Differentiation Of Trophoblast Stem Cells Research Articles

NOTUM-mediated WNT silencing drives extravillous trophoblast cell lineage development

Trophoblast stem (TS) cells have the unique capacity to differentiate into specialized cell types, including extravillous trophoblast (EVT) cells. EVT cells invade into and transform the uterus where they act to remodel the vasculature facilitating the redirection of maternal nutrients to the developing fetus. Disruptions in EVT cell development and function are at the core of pregnancy-related disease. WNT-activated signal transduction is a conserved regulator of morphogenesis of many organ systems, including the placenta. In human TS cells, activation of canonical WNT signaling is critical for maintenance of the TS cell stem state and its downregulation accompanies EVT cell differentiation. We show that aberrant WNT signaling undermines EVT cell differentiation. Notum, palmitoleoyl-protein carboxylesterase (NOTUM), a negative regulator of canonical WNT signaling, was prominently expressed in first-trimester EVT cells developing in situ and up-regulated in EVT cells derived from human TS cells. Furthermore, NOTUM was required for optimal human TS cell differentiation to EVT cells. Activation of NOTUM in EVT cells is driven, at least in part, by endothelial Per-Arnt-Sim (PAS) domain 1 (also called hypoxia-inducible factor 2 alpha). Collectively, our findings indicate that canonical Wingless-related integration site (WNT) signaling is essential for maintenance of human trophoblast cell stemness and regulation of human TS cell differentiation. Downregulation of canonical WNT signaling via the actions of NOTUM is required for optimal EVT cell differentiation.

Wnt signaling activation confers a syncytiotrophoblast progenitor state on trophoblast stem cells of cynomolgus monkey.

Trophoblast stem cells (TSCs), derived from the trophectoderm of the blastocyst, are used as an in vitro model to reveal the mechanisms underlying placentation in mammals. In humans, suitable culture conditions for TSC derivation have recently been established. The established human TSCs (hTSCs) differentiate efficiently toward two trophoblast subtypes: syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). However, the efficiency of differentiation is lower in macaque TSCs than in hTSCs. Here, we demonstrate that the activation of Wnt signaling downregulated the expression of inhibitory G protein and induced trophoblastic lineage switching to the STB progenitor state. The treatment of macaque TSCs with a GSK-3 inhibitor, CHIR99021, upregulated STB progenitor markers and enhanced proliferation. Under the Wnt signaling-activated conditions, macaque TSCs effectively differentiated to STBs upon dbcAMP and forskolin treatment. RNA-seq analyses revealed the downregulation of inhibitory G protein, which may make macaque TSCs responsive to forskolin. Interestingly, this lineage switching appeared to be reversible as the macaque TSCs lost responsiveness to forskolin upon the removal of CHIR99021. The ability to regulate the direction of macaque TSC differentiation would be advantageous in elucidating the mechanisms underlying placentation in non-human primates.

Identification of transcriptional signature change and critical transcription factors involved during the differentiation of mouse trophoblast stem cell into maternal blood vessel associated trophoblast giant cell

The placenta is essential organ for oxygen and nutrient exchange between the mother and the developing fetus. Trophoblast lineage differentiation is closely related to the normal function of the placenta. Trophoblast stem cells (TSCs) can differentiate into all placental trophoblast subtypes and are widely used as in vitro stem cell models to study placental development and trophoblast lineage differentiation. Although extensive research has been conducted on the differentiation of TSCs, the possible parallels between trophoblast giant cells (TGCs) that are differentiated from TSCs in vitro and the various subtypes of TGC lineages in vivo are still poorly understood. In this study, mouse TSCs (mTSCs) were induced to differentiate into TGCs, and our mRNA sequencing (RNA-seq) data revealed that mTSCs and TGCs have distinct transcriptional signatures. We conducted a comparison of mTSCs and TGCs transcriptomes with the published transcriptomes of TGC lineages in murine placenta detected by single-cell RNA-seq and found that mTSCs tend to differentiate into maternal blood vessel-associated TGCs in vitro. Moreover, we identified the transcription factor (TF) ZMAT1, which may be responsible for the differentiation of mTSCs into sinusoid TGCs, and the TFs EGR1 and MITF, which are likely involved in the differentiation of mTSCs into spiral artery-associated TGCs. Thus, our findings provide a valuable resource for the mechanisms of trophoblast lineage differentiation and placental deficiency-associated diseases development.

Vitamin C inactivates c-Jun N-terminal kinase to stabilize heart and neural crest derivatives expressed 1 (Hand1) in regulating placentation and maintenance of pregnancy

Vitamin C (VC) serves as a pivotal nutrient for anti-oxidation process, metabolic responses, and stem cell differentiation. However, its precise contribution to placenta development and gestation remains obscure. Here, we demonstrated that physiological levels of VC act to stabilize Hand1, a key bHLH transcription factor vital for the development trajectory of trophoblast giant cell (TGC) lineages, thereby promoting the differentiation of trophoblast stem cells into TGC. Specifically, VC administration inactivated c-Jun N-terminal kinase (JNK) signaling, which directly phosphorylates Hand1 at Ser48, triggering the proteasomal degradation of Hand1. Conversely, a loss-of-function mutation at Ser48 on Hand1 not only significantly diminished both intrinsic and VC-induced stabilization of Hand1 but also underscored the indispensability of this residue. Noteworthy, the insufficiency of VC led to severe defects in the differentiation of diverse TGC subtypes and the formation of labyrinth's vascular network in rodent placentas, resulting in failure of maintenance of pregnancy. Importantly, VC deficiency, lentiviral knockdown of JNK or overexpression of Hand1 mutants in trophectoderm substantially affected the differentiation of primary and secondary TGC in E8.5 mouse placentas. Thus, these findings uncover the significance of JNK inactivation and consequential stabilization of Hand1 as a hitherto uncharacterized mechanism controlling VC-mediated placentation and perhaps maintenance of pregnancy.Graphical

A quantitative image analysis platform for assessing trophoblast differentiation

Immunofluorescence microscopy is extensively used in characterization of trophoblast differentiation in vitro. However, such data is primarily used to confirm the presence of protein markers or qualitatively compare levels of protein markers across experimental conditions. Imaging data, when processed and analyzed appropriately can provide quantitative and spatial information, and provide biological insight. Towards this end, here we present MATroph, an open-source MATLAB-based computational tool to process images generated by immunofluorescent microscopy. MATroph automatically executes a series of image processing operations, including the classification of red, blue, and green channels from images, background extraction, morphological operations, and image filtering. From the isolated blue channels corresponding to nuclear staining, this tool generates numerical values for cell number. Additionally, relative levels and spatial location of proteins are obtained by mapping red and green channel pixels to blue pixels by assigning minimum pixel distance between the blue and other color objects. Thus, this tool provides information about intracellular protein accumulation areas. Additionally, this tool can also classify cells as single cells or part of colonies, and extract information on protein levels for each; this is particularly useful for quantitative studies on extravillous trophoblast maturation. We provide a user-guide to analyze the relative levels of markers relevant to human trophoblast stem cell self-renewal and differentiation. Importantly, MATroph is composed of a simple MATLAB algorithm, and its implementation requires minimal expertise in programming.

Human CD56+CD39+ dNK cells support fetal survival through controlling trophoblastic cell fate: immune mechanisms of recurrent early pregnancy loss.

Decidual natural killer (dNK) cells are the most abundant immune cells at the maternal-fetal interface during early pregnancy in both mice and humans, and emerging single-cell transcriptomic studies have uncovered various human dNK subsets that are disrupted in patients experiencing recurrent early pregnancy loss (RPL) at early gestational stage, suggesting a connection between abnormal proportions or characteristics of dNK subsets and RPL pathogenesis. However, the functional mechanisms underlying this association remain unclear. Here, we established a mouse model by adoptively transferring human dNK cells into pregnant NOG (NOD/Shi-scid/IL-2Rγnull) mice, where human dNK cells predominantly homed into the uteri of recipients. Using this model, we observed a strong correlation between the properties of human dNK cells and pregnancy outcome. The transfer of dNK cells from RPL patients (dNK-RPL) remarkably worsened early pregnancy loss and impaired placental trophoblast cell differentiation in the recipients. These adverse effects were effectively reversed by transferring CD56+CD39+ dNK cells. Mechanistic studies revealed that CD56+CD39+ dNK subset facilitates early differentiation of mouse trophoblast stem cells (mTSCs) towards both invasive and syncytial pathways through secreting macrophage colony-stimulating factor (M-CSF). Administration of recombinant M-CSF to NOG mice transferred with dNK-RPL efficiently rescued the exacerbated pregnancy outcomes and fetal/placental development. Collectively, this study established a novel humanized mouse model featuring functional human dNK cells homing into the uteri of recipients and uncovered the pivotal role of M-CSF in fetal-supporting function of CD56+CD39+ dNK cells during early pregnancy, highlighting that M-CSF may be a previously unappreciated therapeutic target for intervening RPL.

Dynamic and distinct histone modifications facilitate human trophoblast lineage differentiation

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

Bisphenol S moderately decreases the expression of syncytiotrophoblast marker genes and induces apoptosis in human trophoblast lineages

Bisphenol S (BPS) is currently used in the manufacturing of several household equipment such as water pipes and food containers. Hence, its entrance into the human body is almost inevitable. The presence of BPS in body fluids has been reported. However, its potential toxicity, especially on human placenta development and pregnancy progression, has not been explored. In this study, we assessed the impacts of BPS on the self-renewal and differentiation potentials of placental stem cells, also known as trophoblast stem cells (TSCs), by exposing them to three different BPS concentrations during their self-renewal and differentiation into syncytiotrophoblast (ST), extravillous trophoblast (EVT), and trophoblast organoids. Interestingly, BPS treatment did not affect the stemness, cell cycle and proliferation of the TSCs but it induced apoptosis in each trophoblast lineage. BPS altered the expression of several fusion-related genes. However, this alteration did not translate into significant morphological defects in the STs and organoids. Moreover, BPS did not impair the differentiation of TSCs into EVTs. These findings suggest that the presence of BPS at the feto-maternal interface may exaggerate trophoblast apoptosis and moderately inhibit the trophoblast fusion pathway to affect placenta development and pregnancy. Our study offers valuable insights into the potential toxicity of BPS on human placenta development, emphasizing the need for epidemiological assessment of the relationship between maternal serum levels of BPS and pregnancy complications.

NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

Failures in growth and differentiation of the early human placenta are associated with severe pregnancy disorders such as pre-eclampsia and fetal growth restriction. However, regulatory mechanisms controlling development of placental epithelial cells, the trophoblasts, remain poorly elucidated. Using trophoblast stem cells (TSCs), trophoblast organoids (TB-ORGs) and primary cytotrophoblasts (CTBs) of early pregnancy, we herein show that autocrine NOTCH3 signalling controls human placental expansion and differentiation. The NOTCH3 receptor was specifically expressed in proliferative CTB progenitors and its active form, the nuclear NOTCH3 intracellular domain (NOTCH3-ICD), interacted with the transcriptional co-activator mastermind-like 1 (MAML1). Doxycycline-inducible expression of dominant-negative MAML1 in TSC lines provoked cell fusion and upregulation of genes specific for multinucleated syncytiotrophoblasts, which are the differentiated hormone-producing cells of the placenta. However, progenitor expansion and markers of trophoblast stemness and proliferation were suppressed. Accordingly, inhibition of NOTCH3 signalling diminished growth of TB-ORGs, whereas overexpression of NOTCH3-ICD in primary CTBs and TSCs showed opposite effects. In conclusion, the data suggest that canonical NOTCH3 signalling plays a key role in human placental development by promoting self-renewal of CTB progenitors.

Role of Endometrial Extracellular Vesicles in Mediating Cell-to-Cell Communication in the Uterus: A Review.

There are several critical events that occur in the uterus during early pregnancy which are necessary for the establishment and maintenance of pregnancy. These events include blastocyst implantation, uterine decidualization, uterine neoangiogenesis, differentiation of trophoblast stem cells into different trophoblast cell lineages, and formation of a placenta. These processes involve several different cell types within the pregnant uterus. Communication between these cell types must be intricately coordinated for successful embryo implantation and the formation of a functional maternal-fetal interface in the placenta. Understanding how this intricate coordination transpires has been a focus of researchers in the field for many years. It has long been understood that maternal endometrial tissue plays a key role in intercellular signaling during early pregnancy, sending signals to nearby tissues in a paracrine manner. Recently, insights have been obtained into the mechanisms by which these signaling events occur. Notably, the endometrium has been shown to secrete extracellular vesicles (EVs) that contain crucial cargo (proteins, lipids, RNA, miRNA) that are taken up by recipient cells to initiate a response leading to the occurrence of critical events during implantation and placentation. In this review, we aim to summarize the role that endometrium-derived EVs play in mediating cell-to-cell communications within the pregnant uterus to orchestrate the events that must occur to establish and maintain pregnancy. We will also discuss how aberrant endometrial EV signaling may lead to pathophysiological conditions, such as endometriosis and infertility.

Adrenomedullin has a pivotal role in trophoblast differentiation: A promising nanotechnology-based therapeutic target for early-onset preeclampsia.

Early-onset preeclampsia (EOPE) is a severe pregnancy complication associated with defective trophoblast differentiation and functions at implantation, but manifestation of its phenotypes is in late pregnancy. There is no reliable method for early prediction and treatment of EOPE. Adrenomedullin (ADM) is an abundant placental peptide in early pregnancy. Integrated single-cell sequencing and spatial transcriptomics confirm a high ADM expression in the human villous cytotrophoblast and syncytiotrophoblast. The levels of ADM in chorionic villi and serum were lower in first-trimester pregnant women who later developed EOPE than those with normotensive pregnancy. ADM stimulates differentiation of trophoblast stem cells and trophoblast organoids in vitro. In pregnant mice, placenta-specific ADM suppression led to EOPE-like phenotypes. The EOPE-like phenotypes in a mouse PE model were reduced by a placenta-specific nanoparticle-based forced expression of ADM. Our study reveals the roles of trophoblastic ADM in placental development, EOPE pathogenesis, and its potential clinical uses.

HtrA4 is required for differentiation of human trophoblast stem cells into syncytiotrophoblast

HtrA4 is required for differentiation of human trophoblast stem cells into syncytiotrophoblast

Human trophoblast stem cell culture and differentiation: the devil is in the details

Human trophoblast stem cell culture and differentiation: the devil is in the details

Extracellular Matrix Influences Gene Expression and Differentiation of Mouse Trophoblast Stem Cells.

Trophoblast stem (TS) cells were first isolated from the mouse placenta; however, little is known about their maintenance and niche in vivo. TS cells, like other stem cells, have a unique microenvironment in which the extracellular matrix (ECM) is a component. Placental pathology is associated with ECM change. However, how these changes and the individual ECM components impact the maintenance or differentiation of TS cells has not been established. This study identified which ECM component(s) maintain the greatest expression of markers associated with undifferentiated mouse trophoblast stem (mTS) cells and which alter the profile of markers of differentiation based on mRNA analysis. mTS cells cultured on individual ECM components and subsequent quantitative polymerase chain reaction analysis revealed that laminin promoted the expression of markers associated with undifferentiated TS cells, fibronectin promoted gene expression associated with syncytiotrophoblast (SynT) layer II cells, and collagen IV promoted the expression of genes associated with differentiated trophoblast. To investigate whether pathological placental ECM influenced the expression of genes associated with different trophoblast subtypes, the mouse model of streptozotocin (STZ)-induced pancreatic β cell ablation and diabetes was used. Female mice administered STZ (blood glucose ≥300 mg/dL) or control (blood glucose ≤150 mg/dL) were mated. Placental pathology at embryonic day (E)14.5 was confirmed with reduced fetal blood space area, reduced expression of the pericyte marker αSMA, and decreased expression of ECM proteins. mTS cells cultured on ECM isolated from STZ placenta were associated with reduced expression of undifferentiated mTS markers and increased expression of genes associated with terminally differentiated trophoblast [Gcm-1 and SynA (SynT) and junctional zone Tpbpa and Prl2c2]. Altogether, these results support the value of using ECM isolated from the placenta as a tool for understanding trophoblast contribution to placental pathology.

The transcriptional activator Klf5 recruits p300-mediated H3K27ac for maintaining trophoblast stem cell pluripotency.

The effective proliferation and differentiation of trophoblast stem cells (TSCs) is indispensable for the development of the placenta, which is the key to maintaining normal fetal growth during pregnancy. Kruppel-like factor 5 (Klf5) is implicated in the activation of pluripotency gene expression in embryonic stem cells (ESCs), yet its function in TSCs is poorly understood. Here, we showed that Klf5 knockdown resulted in the downregulation of core TSC-specific genes, consequently causing rapid differentiation of TSCs. Consistently, Klf5-depleted embryos lost the ability to establish TSCs in vitro. At the molecular level, Klf5 preferentially occupied the proximal promoter regions and maintained an open chromatin architecture of key TSC-specific genes. Deprivation of Klf5 impaired the enrichment of p300, a major histone acetyl transferase of H3 lysine 27 acetylation (H3K27ac), and further reduced the occupancy of H3K27ac at promoter regions, leading to decreased transcriptional activity of TSC pluripotency genes. Thus, our findings highlight a novel mechanism of Klf5 in regulating the self-renewal and differentiation of TSCs and provide a reference for understanding placental development and improving pregnancy rates.

Cell surface associated protein mucin 15 (MUC15) is elevated in preeclampsia

BackgroundMucins are a family of proteins that protect the epithelium. A particular type of mucin, MUC15 is highly expressed in the placenta. This study aimed to characterise MUC15 in preeclampsia and investigate its role in placental stem cell biology. MethodsMUC15 mRNA and protein were measured in placentas from patients with early onset (<34 weeks’ gestation) preeclampsia. Circulating serum MUC15 was measured via ELISA. MUC15 was localised in the placenta using in situ hybridisation. MUC15 mRNA expression was measured across differentiation of human trophoblast stem cells (hTSCs) to syncytiotrophoblast and extravillous trophoblasts. MUC15 was measured after syncytialised hTSCs were cultured in hypoxic (1% O2) and proinflammatory (TNF α, IL-6) conditions. MUC15 secretion was assessed when syncytialised hTSCs were treated with brefeldin A (impairs protein trafficking) and batimastat (inhibits matrix metalloproteinases). ResultsMUC15 protein was significantly increased in the placenta (P = 0.0003, n = 32 vs n = 20 controls) and serum (P = 0.016, n = 32 vs n = 22 controls) of patients with preeclampsia, whilst MUC15 mRNA remained unchanged (n = 61 vs n = 18 controls). MUC15 mRNA (P = 0.005) and protein secretion (P = 0.006) increased following differentiation to syncytiotrophoblast cells. In situ hybridisation confirmed MUC15 localised to the syncytiotrophoblast cell within the placenta. Neither hypoxic or inflammatory conditions changed MUC15 mRNA expression or secretion. Brefeldin A treated hTSCs did not alter MUC15 secretion, whilst batimastat reduced MUC15 secretion (P = 0.044). ConclusionsMUC15 is increased in early onset preeclampsia and is cleaved by matrix metalloproteinases. Increased MUC15 may reflect a protective mechanism associated with placental dysfunction. Further research will aid in confirming this.

EP300 facilitates human trophoblast stem cell differentiation

Early placenta development involves cytotrophoblast differentiation into extravillous trophoblast (EVT) and syncytiotrophoblast (STB). Defective trophoblast development and function may result in severe pregnancy complications, including fetal growth restriction and pre-eclampsia. The incidence of these complications is increased in pregnancies of fetuses affected by Rubinstein-Taybi syndrome, a developmental disorder predominantly caused by heterozygous mutations in CREB-binding protein (CREBBP) or E1A-binding protein p300 (EP300). Although the acetyltransferases CREBBP and EP300 are paralogs with many overlapping functions, the increased incidence of pregnancy complications is specific for EP300 mutations. We hypothesized that these complications have their origin in early placentation and that EP300 is involved in that process. Therefore, we investigated the role of EP300 and CREBBP in trophoblast differentiation, using human trophoblast stem cells (TSCs) and trophoblast organoids. We found that pharmacological CREBBP/EP300 inhibition blocks differentiation of TSCs into both EVT and STB lineages, and results in an expansion of TSC-like cells under differentiation-inducing conditions. Specific targeting by RNA interference or CRISPR/Cas9-mediated mutagenesis demonstrated that knockdown of EP300 but not CREBBP, inhibits trophoblast differentiation, consistent with the complications seen in Rubinstein-Taybi syndrome pregnancies. By transcriptome sequencing, we identified transforming growth factor alpha (TGFA, encoding TGF-α) as being strongly upregulated upon EP300 knockdown. Moreover, supplementing differentiation medium with TGF-α, which is a ligand for the epidermal growth factor receptor (EGFR), likewise affected trophoblast differentiation and resulted in increased TSC-like cell proliferation. These findings suggest that EP300 facilitates trophoblast differentiation by interfering with at least EGFR signaling, pointing towards a crucial role for EP300 in early human placentation.

The Fgf/Erf/NCoR1/2 repressive axis controls trophoblast cell fate

Placental development relies on coordinated cell fate decisions governed by signalling inputs. However, little is known about how signalling cues are transformed into repressive mechanisms triggering lineage-specific transcriptional signatures. Here, we demonstrate that upon inhibition of the Fgf/Erk pathway in mouse trophoblast stem cells (TSCs), the Ets2 repressor factor (Erf) interacts with the Nuclear Receptor Co-Repressor Complex 1 and 2 (NCoR1/2) and recruits it to key trophoblast genes. Genetic ablation of Erf or Tbl1x (a component of the NCoR1/2 complex) abrogates the Erf/NCoR1/2 interaction. This leads to mis-expression of Erf/NCoR1/2 target genes, resulting in a TSC differentiation defect. Mechanistically, Erf regulates expression of these genes by recruiting the NCoR1/2 complex and decommissioning their H3K27ac-dependent enhancers. Our findings uncover how the Fgf/Erf/NCoR1/2 repressive axis governs cell fate and placental development, providing a paradigm for Fgf-mediated transcriptional control.

Epidermal growth factor represses differentiation of mouse trophoblast stem cells into spongiotrophoblast cells via epidermal growth factor receptor

The mouse placenta is composed of three different trophoblast layers that are occupied by particular trophoblast subtypes to maintain placental function and pregnancy. Accurate control of trophoblast differentiation is required for proper placental function; however, the molecular mechanisms underlying cell fate decisions in trophoblast stem cells remain poorly understood. Epidermal growth factor (EGF) signaling is involved in multiple biological processes including cell survival, proliferation, and differentiation. The effect of EGF on trophoblast function has been reported in various species; however, the role of EGF signaling in mouse trophoblast specification remains unclear. In this study, we aimed to elucidate the role of EGF signaling in mouse trophoblast differentiation using mouse trophoblast stem cells (mTSCs) in an in vitro culture system. EGF stimulation at the early stage of differentiation repressed mTSC differentiation into spongiotrophoblast cells (SpT). Gene deletion and inhibitor experiments showed that the effect of EGF exposure went through epidermal growth factor receptor (Egfr) activity in mTSCs. EGF stimuli induced acute downstream activation of MAPK/ERK, PI3K/AKT, and JNK pathways, and inhibition of the MAPK/ERK pathway, but not others, alleviated EGF-mediated repression of SpT differentiation. Moreover, expression of Mash2, a master regulator of SpT differentiation, was repressed by EGF stimulation, and MAPK/ERK inhibition counteracted this repression. The Mash2 overexpression recovered SpT marker expression, indicating that the decrease in Mash2 expression was due to abnormal SpT differentiation in EGF-treated mTSCs. Our findings suggest that the EGF-Egfr-MAPK/ERK-Mash2 axis is a core regulatory mechanism for the EGF-mediated repression of SpT differentiation.

Physiologically relevant culture medium Plasmax improves human placental trophoblast stem cell function

Human trophoblast cultures provide powerful tools to model key processes of placental development. In vitro trophoblast studies to date have relied on commercial media that contains nonphysiological levels of nutrients, and the impact of these conditions on trophoblast metabolism and function is unknown. Here, we show that the physiological medium (Plasmax) with nutrient and metabolite concentrations recapitulating human plasma improves human trophoblast stem cell (hTSC) proliferation and differentiation compared with standard medium (DMEM-F12). hTSCs cultured in Plasmax-based medium also show altered glycolytic and mitochondrial metabolism, as well as reduced S-adenosylmethionine/S-adenosyl-homocysteine ratio compared with DMEM-F12-based medium. These findings demonstrate the importance of the nutritional environment for phenotyping cultured human trophoblasts.