Common and uncommon findings in marmoset and human trophoblast stem cells.

In this issue of Cell Stem Cell, Siriwardena et al. analyze peri- and post-implantation marmoset trophoblast development in detail and establish marmoset trophoblast stem cell (TSC) lines from pluripotent stem cells (PSCs). Comparative analysis of marmoset and human TSCs provides insights into species-specific implantation and placentation strategies.

Rapid increase of MFGE8 secretion from endometrial epithelial cells is an indicator of extracellular vesicle mediated embryo maternal dialogue.

Successful embryo implantation relies on synchronized dialog between the embryo and endometrium, and the role of extracellular vesicles (EVs) in facilitating this cross-talk has been recently established. In our previous study, milk fat globule-EGF factor 8 protein (MFGE8) was identified as increasing in receptive endometrial epithelial cells (EECs) in response to trophoblastic EVs. However, the dynamics of MFGE8 protein in this context are not completely understood. Therefore, we examined its expression and secretion in EECs exposed to estrogen, progesterone, and trophoblastic EVs to gain deeper insights into its potential as an indicator of EV-mediated embryo-maternal dialogue. Our findings revealed that MFGE8 secretion is sensitive to estrogen and progesterone, and that trophoblastic EVs stimulate their release in both receptive and non-receptive EECs. Furthermore, trophoblast EV function was dose and time-dependent. Notably, the secretion of MFGE8 increased within a short timeframe of 30 min after addition of EVs, suggesting the possibility of rapid processes such as binding, fusion or internalization of trophoblastic EVs within EECs. Interestingly, MFGE8 released from EECs was associated with EVs, suggesting increased EV secretion from EECs in response to embryonic signals. In conclusion, increased MFGE8 secretion in this embryo implantation model can serve as an indicator of EV-mediated embryo-maternal dialogue.

Receptor usage of Syncytin-1: ASCT2, but not ASCT1, is a functional receptor and effector of cell fusion in the human placenta.

Syncytin-1, a human fusogenic protein of retroviral origin, is crucial for placental syncytiotrophoblast formation. To mediate cell-to-cell fusion, Syncytin-1 requires specific interaction with its cognate receptor. Two trimeric transmembrane proteins, Alanine, Serine, Cysteine Transporters 1 and 2 (ASCT1 and ASCT2), were suggested and widely accepted as Syncytin-1 cellular receptors. To quantitatively assess the individual contributions of human ASCT1 and ASCT2 to the fusogenic activity of Syncytin-1, we developed a model system where the ASCT1 and ASCT2 double knockout was rescued by ectopic expression of either ASCT1 or ASCT2. We demonstrated that ASCT2 was required for Syncytin-1 binding, cellular entry, and cell-to-cell fusion, while ASCT1 was not involved in this receptor interaction. We experimentally validated the ASCT1-ASCT2 heterotrimers as a possible explanation for the previous misidentification of ASCT1 as a receptor for Syncytin-1. This redefinition of receptor specificity is important for proper understanding of Syncytin-1 function in normal and pathological pregnancy.

Establishment of trophoblast cell line derived from buffalo (Bubalus bubalis) parthenogenetic embryo.

We have established trophoblast cell lines, from parthenogenesis-derived buffalo blastocysts. The buffalo trophoblast cells were cultured continuously over 200 days and 21 passages. These cells were observed by phase-contrast microscopy for their morphology and characterized by reverse transcriptase polymerase chain reaction and immunofluorescence against trophoblast-specific markers and cytoskeletal proteins. Trophoblast cells showed positive staining for CDX2, a marker of these cells at both blastocyst and cell line levels. Epithelial morphology of these cells was revealed by positive staining against cytokeratins and tubulin but not against vimentin and dolichos biflorus agglutinin. Gene expression profiles of many important placenta-specific genes were studied in the primary trophectoderm outgrowths, which were collected on days 0, 5, 9, 12 and 15 of culture and trophoblast cell line at passages 12-15. Therefore, the trophoblast cell line derived can potentially be used for in vitro studies on buffalo embryonic development.

Chimerization of human ESC-derived extraembryonic cells with the mouse blastocyst.

It has been reported that human embryonic stem cells (hESCs) treated with BMP4 and inhibitors of TGFß signaling (A83-01) and FGF signaling (PD173074), called BAP, can efficiently differentiate to extraembryonic (ExE) cells in vitro. Due to restricted access to human embryos, it is ethically impossible to test the developmental potential of ExE cells in vivo. Here, we demonstrate that most ExE cells expressed molecular markers for both trophoblasts (TBs) and amniotic cells (ACs). Following intra-uterine transplantation, ExE cells contributed to the mouse placenta. More interestingly, ExE cells could chimerize with the mouse blastocyst as, after injection into the blastocyst, they penetrated its trophectoderm. After implantation of the injected blastocysts into surrogate mice, human cells were found at E14 in placental labyrinth, junction zones, and even near the uterine decidua, expressed placental markers, and secreted human chorionic gonadotropin. Surprisingly, ExE cells also contributed to cartilages of the chimeric embryo with some expressing the chondrogenic marker SOX9, consistent with the mesodermal potential of TBs and ACs in the placenta. Deleting MSX2, a mesodermal determinant, restricted the contribution of ExE cells to the placenta. Thus, we conclude that hESC-derived ExE cells can chimerize with the mouse blastocyst and contribute to both the placenta and cartilages of the chimera consistent with their heteogenious nature. Intra-uterus and intra-blastocyst injections are novel and sensitive methods to study the developmental potential of ExE cells.

Effects of oxycodone on placental lineages: Evidence from the transcriptome profile of mouse trophoblast giant cells.

Pregnant women are often prescribed or abuse opioid drugs. The placenta is likely the key to understanding how opioids cause adverse pregnancy outcomes. Maternal oxycodone (OXY) exposure of pregnant mice leads to disturbances in the layer of invasive parietal trophoblast giant cells (pTGC) that forms the interface between the placenta and uterus. These cells are analogous to extravillous trophoblasts of the human placenta. They are crucial to coordinating the metabolic needs of the conceptus with those of the mother and are primary participants in the placenta-brain axis. Their large nuclear size, however, has precluded both single-cell (sc) and single-nucleus (sn) RNA-seq analyses beyond embryonic day (E) 8.5. Here, we compared the transcriptomes of placentas from pregnant mice exposed to OXY with unexposed controls at E12.5, with particular emphasis on the pTGC. The nonfluidic Parse snRNA-seq approach permitted characterization of the nuclear transcriptomes of all the major placental cell lineages and their presumed progenitors at E12.5. OXY exposure had a negligible effect on components of the placental labyrinth, including the two syncytial cell layers, but caused transcriptomic changes consistent with metabolic stress throughout the spongiotrophoblast. Most notably, there was loss of the majority of pTGC, whose normal gene expression is consistent with elevated energy demand relating to biosynthesis of multiple secretory products, especially hormones, and endoduplication of DNA. This unusual sensitivity of pTGC presumably puts the pregnancy and future health of the offspring at particular risk to OXY exposure.

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.

Marmoset and human trophoblast stem cells differ in signaling requirements and recapitulate divergent modes of trophoblast invasion.

Early human trophoblast development has remained elusive due to the inaccessibility of the early conceptus. Non-human primate models recapitulate many features of human development and allow access to early postimplantation stages. Here, we tracked the pre- to postimplantation transition of the trophoblast lineage in superficially implanting marmoset embryos in vivo. We differentiated marmoset naive pluripotent stem cells into trophoblast stem cells (TSCs), which exhibited trophoblast-specific transcriptome, methylome, differentiation potential, and long-term self-renewal. Notably, human TSC culture conditions failed to support marmoset TSC derivation, instead inducing an extraembryonic mesoderm-like fate in marmoset cells. We show that combined MEK, TGF-ß/NODAL, and histone deacetylase inhibition stabilizes a periimplantation trophoblast-like identity in marmoset TSCs. By contrast, these conditions differentiated human TSCs toward extravillous trophoblasts. Our work presents a paradigm to harness the evolutionary divergence in implantation strategies to elucidate human trophoblast development and invasion.

Galectin-8 Contributes to Human Trophoblast Cell Invasion.

Galectins are a class of lectins that are extensively expressed in all organisms. Galectins are involved in a range of functions, including early development, tissue regeneration, cancer and inflammation. It has been shown that galectin-8 is expressed in the villous and extravillous trophoblast (EVT) cells of the human placenta; however, its physiological role in pregnancy establishment has not been elucidated. Taking these factors into account, we investigated the functional role of galectin-8 in HTR-8/SVneo cells-a human EVT cell line-and human primary cytotrophoblast cells isolated from a first-trimester placenta. We analyzed the effects of recombinant human galectin-8 (rh galectin-8) on the adhesion, migration and invasion of HTR-8/SVneo cells. We used qPCR, cell-based ELISA (cELISA) and gelatin zymography to study the effects of galectin-8 on mediators of these processes, such as integrin subunits alpha-1 and beta-1 and matrix metalloproteinases (MMPs)-2 and -9, on the mRNA and protein levels. Further, we studied the effects of galectin-8 on primary cytotrophoblast cells' invasion. Galectin-8 stimulated the adhesion, migration and invasion of HTR-8/SVneo cells, as well as the invasion of primary cytotrophoblasts. In addition, the MMP-2 and -9 levels were increased, while the expression of integrins alpha-1 and beta-1 was not affected. Galectin-8 has the ability to positively affect EVTs' invasion, so it can be considered a significant factor in the trophoblast cell invasion process.

Transcription factor-based transdifferentiation of human embryonic to trophoblast stem cells.

During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.

Temporal BMP4 effects on mouse embryonic and extraembryonic development.

The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamics of the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and for allantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro.

Charting the Dynamic Trophoblast Plasma Membrane Identifies LYN As a Functional Regulator of Syncytialization.

The differentiation of placental cytotrophoblasts (CTBs) into the syncytiotrophoblast (STB) layer results in a significant remodeling of the plasma membrane proteome. Here, we use a peroxidase-catalyzed proximity labeling strategy to map the dynamic plasma membrane proteomes of CTBs and STBs. Coupled with mass-spectrometry-based proteomics, we identify hundreds of plasma membrane proteins and observe relative changes in protein abundance throughout differentiation, including the upregulation of the plasma-membrane-localized nonreceptor tyrosine kinase LYN. We show that both siRNA-mediated knockdown and small molecule inhibition of LYN kinase function impairs CTB fusion and reduces the expression of syncytialization markers, presenting a function for LYN outside of its canonical role in immunological signaling. Our results demonstrate the use of the proximity labeling platform to discover functional regulators within the plasma membrane and provide new avenues to regulate trophoblast differentiation.

25-Hydroxycholesterol induces oxidative stress, leading to apoptosis and ferroptosis in extravillous trophoblasts.

25-hydroxycholesterol (25HC) is an oxysterol derived from cholesterol and plays a role in various cellular processes, such as lipid metabolism, inflammatory responses, and cell survival. Extravillous trophoblasts (EVTs) are a major cell type found in the placenta, which are highly energetic cells with proliferative and invasive properties. EVT dysfunction can lead to pregnancy complications, including preeclampsia and intrauterine growth restriction. This study investigated the effects and underlying mechanisms of action of 25HC on EVT proliferation. Swan 71 cells, an EVT cell line, were treated with different concentrations of 25HC. Next, cell proliferation was assessed. The mitochondrial reactive oxygen species (mtROS), mitochondrial membrane potentials (MMPs), lipid peroxidation (LPO), and glutathione (GSH) levels were measured. Apoptosis, ferroptosis, and autophagy were evaluated by western blotting and flow cytometry. The results revealed that 25HC significantly inhibited proliferation and decreased the metabolic activity of EVTs. Moreover, 25HC caused oxidative stress by altering mtROS, LPO, MMPs, and GSH levels. Additionally, 25HC induces apoptosis, ferroptosis, and autophagy through the modulation of relevant protein levels. Interestingly, pretreatment with Z-VAD-FMK, an apoptosis inhibitor, and ferrostatin-1, a ferroptosis inhibitor, partially restored the effects of 25HC on cell proliferation, oxidative stress, and cell death. In summary, our findings suggest that 25HC treatment inhibits EVT proliferation and triggers apoptosis, ferroptosis, and autophagy, which are attributable to oxidative stress.

The role of TEAD4 in trophectoderm commitment and development is not conserved in non-rodent mammals.

The first lineage differentiation in mammals gives rise to the inner cell mass and the trophectoderm (TE). In mice, TEAD4 is a master regulator of TE commitment, as it regulates the expression of other TE-specific genes and its ablation prevents blastocyst formation, but its role in other mammals remains unclear. Herein, we have observed that TEAD4 ablation in two phylogenetically distant species (bovine and rabbit) does not impede TE differentiation, blastocyst formation and the expression of TE markers, such as GATA3 and CDX2, although a reduced number of cells in the inner cell mass was observed in bovine TEAD4 knockout (KO) blastocysts. Transcriptional analysis in bovine blastocysts revealed no major transcriptional effect of the ablation, although the expression of hypoblast and Hippo signalling-related genes tended to be decreased in KO embryos. Experiments were conducted in the bovine model to determine whether TEAD4 was required for post-hatching development. TEAD4 KO spherical conceptuses showed normal development of the embryonic disc and TE, but hypoblast migration rate was reduced. At later stages of development (tubular conceptuses), no differences were observed between KO and wild-type conceptuses.

TFEB controls expression of human syncytins during cell-cell fusion.

During human development, a temporary organ is formed, the placenta, which invades the uterine wall to support nutrient, oxygen, and waste exchange between the mother and fetus until birth. Most of the human placenta is formed by a syncytial villous structure lined by syncytialized trophoblasts, a specialized cell type that forms via cell-cell fusion of underlying progenitor cells. Genetic and functional studies have characterized the membrane protein fusogens Syncytin-1 and Syncytin-2, both of which are necessary and sufficient for human trophoblast cell-cell fusion. However, identification and characterization of upstream transcriptional regulators regulating their expression have been limited. Here, using CRISPR knockout in an in vitro cellular model of syncytiotrophoblast development (BeWo cells), we found that the transcription factor TFEB, mainly known as a regulator of autophagy and lysosomal biogenesis, is required for cell-cell fusion of syncytiotrophoblasts. TFEB translocates to the nucleus, exhibits increased chromatin interactions, and directly binds the Syncytin-1 and Syncytin-2 promoters to control their expression during differentiation. Although TFEB appears to play a critical role in syncytiotrophoblast differentiation, ablation of TFEB largely does not affect lysosomal gene expression or lysosomal biogenesis in differentiating BeWo cells, suggesting a previously uncharacterized role for TFEB in controlling the expression of human syncytins.

An old dog with new tricks: TFEB promotes syncytin expression and cell fusion in the human placenta.

In the human placenta, cell fusion is crucial for forming the syncytiotrophoblast, a multinucleated giant cell essential for maintaining pregnancy and ensuring fetal health. The formation of the syncytiotrophoblast is catalyzed by the evolutionarily modern fusogens syncytin-1 and syncytin-2. In this issue of Genes & Development, Esbin and colleagues (doi:10.1101/gad.351633.124) reveal a critical role for the transcription factor TFEB in the regulation of syncytin expression and the promotion of trophoblast fusion. Notably, TFEB's pro-fusion role operates independently of its well-known functions in lysosome biogenesis and autophagy, suggesting that TFEB has acquired additional functions to promote cell fusion in the human placenta.

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.

Rapid retinoic acid-induced trophoblast cell model from human induced pluripotent stem cells.

A limited number of accessible and representative models of human trophoblast cells currently exist for the study of placentation. Current stem cell models involve either a transition through a naïve stem cell state or precise dynamic control of multiple growth factors and small-molecule cues. Here, we demonstrated that a simple five-day treatment of human induced pluripotent stem cells with two small molecules, retinoic acid (RA) and Wnt agonist CHIR 99021 (CHIR), resulted in rapid, synergistic upregulation of CDX2. Transcriptomic analysis of RA + CHIR-treated cells showed high similarity to primary trophectoderm cells. Multipotency was verified via further differentiation towards cells with syncytiotrophoblast or extravillous trophoblast features. RA + CHIR-treated cells were also assessed for the established criteria defining a trophoblast cell model, and they possess all the features necessary to be considered valid. Collectively, our data demonstrate a facile, scalable method for generating functional trophoblast-like cells in vitro to better understand the placenta.

Single-cell RNA sequencing reveals placental response under environmental stress.

The placenta is crucial for fetal development, yet the impact of environmental stressors such as arsenic exposure remains poorly understood. We apply single-cell RNA sequencing to analyze the response of the mouse placenta to arsenic, revealing cell-type-specific gene expression, function, and pathological changes. Notably, the Prap1 gene, which encodes proline-rich acidic protein 1 (PRAP1), is significantly upregulated in 26 placental cell types including various trophoblast cells. Our study shows a female-biased increase in PRAP1 in response to arsenic and localizes it in the placenta. In vitro and ex vivo experiments confirm PRAP1 upregulation following arsenic treatment and demonstrate that recombinant PRAP1 protein reduces arsenic-induced cytotoxicity and downregulates cell cycle pathways in human trophoblast cells. Moreover, PRAP1 knockdown differentially affects cell cycle processes, proliferation, and cell death depending on the presence of arsenic. Our findings provide insights into the placental response to environmental stress, offering potential preventative and therapeutic approaches for environment-related adverse outcomes in mothers and children.

In utero morphological and functional properties of bovine trophoblastic vesicles.

In many mammals, including ruminants, pregnancy requires pregnancy recognition signaling molecules secreted by the conceptus; however, the mechanism underlying pregnancy establishment in cattle remains unknown. Trophoblastic vesicles (TVs) are artificially produced from the extraembryonic tissues of the elongating conceptus and may be useful tools for understanding conception. This study investigated the morphological and functional properties of TVs in comparison to those of intact conceptuses. TVs were prepared from the extraembryonic tissues of conceptuses collected 14 days after artificial insemination (AI), cryopreserved immediately after dissection, and cultured after thawing for subsequent transplantation into the uterus. The transferred TVs were collected 7 days after transplantation and compared with extraembryonic tissue samples collected from conceptuses at 21 days post-AI. The recovered TVs were 40 times longer than those of their pre-transplant counterparts. Microscopic evaluation revealed that their membrane structures consisted of trophoblast and hypoblast layers. The expression patterns of the cell differentiation markers, CDX2, SOX2, and GATA6, and interferon tau (IFNT) protein expression levels in the TVs were similar to those in control extraembryonic tissue samples. These findings suggest that TVs are capable of morphological elongation and maintain IFNT production in a similar way as original trophoblasts.