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Immunoperoxidase staining for vimentin (a and b) and ACT (c and d) in intact term placenta (a and c) and Percoll gradient-purified cells (b and d). a) Antivimentin stains the endothelial cells of capillaries (large arrowheads) and fibroblasts (arrow), but not the syncytiotrophoblasts or cytotrophoblasts (small arrowhead), b) Percoll gradient-purified cells do not stain for vimentin. c) Anti-ACT stains the macrophages (Hofbauer cells) of the villous core (large arrowheads), but not the syncytiotrophoblasts or cytotrophoblasts (small arrowheads), d) Percoll gradient-purified cells do not stain for anti-ACT. All micrographs, X750.
Source publication
Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmiss...
Contexts in source publication
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... Rare single cells exhibited staining (see text). 6 See Fig. 2. terminated by rapid freezing in an acetone-dry ice bath. Cells and media were analyzed for steroid contents by ...
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... content analysis using 4',6-diamidino-2-pheny- lindole-stained cells revealed the presence of an appre- ciable mitotic population. In one study, of 71,830 cells counted, 84.8% were in G o or d (2N ploidy), 15.1% were in G2 or mitosis, and 0.1% were in the S phase of the cell cycle. These data suggest that a significant fraction (15%) of the cells was actively dividing or ready to divide. ...
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... either the cyto-or syncytiotrophoblasts or the Percoll-purified cells (Fig. 2, a and b). This ruled out the possibility that our isolated cells were fibroblasts or endothelial cells. To determine if the isolated mononuclear cells were macrophages (27), we performed immunochemical stain- ing for ACT, a known marker for macrophages (28). Anti-ACT antibody stained only mesenchymal cells in the villi of the starting term ...
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... cells were fibroblasts or endothelial cells. To determine if the isolated mononuclear cells were macrophages (27), we performed immunochemical stain- ing for ACT, a known marker for macrophages (28). Anti-ACT antibody stained only mesenchymal cells in the villi of the starting term placentae, but did not stain the isolated mononuclear cells (Fig. 2, c and d). There- fore, the isolated cells are not ...
Citations
... Additionally, the effect of trophoblast differentiation on pathway expression was examined in BeWo and PHT cells. These cells are the only placental cell models to undergo trophoblast differentiation spontaneously (PHT cells) 27 or upon stimulation with syncytializing agents such as forskolin (BeWo) 28 . We observed that the expression of TH, DDC, PNMT, and COMT, is not regulated by the transition to differentiated STB (Fig. 3A-D). ...
Catecholamines norepinephrine and dopamine have been implicated in numerous physiological processes within the central nervous system. Emerging evidence has highlighted the importance of tightly regulated monoamine levels for placental functions and fetal development. However, the complexities of synthesis, release, and regulation of catecholamines in the fetoplacental unit have not been fully unraveled. In this study, we investigated the expression of enzymes and transporters involved in synthesis, degradation, and transport of norepinephrine and dopamine in the human placenta and rat fetoplacental unit. Quantitative PCR and Western blot analyses were performed in early-to-late gestation in humans (first trimester vs. term placenta) and mid-to-late gestation in rats (placenta and fetal brain, intestines, liver, lungs, and heart). In addition, we analyzed the gene expression patterns in isolated primary trophoblast cells from the human placenta and placenta-derived cell lines (HRP-1, BeWo, JEG-3). In both human and rat placentas, the study identifies the presence of only PNMT, COMT, and NET at the mRNA and protein levels, with the expression of PNMT and NET showing gestational age dependency. On the other hand, rat fetal tissues consistently express the catecholamine pathway genes, revealing distinct developmental expression patterns. Lastly, we report significant transcriptional profile variations in different placental cell models, emphasizing the importance of careful model selection for catecholamine metabolism/transport studies. Collectively, integrating findings from humans and rats enhances our understanding of the dynamic regulatory mechanisms that underlie catecholamine dynamics during pregnancy. We identified similar patterns in both species across gestation, suggesting conserved molecular mechanisms and potentially shedding light on shared biological processes influencing placental development.
... However, an important methodological issue to take into consideration is the contamination of the trophoblast culture with other cell types, such as mesenchymal cells or immune cells. In this regard, the introduction of the discontinuous Percoll gradient first described by Harvey Kliman et al. [1], allowed to obtain 95% viable CTBs within the Percoll layer corresponding to a density of 1.048-1.062 g/mL, with very few (1-5%) vimentin-positive (mesenchymal, endothelial) or α1-antichymotrypsin-positive (macrophages) contaminating cells [1,6]. ...
... In this regard, the introduction of the discontinuous Percoll gradient first described by Harvey Kliman et al. [1], allowed to obtain 95% viable CTBs within the Percoll layer corresponding to a density of 1.048-1.062 g/mL, with very few (1-5%) vimentin-positive (mesenchymal, endothelial) or α1-antichymotrypsin-positive (macrophages) contaminating cells [1,6]. ...
Since the early 1960s, researchers began culturing placental cells to establish an in vitro model to study the biology of human trophoblasts, including their ability to differentiate into syncytiotrophoblasts and secrete steroid and peptide hormones that help sustain a viable pregnancy. This task was addressed by testing different serum concentrations, cell culture media, digestive enzymes, growth factors, substrate coating with diverse proteins from the extracellular matrix, and so on. Among the many methodological challenges, the contamination of trophoblasts with other cell types, such as immune and stromal cells, was a matter of concern. However, introducing the Percoll gradient to isolate cytotrophoblasts was an excellent contribution, and later, the depletion of contaminating cells by using magnetic bead-conjugated antibodies also helped increase the purity of cytotrophoblasts. Herein, with some modifications, we describe a rapid and easy method for cytotrophoblast isolation from the term human placenta based on the previously reported method by Harvey Kliman et al. (Endocrinology 118:1567–1582, 1986). This method yields about 40–90 million cells from a single placenta, with a purity of around 85–90%.
... In the 1960s, vCTBs were hypothesized to be precursors for STB (Richart, 1961;Midgley et al., 1963). This has been further revealed using trophoblast cell lines (Kliman et al., 1986) and organoid cultures (Haider et al., 2018;. Combining our current knowledge, STB is derived from asymmetrical cell division and differentiation of vCTBs followed by fusion with pre-existing syncytium (Kn€ ofler et al., 2019). ...
BACKGROUND
With increasing significance of developmental programming effects associated with placental dysfunction, more investigations are devoted to improving the characterization and understanding of placental signatures in health and disease. The placenta is a transitory but dynamic organ adapting to the shifting demands of fetal development and available resources of the maternal supply throughout pregnancy. Trophoblasts (cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts) are placental-specific cell types responsible for the main placental exchanges and adaptations. Transcriptomic studies with single-cell resolution have led to advances in understanding the placenta’s role in health and disease. These studies, however, often show discrepancies in characterization of the different placental cell types.
OBJECTIVE AND RATIONALE
We aim to review the knowledge regarding placental structure and function gained from the use of single-cell RNA sequencing (scRNAseq), followed by comparing cell-type-specific genes, highlighting their similarities and differences. Moreover, we intend to identify consensus marker genes for the various trophoblast cell types across studies. Finally, we will discuss the contributions and potential applications of scRNAseq in studying pregnancy-related diseases.
SEARCH METHODS
We conducted a comprehensive systematic literature review to identify different cell types and their functions at the human maternal–fetal interface, focusing on all original scRNAseq studies on placentas published before March 2023 and published reviews (total of 28 studies identified) using PubMed search. Our approach involved curating cell types and subtypes that had previously been defined using scRNAseq and comparing the genes used as markers or identified as potential new markers. Next, we reanalyzed expression matrices from the six available scRNAseq raw datasets with cell annotations (four from first trimester and two at term), using Wilcoxon rank-sum tests to compare gene expression among studies and annotate trophoblast cell markers in both first trimester and term placentas. Furthermore, we integrated scRNAseq raw data available from 18 healthy first trimester and nine term placentas, and performed clustering and differential gene expression analysis. We further compared markers obtained with the analysis of annotated and raw datasets with the literature to obtain a common signature gene list for major placental cell types.
OUTCOMES
Variations in the sampling site, gestational age, fetal sex, and subsequent sequencing and analysis methods were observed between the studies. Although their proportions varied, the three trophoblast types were consistently identified across all scRNAseq studies, unlike other non-trophoblast cell types. Notably, no marker genes were shared by all studies for any of the investigated cell types. Moreover, most of the newly defined markers in one study were not observed in other studies. These discrepancies were confirmed by our analysis on trophoblast cell types, where hundreds of potential marker genes were identified in each study but with little overlap across studies. From 35 461 and 23 378 cells of high quality in the first trimester and term placentas, respectively, we obtained major placental cell types, including perivascular cells that previously had not been identified in the first trimester. Importantly, our meta-analysis provides marker genes for major placental cell types based on our extensive curation.
WIDER IMPLICATIONS
This review and meta-analysis emphasizes the need for establishing a consensus for annotating placental cell types from scRNAseq data. The marker genes identified here can be deployed for defining human placental cell types, thereby facilitating and improving the reproducibility of trophoblast cell annotation.
... Placentas from healthy women aged 20-35 who delivered at full term were collected from the Capital Medical University-affiliated Fuxing Hospital. According to a previously published study, primary human trophoblast (PHT) cells were isolated from human-term placentas for subsequent studies [49,50]. The placentas were manually cut into pieces and digested three times in DMEM (HyClone, Logan, UT, USA) with 0.125% trypsin (Gibco, 25200-072, New York, NY, USA) and 0.02% DNase I (Sigma Aldrich, St. Louis, MO, USA, DN25-100 mg). ...
Previous studies have shown that nuclear binding protein 2 (NUCB2) is expressed in the human placenta and increases with an increase in the syncytialization of trophoblast cells. This study aimed to investigate the role of NUCB2 in the differentiation and fusion of trophectoderm cells. In this study, the expression levels of NUCB2 and E-cadherin in the placentas of rats at different gestation stages were investigated. The results showed that there was an opposite trend between the expression of placental NUCB2 and E-cadherin in rat placentas in different trimesters. When primary human trophoblast (PHT) and BeWo cells were treated with high concentrations of Nesfatin-1, the trophoblast cell syncytialization was significantly inhibited. The effects of NUCB2 knockdown in BeWo cells and Forskolin-induced syncytialization were investigated. These cells showed a significantly decreased cell fusion rate. The mechanism underlying NUCB2-regulated trophoblast cell syncytialization was explored using RNA-Seq and the results indicated that the epidermal growth factor receptor (EGFR)-phospholipase C gamma 1 (PLCG1)-calmodulin-dependent protein kinase IV (CAMK4) pathway might be involved. The results suggested that the placental expression of NUCB2 plays an important role in the fusion of trophoblasts during differentiation via the EGFR-PLCG1-CAMK4 pathway.
... However, recent studies have revealed the presence of foetal cells in cervical mucus. (Kliman et al. 1986, Griffith-Jones et al. 1992. The dynamics of these cells, including their shedding into the uterine cavity and replacement of epithelial cells in blood vessels and glands (Arutyunyan et al. 2023, Moser et al. 2010. ...
Background
The detection of foetal DNA and extravillus trophoblasts (EVTs) in early pregnancy in cervical and uterine samples offers a potential pathway for non-invasive prenatal diagnostics. However, the challenge lies in effectively quantifying these samples. This study introduces a novel approach using the Ras association domain family 1 A (RASSF1A), which exhibits hypermethylation in foetal cells and hypomethylation in maternal cells. The differential methylation pattern of RASSF1A provides a unique biomarker for quantifying foetal cells in cervical and intrauterine samples.
Methods
This study was conducted between September 2022 and May 2023. In total, 23 samples (12 cervical cell samples and 11 intrauterine samples) were collected from women in the Sichuan Jinxin Women & Children Hospital, Jingxiu District, Chengdu, China. The cervical cell samples were collected via lavage and brush techniques, and the intrauterine cell samples were obtained via uterine lavage. These samples were collected as part of a broader effort to advance our understanding of foetal cell dynamics during early pregnancy. The sampling methods were chosen for their minimally invasive nature and their potential in capturing a representative cell population from the respective sites. After digestion of the cell samples using a methylation-sensitive restriction enzyme cocktail, a critical step to differentiate between maternal and foetal DNA, the quantitative polymerase chain reaction (qPCR) of RASSF1A and β-actin (ACTB) were employed to measure foetal DNA and cell concentrations. Immunofluorescence techniques targeting histocompatibility complex, class I G (HLA-G) and GATA binding protein 3 (GATA-3) were employed to detect EVTs in the cell samples and in decidual tissue, with the latter providing an additional layer of confirmation for the presence of foetal cells.
Results
The results showed no hypermethylated RASSF1A was detected in any of the cervical samples, irrespective of whether the samples were obtained by brush or lavage. However, an average of 17,236 ± 7490 foetal cells per sample were detected in the uterine lavage samples. Foetal cells accounted for approximately 0.14% ± 0.10% of the total cell population in these samples. The presence of EVTs in these samples was confirmed by their expression of both HLA-G and GATA-3.
Conclusion
The detection of foetal cells in uterine cavity samples based on hypermethylation of RASSF1A and quantification of foetal cells can be used to prenatal screening. GATA-3 can be used to label EVTs.
... Only after invasion can trophoblast cells successfully degrade and migrate through the extracellular matrix to interact closely with the endothelial cells of the uterine spiral arteries and further replace them (4,5). Insufficient trophoblastic invasion of the decidua and spiral arteries is considered to be the first stage of PE development (6,7). For researchers in reproductive medicine, the question of how to promote trophoblast cell function is of particular interest. ...
... The present study demonstrated that DCA could significantly improve the migration function of LPS-stimulated trophoblast cells. Previous studies have demonstrated that impaired trophoblast cell function impeded spiral artery transformation, which may result in pre-eclampsia (6,7). Accordingly, DCA might have a therapeutic potential to prevent pregnancy complications with trophoblast disorder, such as pre-eclampsia, which remains to be validated in future studies. ...
Objective(s)
Inadequate cytotrophoblast migration and invasion are speculated to result in preeclampsia, which is a pro-inflammatory condition. Sodium dichloroacetate (DCA) exerts anti-inflammatory actions. Thus,we sought to investigate the effect of DCA on the migration function of the lipopolysaccharide (LPS)-stimulated human-trophoblast-derived cell line (HTR-8/SVneo).
Materials and Methods
HTR-8/SVneo cells were treated with LPS to suppress cell migration. Cell migration was examined by both scratch wound healing assay and transwell migration assay. Western blotting was used to analyze the expression levels of toll-like receptor-4 (TLR4), nuclear factor-κB (NF-κB), TNF-α, IL-1β, and IL-6 in the cells.
Results
DCA reversed LPS-induced inhibition of migration in HTR-8/SVneo cells. Furthermore, DCA significantly suppressed LPS-induced activation of TLR4, phosphorylation of NF-κB (p65), translocation of p65 into the nucleus, and the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). Treatment with inhibitors of TLR4 signal transduction (CLI095 or MD2-TLR-4-IN-1) reduced LPS-induced overexpression of pro-inflammatory cytokines, and a synergistic effect was found between TLR4 inhibitors and DCA in HTR-8/SVneo cells.
Conclusion
DCA improved trophoblast cell migration function by suppressing LPS-induced inflammation, at least in part, via the TLR4/NF-κB signaling pathway. This result indicates that DCA might be a potential therapeutic candidate for human pregnancy-related complications associated with trophoblast disorder.
... PHT cells were isolated from term placentas using the Kliman method 22 involving DNAse/trypsin digestion and purification on a Percoll gradient as previously described. 12 Cells were plated at a density of 2 x 10 6 per well for amino acid uptake assays or 2.75 × 10 6 cells per well in a 6-well dish for siRNA gene silencing and subsequent experiments. ...
Normal fetal development is critically dependent on optimal nutrient supply by the placenta, and placental amino acid transport has been demonstrated to be positively associated with fetal growth. Mechanistic target of rapamycin (mTOR) is a positive regulator of placental amino acid transporters, such as System A. Oleic acid (OA) has been previously shown to have a stimulatory role on placental mTOR signaling and System A amino acid uptake in primary human trophoblast (PHT) cells. We investigated the mechanistic link between OA and System A activity in PHT. We found that inhibition of mTOR complex 1 or 2, using small interfering RNA to knock down raptor or rictor, prevented OA‐stimulated System A amino acid transport indicating the interaction of OA with mTOR. Phosphatidic acid (PA) is a key intermediary for phospholipid biosynthesis and a known regulator of the mTOR pathway; however, phospholipid biosynthetic pathways have not been extensively studied in placenta. We identified placental isoforms of acyl transferase enzymes involved in de novo phospholipid synthesis. Silencing of 1‐acylglycerol‐3‐phosphate‐O‐acyltransferase‐4, an enzyme in this pathway, prevented OA mediated stimulation of mTOR and System A amino acid transport. These data indicate that OA stimulates mTOR and amino acid transport in PHT cells mediated through de novo synthesis of PA. We speculate that fatty acids in the maternal circulation, such as OA, regulate placental functions critical for fetal growth by interaction with mTOR and that late pregnancy hyperlipidemia may be critical for increasing nutrient transfer to the fetus.
... To investigate the effect of VD upon RAS components in the human placenta, we cultured primary trophoblast from healthy term placentas. These cells undergo spontaneous differentiation within 24-48 hours of culture, fusing into syncytiotrophoblasts (STB), which represent the endocrinologically most active placental cell lineage [33]. Cultured trophoblast cells from term placenta were allowed to differentiate into syncytiotrophoblasts within 24 hours of culture and further incubated for an additional 24 hours in the presence of increasing calcitriol concentrations (0.1 -100 nM) or 0.1 % ethanol as its vehicle. ...
... The placentas from uncomplicated pregnancies were processed and cultured as previously described [33,36]. Briefly, villous tissue was enzymatically treated and trophoblasts were separated on Percoll gradients. ...
Angiotensin-converting enzyme (ACE)1, ACE2, and renin are components of the renin-angiotensin system (RAS), which regulates blood pressure. ACE2 also serves as a receptor for SARS-CoV-2 and together with the transmembrane serine protease 2 (TMPRSS-2), mediates viral cell-endocytosis. As the placenta expresses all these factors, it acts as a target for SARS-CoV-2 and also as a source of blood pressure modulators. An ACE1/ACE2 ratio imbalance can lead to RAS dysregulation and a bad prognosis in COVID-19 patients. Calcitriol, the vitamin D active metabolite, negatively regulates RAS, reduces inflammation, and enhances antiviral immunity, thereby playing a protective role against COVID-19 severity. Placental calcitriol has been inversely correlated with maternal blood pressure; however, its regulatory role in RAS components and SARS-CoV-2 receptors within the fetomaternal unit has been barely explored. Therefore, we investigated the effects of calcitriol on placental RAS components. Calcitriol downregulated ACE1, ACE2, TMPRSS-2, and renin gene expression in cultured syncytiotrophoblasts and the extravillous trophoblast cell line HTR-8/SVneo. The ACE1/ACE2 ratio was also downregulated by calcitriol. Similar results were obtained in syncytiotrophoblasts treated with calcidiol, the precursor of calcitriol. Altogether, these results support that vitamin D is essential in restricting SARS-CoV-2 placental infection while helping to regulate maternal blood pressure during pregnancy.
... Following collection of the placenta, primary human trophoblast (PHT) cells were isolated using a well-established protocol involving sequential trypsin digestion and Percoll purification (34). After the isolation, cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO) and Ham's F-12 nutrient mixture (Life Technologies, Carlsbad, CA) containing 10 % of fetal bovine serum (FBS, Atlanta Biological, Atlanta, GA), 50 μg/ml gentamicin, 60 μg/ml benzyl penicillin and 100 μg/ml streptomycin (Sigma-Aldrich). ...
The System L amino acid transporter, particularly the isoform Large Neutral Amino Acid Transporter Small Subunit 1 (LAT1) encoded by SLC7A5, is believed to mediate the transfer of essential amino acids in the human placenta. Placental System L amino acid transporter expression and activity is decreased in pregnancies complicated by IUGR and increased in fetal overgrowth. However, it remains unknown if changes in the expression of LAT1 are mechanistically linked to System L amino acid transport activity. Here we combined overexpression approaches with protein analysis and functional studies in cultured primary human trophoblast (PHT) cells to test the hypothesis that SLC7A5 overexpression increases the uptake of essential amino acids and activates mTOR signaling in PHT cells. Overexpression of SLC7A5 resulted in a marked increase in protein expression of LAT1 in the PHT cells microvillous plasma membrane and System L amino acid transporter activity. Moreover, mTOR signaling was activated, and System A amino acid transporter activity increased following SLC7A5 overexpression, suggesting coordination of trophoblast amino transporter expression and activity to ensure balanced nutrient flux to the fetus. This is the first report showing that overexpression of LAT1 is sufficient to increase the uptake of essential amino acids in PHT cells, which activates mTOR, a master regulator of placental function. The decreased placental System L activity in human IUGR and the increased placental activity of this transporter system in some cases of fetal overgrowth may directly contribute to changes in fetal amino acid availability and altered fetal growth in these pregnancy complications.
... PHT cells were isolated from human placenta as previously described. 42 Briefly, minced placental tissues were digested with trypsin (Sigma-Aldrich) and DNase I (Sigma-Aldrich). The supernatant was subsequently centrifuged, and the resulting cell suspension was separated using a Percoll density gradient (GE Healthcare BioSciences AB). ...
Maintaining placental endocrine homeostasis is crucial for a successful pregnancy. Pre‐eclampsia (PE), a gestational complication, is a leading cause of maternal and perinatal morbidity and mortality. Aberrant elevation of testosterone (T 0 ) synthesis, reduced estradiol (E 2 ), and melatonin productions have been identified in preeclamptic placentas. However, the precise contribution of disrupted homeostasis among these hormones to the occurrence of PE remains unknown. In this study, we established a strong correlation between suppressed melatonin production and decreased E 2 as well as elevated T 0 synthesis in PE placentas. Administration of the T 0 analog testosterone propionate (TP; 2 mg/kg/day) to pregnant mice from E7.5 onwards resulted in PE‐like symptoms, along with elevated T 0 production and reduced E 2 and melatonin production. Notably, supplementation with melatonin (10 mg/kg/day) in TP‐treated mice had detrimental effects on fetal and placental development and compromised hormone synthesis. Importantly, E 2 , but not T 0 , actively enhanced melatonin synthetase AANAT expression and melatonin production in primary human trophoblast (PHT) cells through GPER1‐PKA‐CREB signaling pathway. On the other hand, melatonin suppressed the level of estrogen synthetase aromatase while promoting the expressions of androgen synthetic enzymes including 17β‐HSD3 and 3β‐HSD1 in PHT cells. These findings reveal an orchestrated feedback mechanism that maintains homeostasis of placental sex hormones and melatonin. It is implied that abnormal elevation of T 0 synthesis likely serves as the primary cause of placental endocrine disturbances associated with PE. The suppression of melatonin may represent an adaptive strategy to correct the imbalance in sex hormone levels within preeclamptic placentas. The findings of this study offer novel evidence that identifies potential targets for the development of innovative therapeutic strategies for PE.
