorooctanoic acid (PFOA; 72% closure), perfluorooctanesulfonic acid (PFOS; 57% closure), or ammonium perfluoro-2-methyl-3-oxahexanoate (GenX; 79% closure). PFOA and GenX were subsequently evaluated for disrupted expression of 46 genes reported to be vital to trophoblast health. Disrupted regulation of oxidative stress was suggested by altered expression of GPEX1 (300 µM GenX and 3 µM GenX), GPER1 (300 µM GenX), and SOD1 and altered cellular response to xenobiotic stress was indicated by upregulation of the placental efflux transporter, ABCG2 (300 µM GenX, 3 µM GenX, and 100 µM PFOA). These findings suggest the placenta is potentially a direct target of PFAS exposure and indicate that trophoblast cell gene expression and function are disrupted at PFAS levels well below the calculated cytotoxicity threshold (EC50). Future work is needed to determine the mechanism(s) of action of PFAS towards placental trophoblasts.

Perfluorooctanoic acid exposure increases both proliferation and apoptosis of human placental trophoblast cells mediated by ER stress-induced ROS or UPR pathways

Perfluorooctanoate acid (PFOA) is a highly persistent and widespread chemical in the environment. PFOA serum levels in pregnant women are positively associated with an increased risk of placenta-related disorders. However, the mechanism of PFOA cytotoxicity involved in placental cells and cellular responses such as ER stress remains poorly understood. In this study, we studied the cellular toxicity of PFOA with a focus on proliferation and apoptosis in a human placental trophoblast cell line. Cell viability, number, apoptosis, stress response, activation of the involved signaling pathways were assessed. Our results showed PFOA affected cell viability, proliferation and also resulted in apoptosis. Besides, both pro-proliferation and pro-apoptosis effects were attenuated by endoplasmic reticulum (ER) stress inhibitors. Further experiments demonstrated that two different signaling pathways were activated by PFOA-induced ER stress and involved in PFOA toxicity: the reactive oxygen species (ROS)-dependent ERK signaling triggered trophoblast proliferation, while the ATF4-dependent C/EBP homologous protein (CHOP) signaling was the trigger of apoptosis. We conclude that PFOA-induced ER stress is the trigger of proliferation and apoptosis of trophoblast via ROS or UPR signaling pathway, which leads to the altered balance critical to the normal development and function of the placenta.