Search

Preeclampsia (PE) is a prevalent obstetric complication affecting approximately 3–5% of pregnancies worldwide and is a major cause of maternal and perinatal morbidity and mortality. Preeclampsia is considered a disease of the endothelial system that can progress to eclampsia, characterized by seizures. Early diagnosis and appropriate management are crucial to improving maternal and fetal outcomes, as preeclampsia can lead to severe complications such as placental abruption, fetal growth restriction, and stroke. The pathophysiology of PE is complex, involving a combination of genetic, acquired, and immunological factors. A central feature of the condition is inadequate placentation and impaired uteroplacental perfusion, leading to local hypoxia, endothelial dysfunction, vasoconstriction, and immunological dysregulation. Recent evidence suggests that dysregulation of ion transporters may play a significant role in the adaptation of uterine circulation during placentation. These transporters are essential for maintaining maternal-fetal homeostasis, influencing processes such as nutrient exchange, hormone synthesis, trophoblast cell migration, and the function of smooth muscle cells in blood vessels. In preeclampsia, adverse conditions like hypoxia and oxidative stress result in the downregulation of ion, solute, and water transporters, impairing their function. This review focuses on membrane transporters involved in PE, discussing functional alterations and their physiological implications. The goal of this investigation is to enhance understanding of how dysregulation of ion and small molecule transporters contributes to the development and progression of preeclampsia, underscoring the importance of exploring these signaling pathways for potential therapeutic interventions. Visual representation of the location of the main ion, solute, and water transporters in placental tissue and their functions related to placentation. Alterations in the expression and/or function of these components affect the regulation of vascular tone, trophoblast invasion, cell proliferation, and water balance, contributing to PE development and progression. [Display omitted] • Changes in ion and small molecule transporters can impair normal placental function. • Ion transporters are crucial for placental function; dysfunction may contribute to PE. • Potassium transporters impact vascular tone and angiogenesis, playing a role in PE. • Sodium transporter ENaC is vital for placentation; reduced expression links to PE. • Targeting ion transporters like nAChRs may offer PE treatment options. [ABSTRACT FROM AUTHOR]

Background: As one of the most common and abundant internal modifications of eukaryotic mRNA, N6-methyladenosine (m6A) modifications are closely related to placental development. Ferroptosis is a newly discovered form of programmed cell death. During placental development, placental trophoblasts are susceptible to ferroptosis. However, the interactions of m6A and ferroptosis in trophoblast physiology and injury are unclear. Methods: Recurrent miscarriage (RM) was selected as the main gestational disease in this study. Published data (GSE76862) were used to analyze the gene expression profiles in patients with RM. The extent of m6A modification in total RNA of villous tissues between patients with RM and healthy controls (HC) was compared. ALKBH5 (encoding AlkB homolog 5, RNA demethylase) was selected as the candidate gene for further research. Quantitative real-time reverse transcription PCR, western blotting, and immunohistochemistry (IHC) confirmed the elevated expression of ALKBH5 in the cytotrophoblasts of patients with RM. Then, cell counting kit-8 assays, glutathione disulfide/glutathione quantification, 2′,7′-dichlorfluorescein-diacetate staining, and malonaldehyde assays were used to explore the alterations of ferroptosis-related characteristics following RAS-selective lethal (RSL3) stimulation after overexpression of ALKBH5. Thereafter, we re-analyzed the published RNA sequencing data upon knockdown of ALKBH5, combined with published tissue RNA-seq data, and FTL (encoding ferritin light chain) was identified as the ferroptosis-related gene in cytotrophoblasts of patients with RM that is regulated by ALKBH5. Finally, western blotting and IHC confirmed the increased expression of FTL in the cytotrophoblasts from patients with RM. Results: Total m6A levels were decreased in patients with RM. The most significant differentially m6A-related gene was ALKBH5, which was increased in patients with RM. In vitro cell experiments showed that treatment with RSL3 resulted in increased cell death and upregulated ALKBH5 expression. Overexpression of ALKBH5 alleviated RSL3-induced HTR8 cell death and caused decreased levels of intracellular oxidation products. Published transcriptome sequencing revealed that FTL was the major ferroptosis-related gene regulated by ALKBH5 in the villous tissues of patients with RM. Consistent with the expression of ALKBH5, FTL was increased by RSL3-induction and increased in patients with RM. Conclusion: Elevated ALKBH5 alleviated RSL3-induced cytotrophoblast cell death by promoting the expression of FTL in patients with RM. Our results supported the view that ALKBH5 is an important regulator of the ferroptosis-related etiology of RM and suggested that ALKBH5 could be responsible for epigenetic aberrations in RM pathogenesis. [ABSTRACT FROM AUTHOR]

There are no effective therapies to prevent preeclampsia (PE). Pravastatin shows promise by attenuating processes associated with PE such as decreased cytotrophoblast (CTB) migration, aberrant angiogenesis, and increased oxidative stress. This study assesses the effects of pravastatin on hyperglycemia-induced CTB dysfunction. Methods: Human CTB cells were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 h. Some cells were pretreated with pravastatin (1 µg/mL), while others were cotreated with pravastatin and glucose. The expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) mRNA, vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble endoglin (sEng) were measured. CTB migration was assayed using a CytoSelect migration assay kit. Statistical comparisons were performed using an analysis of variance with Duncan's post hoc test. Results: The hyperglycemia-induced downregulation of uPA was attenuated in CTB cells pretreated with pravastatin at glucose levels > 200 mg/dL and cotreated at glucose levels > 300 mg/dL (p < 0.05). Hyperglycemia-induced decreases in VEGF and PlGF and increases in sEng and sFlt-1 were attenuated in both the pretreatment and cotreatment samples regardless of glucose dose (p < 0.05). Pravastatin attenuated hyperglycemia-induced dysfunction of CTB migration. Conclusions: Pravastatin mitigates stress signaling responses in hyperglycemic conditions, weakening processes leading to abnormal CTB migration and invasion associated with PE in pregnancy. [ABSTRACT FROM AUTHOR]

Non-invasive prenatal testing (NIPT) investigates placental DNA and may detect confined placental mosaicism (CPM). The aim of this study was to confirm CPM in the term placenta in cases with abnormal NIPT but normal follow-up cytogenetic studies of fetus and mother. Additionally we examined the distribution of abnormal cells over the placenta. Four chorionic villus (CV) biopsies from four placental quadrants were requested in cases where CPM was assumed. Both cell lineages of the CV, cytotrophoblast (CTB) and mesenchymal core (MC), were analyzed separately with SNP array. The chromosome aberration was confirmed in 67 % of the placentas. Three quarters of the CTB and MC biopsies from these mosaic placentas were uniformly normal (57 %) or abnormal (20 %), and a minority showed mosaicism. Among 16 cases of CPM where first trimester CV were examined as well, 11 had chromosomally normal results during pregnancy. Cytogenetic investigations of term placental biopsies suspected to be affected with CPM did not reveal the chromosome aberration in one third of the placentas. This is caused by the patchy pattern in which chromosomally abnormal cells are distributed over the placenta with the majority of the biopsies being uniformly normal. Further CPM research, including its clinical impact, requires the analysis of more than four biopsies to get insight into the extent of the affected part. Moreover, a subset of CPM type 1 and 3 seems to be only detectable with NIPT and not with first trimester CVS. • Confirming confined placental mosaicism (CPM) in term placentas is challenging. • CPM exhibits a patchy pattern over the placenta with most biopsies uniformly normal. • A subset of CPM type 1 and 3 seems to be only detectable with NIPT and not with CVS. [ABSTRACT FROM AUTHOR]

Objectives: Necroptosis, a form of programmed cell death, can occur in the placenta of patients with preeclampsia (PE). Hydrogen sulfide (H2S) can inhibit necroptosis of human umbilical vein endothelial cells under the high glucose-induced injury. Whether H2S can protect trophoblasts against necroptosis underlying PE has not been elucidated. This study aimed to explore the protective role of H2S in trophoblast cells against necroptosis underlying PE. Design: This is an in vitro experimental study. Participants: A total of 10 pregnant women with severe PE and 10 matched control normotensive pregnant women were included. The placenta tissues were extracted from participators. The human JEG-3 trophoblasts were commercially available. Methods: The expression and localization of necrotic proteins were assayed in human placenta samples, and the effect of necrotic cell death on the proliferation and apoptosis of human JEG-3 trophoblasts was evaluated. The component expressions of inflammatory cytokine and p38MAPK signaling pathway were measured in samples pretreated with or without NaHS (H2S donor) and SB203580 (p38 inhibitor). Results: RIPA1, RIPA3, and p-p38 levels were significantly higher in PE placental tissue, whereas cystathionine β-synthase expression was decreased. In JEG-3 trophoblasts, necroptosis increased apoptotic cell numbers, suppressed cell proliferation, increased inflammatory cytokine expression, and increased p38MAPK activation, which can be prevented by NaHS. Limitations: In the present study, we did not provide sufficient evidence that necroptosis was a part of the pathogenesis of PE. Conclusions: We proposed the putative role of necroptosis in early-onset PE, reflected by the blockage of caspase-8/3 and increased expression of RIPA1 and RIPA3 in PE placenta tissues. Furthermore, we demonstrated that exogenous H2S protected cytotrophoblasts against ceramide-induced necroptosis via the p38MAPK pathway. [ABSTRACT FROM AUTHOR]

Malaria during pregnancy has been associated with significant risks to both the mother and the fetus, leading to complications such as anemia, low birth weight, and increased infant mortality. The trophoblast cells, a key component of the placenta, are crucial for nutrient and oxygen exchange between mother and fetus. The differentiation of cytotrophoblasts (CTBs) into syncytiotrophoblasts (STBs) is critical for proper pregnancy development. These cells form the bi-stratified epithelium surrounding the placental villi. While previous studies have described an inflammatory activation of STB cells exposed to Plasmodium falciparum-infected erythrocytes (P. falciparum-IE) or components such as hemozoin (HZ), little is known about the direct effect this parasite may have on the epithelial turnover and function of trophoblast cells. This study aims to contribute to understanding mechanisms leading to placental damage during placental malaria using a BeWo cell line as a differentiation model. It was found that P. falciparum-IE interferes with the fusion of BeWo cells, affecting the differentiation process of trophoblast. A reduction in syncytialization could be associated with the adverse effects of infection in fetal health, altering the remodeling of the trophoblast epithelial barrier and reducing their capacity to exchange substances. However, further studies are necessary to assess alterations in the functionality of this epithelium. [ABSTRACT FROM AUTHOR]

AbstractIntroduction: We investigated the role of E-cadherin and Ber-EP4 in tubal pregnancy by comparing their expressions in epithelial and trophoblastic cells both in ectopic tubal and intrauterine pregnancies. Methods: The Formalin-fixed paraffin embedded blocks of 17 intrauterine and 17 tubal pregnancies were immunohistochemically stained with E-cadherin and Ber-EP4. Results: E-cadherin was expressed in the epithelium, villous and extravillous trophoblast in tubal and intrauterine pregnancies but not in the syncytiotrophoblast. The staining intensity was lower in the extra-villous trophoblast in tubal ectopic pregnancies compared with intrauterine pregnancies. Ber-EP4 was expressed in the epithelium of tubal and intrauterine pregnancies and only in villous cytotrophoblast. The intensity of staining in tubal pregnancy was higher than in intrauterine pregnancy. Discussion: The loss of E-cadherin expression in extra-villous trophoblast and increased expression of Ber-EP4 in the villous cytotrophoblast may play a role in the formation of tubal pregnancy by allowing the blastocyst to attach to the tubal epithelium. [ABSTRACT FROM AUTHOR]

CYP19A1 encodes aromatase, which converts testosterone to estrogen, and is induced during placental maturation. To elucidate the molecular mechanism underlying this function, histone methylation was analyzed using the placental cytotrophoblast cell line, JEG3. Treatment of JEG3 cells with 3-deazaneplanocin A, an inhibitor of several methyltransferases, resulted in increased CYP19A1 expression, accompanied by removal of the repressive mark H3K27me3 from the CYP19A1 promoter. However, this increase was not observed in cells treated with GSK126, another specific inhibitor for H3K27me3 methylation. Expression of TFAP2C , which encodes AP-2γ, a transcription factor that regulates CYP19A1 , was also elevated on 3-deazaneplanocin A treatment. Interestingly, TFAP2C messenger RNA (mRNA) was readily degraded in JEG3 cells but protected from degradation in the presence of 3-deazaneplanocin A. TFAP2C mRNA contained N6-methyladenosines, which were reduced on drug treatment. These observations indicate that the TFAP2C mRNA undergoes adenosine methylation and rapid degradation, whereas 3-deazaneplanocin A suppresses methylation, resulting in an increase in AP-2γ levels. We conclude that the increase in AP-2γ expression via stabilization of the TFAP2C mRNA is likely to underlie the increased CYP19A1 expression. [ABSTRACT FROM AUTHOR]

Background As one of the most common and abundant internal modifications of eukaryotic mRNA, N6-methyladenosine (m6A) modifications are closely related to placental development. Ferroptosis is a newly discovered form of programmed cell death. During placental development, placental trophoblasts are susceptible to ferroptosis. However, the interactions of m6A and ferroptosis in trophoblast physiology and injury are unclear. Methods Recurrent miscarriage (RM) was selected as the main gestational disease in this study. Published data (GSE76862) were used to analyze the gene expression profiles in patients with RM. The extent of m6A modification in total RNA of villous tissues between patients with RM and healthy controls (HC) was compared. ALKBH5 (encoding AlkB homolog 5, RNA demethylase) was selected as the candidate gene for further research. Quantitative real-time reverse transcription PCR, western blotting, and immunohistochemistry (IHC) confirmed the elevated expression of ALKBH5 in the cytotrophoblasts of patients with RM. Then, cell counting kit-8 assays, glutathione disulfide/glutathione quantification, 2′,7′-dichlorfluorescein-diacetate staining, and malonaldehyde assays were used to explore the alterations of ferroptosis-related characteristics following RAS-selective lethal (RSL3) stimulation after overexpression of ALKBH5. Thereafter, we re-analyzed the published RNA sequencing data upon knockdown of ALKBH5, combined with published tissue RNA-seq data, and FTL (encoding ferritin light chain) was identified as the ferroptosis-related gene in cytotrophoblasts of patients with RM that is regulated by ALKBH5. Finally, western blotting and IHC confirmed the increased expression of FTL in the cytotrophoblasts from patients with RM. Results Total m6A levels were decreased in patients with RM. The most significant differentially m6A-related gene was ALKBH5, which was increased in patients with RM. In vitro cell experiments showed that treatment with RSL3 resulted in increased cell death and upregulated ALKBH5 expression. Overexpression of ALKBH5 alleviated RSL3-induced HTR8 cell death and caused decreased levels of intracellular oxidation products. Published transcriptome sequencing revealed that FTL was the major ferroptosis-related gene regulated by ALKBH5 in the villous tissues of patients with RM. Consistent with the expression of ALKBH5, FTL was increased by RSL3-induction and increased in patients with RM. Conclusion Elevated ALKBH5 alleviated RSL3-induced cytotrophoblast cell death by promoting the expression of FTL in patients with RM. Our results supported the view that ALKBH5 is an important regulator of the ferroptosis-related etiology of RM and suggested that ALKBH5 could be responsible for epigenetic aberrations in RM pathogenesis.

The trophoblast cells are responsible for the transfer of nutrients between the mother and the foetus and play a major role in placental endocrine function by producing and releasing large amounts of hormones and growth factors. Syncytiotrophoblast cells (STB), formed by the fusion of mononuclear cytotrophoblasts (CTB), constitute the interface between the foetus and the mother and are essential for all of these functions. We performed transcriptome analysis of human placental samples from two control groups—live births (LB), and stillbirths (SB) with a clinically recognised cause—and from our study group, idiopathic stillbirths (iSB). We identified 1172 DEGs in iSB, when comparing with the LB group; however, when we compared iSB with the SB group, only 15 and 12 genes were down- and upregulated in iSB, respectively. An assessment of these DEGs identified 15 commonly downregulated genes in iSB. Among these, several syncytiotrophoblast markers, like genes from the PSG and CSH families, as well as ALPP, KISS1, and CRH, were significantly downregulated in placental samples from iSB. The transcriptome analysis revealed underlying differences at a molecular level involving the syncytiotrophoblast. This suggests that defects in the syncytial layer may underlie unexplained stillbirths, therefore offering insights to improve clinical obstetrics practice. [ABSTRACT FROM AUTHOR]

Trophoblast differentiation is a crucial process in the formation of the placenta where cytotrophoblasts (CTs) differentiate and fuse to form the syncytiotrophoblast (ST). The bioactive components of cannabis, such as Δ9-THC, are known to disrupt trophoblast differentiation and fusion, as well as mitochondrial dynamics and respiration. However, less is known about the impact of cannabidiol (CBD) on trophoblast differentiation. Due to the central role of mitochondria in stem cell differentiation, we evaluated the impact of CBD on trophoblast mitochondrial function and differentiation. Using BeWo b30 cells, we observed decreased levels of mRNA for markers of syncytialization (GCM1, ERVW1, hCG) following 20 µM CBD treatment during differentiation. In CTs, CBD elevated transcript levels for the mitochondrial and cellular stress markers HSP60 and HSP70, respectively. Furthermore, CBD treatment also increased the lipid peroxidation and oxidative damage marker 4-hydroxynonenal. Mitochondrial membrane potential, basal respiration and ATP production were diminished with the 20 µM CBD treatment in both sub-lineages. mRNA levels for endocannabinoid system (ECS) components (FAAH, NAPEPLD, TRPV1, CB1, CB2, PPARγ) were altered differentially by CBD in CTs and STs. Overall, we demonstrate that CBD impairs trophoblast differentiation and fusion, as well as mitochondrial bioenergetics and redox homeostasis. [ABSTRACT FROM AUTHOR]

ABSTRACT The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with Toxoplasma gondii. We grew human trophoblast stem cells (TSCT) under conditions leading to either syncytiotrophoblast (TSSYN) or cytotrophoblast (TSCYT) and infected them with T. gondii. We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TSSYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by transmission electron microscopy and scanning electron microscopy (SEM), a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TSSYNs were highly refractory to parasite adhesion and replication, while TSCYTs were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TSSC-derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes. We demonstrate that TSSYNs are highly resistant to L. monocytogenes, while TSCYTs are not. Like T. gondii, TSSYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell-derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.IMPORTANCECongenital toxoplasmosis can cause a devastating consequence to the fetus. To reach the fetus’s tissues, Toxoplasma gondii must cross the placenta barrier. However, how this parasite crosses the placenta and the precise molecular mechanisms of placental resistance to this parasite are still unknown. In this study, we aimed to characterize a new cellular model of human trophoblast stem cells to determine their resistance, susceptibility, and response to T. gondii. Syncytiotrophoblast derived from trophoblast stem cells recapitulate the resistance profile similarly to placenta cells. We also showed that these cells are highly resistant to Listeria monocytogenes, at the level of bacterial adhesion. Our results suggest that resisting pathogen adhesion/attachment may be a generalized mechanism of syncytiotrophoblast resistance, and trophoblast stem cells represent a promising model to investigate cell-intrinsic mechanisms of resistance to pathogen adhesion and replication.

There are no effective therapies to prevent preeclampsia (PE). Pravastatin shows promise by attenuating processes associated with PE such as decreased cytotrophoblast (CTB) migration, aberrant angiogenesis, and increased oxidative stress. This study assesses the effects of pravastatin on hyperglycemia-induced CTB dysfunction. Methods: Human CTB cells were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 h. Some cells were pretreated with pravastatin (1 µg/mL), while others were cotreated with pravastatin and glucose. The expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) mRNA, vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble endoglin (sEng) were measured. CTB migration was assayed using a CytoSelect migration assay kit. Statistical comparisons were performed using an analysis of variance with Duncan’s post hoc test. Results: The hyperglycemia-induced downregulation of uPA was attenuated in CTB cells pretreated with pravastatin at glucose levels > 200 mg/dL and cotreated at glucose levels > 300 mg/dL (p < 0.05). Hyperglycemia-induced decreases in VEGF and PlGF and increases in sEng and sFlt-1 were attenuated in both the pretreatment and cotreatment samples regardless of glucose dose (p < 0.05). Pravastatin attenuated hyperglycemia-induced dysfunction of CTB migration. Conclusions: Pravastatin mitigates stress signaling responses in hyperglycemic conditions, weakening processes leading to abnormal CTB migration and invasion associated with PE in pregnancy.

Malaria during pregnancy has been associated with significant risks to both the mother and the fetus, leading to complications such as anemia, low birth weight, and increased infant mortality. The trophoblast cells, a key component of the placenta, are crucial for nutrient and oxygen exchange between mother and fetus. The differentiation of cytotrophoblasts (CTBs) into syncytiotrophoblasts (STBs) is critical for proper pregnancy development. These cells form the bi-stratified epithelium surrounding the placental villi. While previous studies have described an inflammatory activation of STB cells exposed to Plasmodium falciparum-infected erythrocytes (P. falciparum-IE) or components such as hemozoin (HZ), little is known about the direct effect this parasite may have on the epithelial turnover and function of trophoblast cells. This study aims to contribute to understanding mechanisms leading to placental damage during placental malaria using a BeWo cell line as a differentiation model. It was found that P. falciparum-IE interferes with the fusion of BeWo cells, affecting the differentiation process of trophoblast. A reduction in syncytialization could be associated with the adverse effects of infection in fetal health, altering the remodeling of the trophoblast epithelial barrier and reducing their capacity to exchange substances. However, further studies are necessary to assess alterations in the functionality of this epithelium.

The trophoblast cells are responsible for the transfer of nutrients between the mother and the foetus and play a major role in placental endocrine function by producing and releasing large amounts of hormones and growth factors. Syncytiotrophoblast cells (STB), formed by the fusion of mononuclear cytotrophoblasts (CTB), constitute the interface between the foetus and the mother and are essential for all of these functions. We performed transcriptome analysis of human placental samples from two control groups—live births (LB), and stillbirths (SB) with a clinically recognised cause—and from our study group, idiopathic stillbirths (iSB). We identified 1172 DEGs in iSB, when comparing with the LB group; however, when we compared iSB with the SB group, only 15 and 12 genes were down- and upregulated in iSB, respectively. An assessment of these DEGs identified 15 commonly downregulated genes in iSB. Among these, several syncytiotrophoblast markers, like genes from the PSG and CSH families, as well as ALPP, KISS1, and CRH, were significantly downregulated in placental samples from iSB. The transcriptome analysis revealed underlying differences at a molecular level involving the syncytiotrophoblast. This suggests that defects in the syncytial layer may underlie unexplained stillbirths, therefore offering insights to improve clinical obstetrics practice.

The placenta develops alongside the embryo and nurtures fetal development to term. During the first stages of embryonic development, due to low blood circulation, the blood and ambient oxygen supply is very low (~1-2% O 2 ) and gradually increases upon placental invasion. While a hypoxic environment is associated with stem cell self-renewal and proliferation, persistent hypoxia may have severe effects on differentiating cells and could be the underlying cause of placental disorders. We find that human trophoblast stem cells (hTSC) thrive in low oxygen, whereas differentiation of hTSC to trophoblast to syncytiotrophoblast (STB) and extravillous trophoblast (EVT) is negatively affected by hypoxic conditions. The pro-differentiation factor GCM1 (human Glial Cell Missing-1) is downregulated in low oxygen, and concordantly there is substantial reduction of GCM1-regulated genes in hypoxic conditions. Knockout of GCM1 in hTSC caused impaired EVT and STB formation and function, reduced expression of differentiation-responsive genes, and resulted in maintenance of self-renewal genes. Treatment with a PI3K inhibitor reported to reduce GCM1 protein levels likewise counteracts spontaneous or directed differentiation. Additionally, chromatin immunoprecipitation of GCM1 showed enrichment of GCM1-specific binding near key transcription factors upregulated upon differentiation including the contact inhibition factor CDKN1C. Loss of GCM1 resulted in downregulation of CDKN1C and corresponding loss of contact inhibition, implicating GCM1 in regulation of this critical process.

Recent advances in the use of trophoblast stem cells and organoid models have markedly enhanced our understanding of placental development and function. These models offer significant improvements over previous systems due to their extended viability in culture and capacity to replicate various trophoblast functions, such as extravillous trophoblast invasion, syncytialisation and 3D architecture. Initially, the generation of trophoblast organoids was confined to first trimester placental tissue; however, it was recently reported that term placentae can also serve as a source of trophoblast stem cells. Here, we report that both 2D proliferative cytotrophoblasts and 3D trophoblast organoids can be effectively derived from cryopreserved term cytotrophoblasts isolated by the 'Kliman' method of sequential trypsin digestion and Percoll density gradient centrifugation, when cultured in specialised medium. This was confirmed by the expression of characteristic trophoblast markers including cytokeratin-7, E-cadherin, and human chorionic gonadotropin beta (β-hCG). The proliferative cytotrophoblasts were induced to differentiate to syncytiotrophoblasts, marked by elevated β-hCG expression, reduced Ki67-positive nuclei, and a fused syncytial phenotype. The protocol described here enables the application of organoid models and in vitro functional studies to stored cytotrophoblast samples for the study of placental function from unique patient cohorts. Moreover, the utilization of term placental sources may alleviate ethical concerns with using cells from pregnancy terminations, thus expanding access for a broader research community., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Ltd.)

The placenta plays a fundamental role in fetal growth and maintenance of pregnancy. Its cellular components include a large multinucleated syncytiotrophoblast (STB) and its progenitor, cytotrophoblasts (CTBs), both of which perform vital functions in the human placenta. Primary cytotrophoblasts isolated from term human placentas that spontaneously fuse and differentiate into syncytiotrophoblast-like cells in vitro have been utilized to investigate the functions of the syncytiotrophoblast and placenta with multiple modifications. Although recent advances have enabled the use of trophoblast stem cell-derived organoids as a model for villous trophoblasts, primary CTBs offer several advantages, including spontaneous differentiation, easy access to materials (from term-delivered human placentas), and simple methodology. Here, we present a precise step-by-step process for isolating pure CTBs from term human placenta based on previously reported placenta digestion, density centrifugation, and CTB purification using anti-HLA-A, B, C antibody. Subsequently, we provide a method to improve CTB viability and differentiation into STB-like cells using epidermal growth factor (EGF) and a ROCK inhibitor (Y-27632) that ensures long-term and stable cultures without altering their proliferation. Because these cells can grow on standard tissue culture plates, this model can be easily utilized for various placental investigations, including innate immune responses, drug resistance, and STB metabolism. Employing this approach considerably enhances our understanding of placental functions, which are key to maternal and offspring health., Competing Interests: Declaration of competing interest The authors declare that there are no potential conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with Toxoplasma gondii . We grew human trophoblast stem cells (TS CT ) under conditions leading to either syncytiotrophoblast (TS SYN ) or cytotrophoblast (TS CYT ) and infected them with T. gondii . We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TS SYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by transmission electron microscopy and scanning electron microscopy (SEM), a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TS SYNs were highly refractory to parasite adhesion and replication, while TS CYTs were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TS SC -derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes . We demonstrate that TS SYNs are highly resistant to L. monocytogenes , while TS CYTs are not. Like T. gondii , TS SYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell-derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.IMPORTANCECongenital toxoplasmosis can cause a devastating consequence to the fetus. To reach the fetus's tissues, Toxoplasma gondii must cross the placenta barrier. However, how this parasite crosses the placenta and the precise molecular mechanisms of placental resistance to this parasite are still unknown. In this study, we aimed to characterize a new cellular model of human trophoblast stem cells to determine their resistance, susceptibility, and response to T. gondii . Syncytiotrophoblast derived from trophoblast stem cells recapitulate the resistance profile similarly to placenta cells. We also showed that these cells are highly resistant to Listeria monocytogenes , at the level of bacterial adhesion. Our results suggest that resisting pathogen adhesion/attachment may be a generalized mechanism of syncytiotrophoblast resistance, and trophoblast stem cells represent a promising model to investigate cell-intrinsic mechanisms of resistance to pathogen adhesion and replication., Competing Interests: The authors declare no conflict of interest.

The intervillous space of human placenta is filled with maternal blood, and villous trophoblasts are constantly exposed to the shear stress generated by maternal blood pressure and flow throughout the entire gestation period. However, the effects of shear stress on villous trophoblasts and their biological significance remain unknown. Here, using our recently established naïve human pluripotent stem cells-derived cytotrophoblast stem cells (nCTs) and a device that can apply arbitrary shear stress to cells, we investigated the impact of shear stress on early-stage trophoblasts. After 72 h of exposure to 10 dyn/cm2 shear stress, nCTs became fused and multinuclear, and mRNA expression of the syncytiotrophoblast (ST) markers, such as glial cell missing 1, endogenous retrovirus group W member 1 envelope, chorionic gonadotropin subunit beta 3, syndecan 1, pregnancy specific beta-1-glycoprotein 3, placental growth factor, and solute carrier family 2 member 1 were significantly upregulated compared to static conditions. Immunohistochemistry showed that shear stress increased fusion index, human chorionic gonadotropin secretion, and human placental lactogen secretion. Increased microvilli formation on the surface of nCTs under flow conditions was detected using scanning electron microscopy. Intracellular cyclic adenosine monophosphate significantly increased under flow conditions. Moreover, transcriptome analysis of nCTs subjected to shear stress revealed that shear stress upregulated ST-specific genes and downregulated CT-specific genes. Collectively, these findings indicate that shear stress promotes the differentiation of nCTs into ST., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Human pluripotent stem cells (hPSCs) form an ideal system to study the formation of placental cells, from an undifferentiated human embryonic stem cell state. The conventional human in vitro model systems to study the human placenta cannot be employed for understanding placental dysfunctions or the development of specialized placental cell types. Hence, human PSCs make an ideal model system to study human placental development and disorders. Here, we describe an efficient and validated protocol to reproducibly study the formation of human cytotrophoblasts (CTBs) and syncytiotrophoblast (STBs) from undifferentiated hPSCs. CTBs are the trophoblast stem cells that can differentiate into specialized placental cell types such as STBs. The multinucleated STB plays vital role in the exchange of nutrients and gases across the placenta and secretes several hormones during pregnancy, such as human chorionic gonadotropin β (hCGβ). Here we describe two methods of seeding the hPSCs: chemical (clumps method) and enzymatic methods (single cells) to differentiate them to CTB and STB, activating BMP (B) signaling and inhibiting ACTIVIN/NODAL and FGF signaling pathways (2i), thus naming our protocol as "B2i" (Sudheer et al., Stem Cells Dev 21:2987-3000, 2012). This protocol forms the perfect model system for understanding in vitro placentation, modeling diseases arising from abnormal placentation that cause complications such as miscarriage, preeclampsia or intrauterine growth restriction (IUGR), and drug discovery for placental disorders., (© 2023. Springer Science+Business Media, LLC.)

Recent developments in the in vitro culture of peri-implantation stage human embryos have expanded opportunities to investigate implantation stage human development and trophoblast differentiation in the absence of maternal tissues. Emerging single cell omics analyses have offered researchers new tools to explore unanswered biological questions to new depths. In order to investigate the dynamics of human trophoblast cell differentiation during implantation at the single-cell resolution, efficient cell dissociation approaches of trophoblasts from embryos are necessary. Here, we describe the protocol for extended culture of peri-implantation stage human embryos with enzymatic digestion and manual collection of individual cells for downstream assays., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Trophoblasts are placenta-specific epithelial cells that play an essential role in conducting nutrient, gas, and waste exchange between the fetus and the mother. Primary culture of human trophoblasts from donated term placentae is an important tool to study placental functions. Currently, there is a lack of general consensus of the optimal culture conditions for maintaining term trophoblast cells in vitro. A key problem with culturing trophoblasts from term placentae is overgrowth of the trophoblasts by rapidly proliferating cellular contaminants. Recently we reported a system to culture trophoblasts from term placentae which differentiate into syncytiotrophoblast-like multinucleated cells that can be maintained in culture for at least 30 days with minimal contamination. This chapter details our optimized approach for long-term, contaminant-free in vitro culture of primary trophoblasts from term placentae., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The human placenta is a transient organ that functions to support the needs of the fetus throughout gestation. Trophoblasts are the major epithelial cells found within the placenta and comprise a variety of distinct cell types with specialized roles in fetal-maternal communication. Our understanding of human trophoblast development remains limited due to ethical and legal restrictions on accessing first-trimester placental tissues, as well as the inability of common animal models to replicate primate placental development. It is therefore important to advance in vitro models of human trophoblast development as a basis for studying pregnancy-associated complications and diseases. In this chapter, we describe a protocol for generating 3D trophoblast organoids from naïve human pluripotent stem cells (hPSCs). The resulting stem-cell-derived trophoblast organoids (SC-TOs) contain distinct cytotrophoblast (CTB), syncytiotrophoblast (STB), and extravillous trophoblast (EVT) cell types, which closely correspond to trophoblast identities in the human post-implantation embryo. We discuss methods for characterizing SC-TOs by immunofluorescence, flow cytometry, mRNA and microRNA expression profiling, and placental hormone secretion. Furthermore, SC-TOs can undergo differentiation into specialized 3D EVT organoids, which display robust invasion when co-cultured with human endometrial cells. Thus, the protocol described herein offers an accessible 3D model system of human placental development and trophoblast invasion., (© 2023. Springer Science+Business Media, LLC.)