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.

An integrated atlas of human placental development delineates essential regulators of trophoblast stem cells.

The trophoblast lineage safeguards fetal development by mediating embryo implantation, immune tolerance, nutritional supply and gas exchange. Human trophoblast stem cells (hTSCs) provide a platform to study lineage specification of placental tissues; however, the regulatory network controlling self-renewal remains elusive. Here, we present a single-cell atlas of human trophoblast development from zygote to mid-gestation together with single-cell profiling of hTSCs. We determine the transcriptional networks of trophoblast lineages in vivo and leverage probabilistic modelling to identify a role for MAPK signalling in trophoblast differentiation. Placenta- and blastoid-derived hTSCs consistently map between late trophectoderm and early cytotrophoblast, in contrast to blastoid-trophoblast, which correspond to trophectoderm. We functionally assess the requirement of the predicted cytotrophoblast network in an siRNA-screen and reveal 15 essential regulators for hTSC self-renewal, including MAZ, NFE2L3, TFAP2C, NR2F2 and CTNNB1. Our human trophoblast atlas provides a powerful analytical resource to delineate trophoblast cell fate acquisition, to elucidate transcription factors required for hTSC self-renewal and to gauge the developmental stage of in vitro cultured cells.

PEG10 viral aspartic protease domain is essential for the maintenance of fetal capillary structure in the mouse placenta.

The therian-specific gene paternally expressed 10 (Peg10) plays an essential role in placenta formation: Peg10 knockout mice exhibit early embryonic lethality as a result of severe placental defects. The PEG10 protein exhibits homology with long terminal repeat (LTR) retrotransposon GAG and POL proteins; therefore, we generated mice harboring a mutation in the highly conserved viral aspartic protease motif in the POL-like region of PEG10 because this motif is essential for the life cycle of LTR retrotransposons/retroviruses. Intriguingly, frequent perinatal lethality, not early embryonic lethality, was observed with fetal and placental growth retardation starting mid-gestation. In the mutant placentas, severe defects were observed in the fetal vasculature, where PEG10 is expressed in the three trophoblast cell layers that surround fetal capillary endothelial cells. Thus, Peg10 has essential roles, not only in early placenta formation, but also in placental vasculature maintenance from mid- to late-gestation. This implies that along the feto-maternal placenta interface an interaction occurs between two retrovirus-derived genes, Peg10 and retrotransposon Gag like 1 (Rtl1, also called Peg11), that is essential for the maintenance of fetal capillary endothelial cells.

Protective and sex-specific effects of moderate dose folic acid supplementation on the placenta following assisted reproduction in mice.

Epigenetic defects induced by assisted reproductive technologies (ART) have been suggested as a potential mechanism contributing to suboptimal placentation. Here, we hypothesize that ART perturbs DNA methylation (DNAme) and gene expression during early placenta development, leading to abnormal placental phenotypes observed at term. Since folic acid (FA) plays a crucial role in epigenetic regulation, we propose that FA supplementation can rescue ART-induced placental defects. Female mice were placed on a control diet (CD), a moderate 4-fold (FAS4) or high dose 10-fold (FAS10) FA-supplemented diet prior to ART and compared to a natural mating group. ART resulted in 41 and 28 differentially expressed genes (DEGs) in E10.5 female and male placentas, respectively. Many DEGs were implicated in early placenta development and associated with DNAme changes; a number clustered at known imprinting control regions (ICR). In females, FAS4 partially corrected alterations in gene expression while FAS10 showed evidence of male-biased adverse effects. DNAme and gene expression for five genes involved in early placentation (Phlda2, EphB2, Igf2, Peg3, L3mbtl1) were followed up in placentas from normal as well as delayed and abnormal embryos. Phlda2 and Igf2 expression levels were lowest after ART in placentas of female delayed embryos. Moreover, ART concomitantly reduced DNAme at the Kcnq1ot1 ICR which regulates Phlda2 expression; FAS4 partially improved DNAme in a sex-specific manner. In conclusion, ART-associated placental DNAme and transcriptome alterations observed at mid-gestation are sex-specific; they may help explain adverse placental phenotypes detected at term and are partially corrected by maternal moderate dose FA supplementation.

Nutraceuticals in Pregnancy: A Special Focus on Probiotics.

The placenta is crucial to fetal development and performs vital functions such as nutrient exchange, waste removal and hormone regulation. Abnormal placental development can lead to conditions such as fetal growth restriction, pre-eclampsia and stillbirth, affecting both immediate and long-term fetal health. Placental development is a highly complex process involving interactions between maternal and fetal components, imprinted genes, signaling pathways, mitochondria, fetal sexomes and environmental factors such as diet, supplementation and exercise. Probiotics have been shown to make a significant contribution to prenatal health, placental health and fetal development, with associations with reduced risk of preterm birth and pre-eclampsia, as well as improvements in maternal health through effects on gut microbiota, lipid metabolism, vaginal infections, gestational diabetes, allergic diseases and inflammation. This review summarizes key studies on the influence of dietary supplementation on placental development, with a focus on the role of probiotics in prenatal health and fetal development.

Ambient fine particulate matter exposure disrupts placental autophagy and fetal development in gestational mice.

Recent studies have shown that some adverse pregnancy outcomes, especially intrauterine growth restriction (IUGR), are associated with gestational exposure to ambient fine particulate matter (PM2.5). However, potential mechanism remains to be elucidated. In the present study, pregnant C57BL/6 mice were randomly assigned to be exposed to either filtered air or ambient PM2.5 in the gestation period via a concentrated whole-body exposure system. We found that gestational PM2.5 exposure exerted no effect on implantation, preterm delivery, as well as fetal resorption and death. However, in utero fetal exposure to PM2.5 showed a significant reduction in body weight and crown-rump length on GD13 and GD18. Meanwhile, maternal blood sinusoid in placenta was markedly reduced along with abnormal expression of placental nutrient transporters and growth hormone in dams exposed to PM2.5. Additional tests showed gestational PM2.5 exposure decreased autophagy-related protein levels and inhibited autophagy flux mainly on GD15. Correspondingly, AMPK/mTOR signaling pathway, a critical negative regulator of autophagy, was activated in placenta on GD15 by PM2.5 exposure as well. These findings provide evidences that placental developmental disorder caused by autophagy inhibition might be an important mechanism for the growth restriction caused by PM2.5 exposure.

Proteoglycans: Systems-Level Insight into Their Expression in Healthy and Diseased Placentas.

Proteoglycan macromolecules play key roles in several physiological processes (e.g., adhesion, proliferation, migration, invasion, angiogenesis, and apoptosis), all of which are important for placentation and healthy pregnancy. However, their precise roles in human reproduction have not been clarified. To fill this gap, herein, we provide an overview of the proteoglycans' expression and role in the placenta, in trophoblast development, and in pregnancy complications (pre-eclampsia, fetal growth restriction), highlighting one of the most important members of this family, syndecan-1 (SDC1). Microarray data analysis showed that of 34 placentally expressed proteoglycans, SDC1 production is markedly the highest in the placenta and that SDC1 is the most upregulated gene during trophoblast differentiation into the syncytiotrophoblast. Furthermore, placental transcriptomic data identified dysregulated proteoglycan genes in pre-eclampsia and in fetal growth restriction, including SDC1, which is supported by the lower concentration of syndecan-1 in maternal blood in these syndromes. Overall, our clinical and in vitro studies, data analyses, and literature search pointed out that proteoglycans, as important components of the placenta, may regulate various stages of placental development and participate in the maintenance of a healthy pregnancy. Moreover, syndecan-1 may serve as a useful marker of syncytialization and a prognostic marker of adverse pregnancy outcomes. Further studies are warranted to explore the role of proteoglycans in healthy and complicated pregnancies, which may help in diagnostic or therapeutic developments.

Human placenta and trophoblast development: key molecular mechanisms and model systems.

Abnormal placentation is considered as an underlying cause of various pregnancy complications such as miscarriage, preeclampsia and intrauterine growth restriction, the latter increasing the risk for the development of severe disorders in later life such as cardiovascular disease and type 2 diabetes. Despite their importance, the molecular mechanisms governing human placental formation and trophoblast cell lineage specification and differentiation have been poorly unravelled, mostly due to the lack of appropriate cellular model systems. However, over the past few years major progress has been made by establishing self-renewing human trophoblast stem cells and 3-dimensional organoids from human blastocysts and early placental tissues opening the path for detailed molecular investigations. Herein, we summarize the present knowledge about human placental development, its stem cells, progenitors and differentiated cell types in the trophoblast epithelium and the villous core. Anatomy of the early placenta, current model systems, and critical key regulatory factors and signalling cascades governing placentation will be elucidated. In this context, we will discuss the role of the developmental pathways Wingless and Notch, controlling trophoblast stemness/differentiation and formation of invasive trophoblast progenitors, respectively.

PPARs and Angiogenesis-Implications in Pathology.

Peroxisome proliferator-activated receptors (PPARs) belong to the family of ligand-activated nuclear receptors. The PPAR family consists of three subtypes encoded by three separate genes: PPARα (NR1C1), PPARß/δ (NR1C2), and PPARγ (NR1C3). PPARs are critical regulators of metabolism and exhibit tissue and cell type-specific expression patterns and functions. Specific PPAR ligands have been proposed as potential therapies for a variety of diseases such as metabolic syndrome, cancer, neurogenerative disorders, diabetes, cardiovascular diseases, endometriosis, and retinopathies. In this review, we focus on the knowledge of PPAR function in angiogenesis, a complex process that plays important roles in numerous pathological conditions for which therapeutic use of PPAR modulation has been suggested.

A trans-homologue interaction between reciprocally imprinted miR-127 and Rtl1 regulates placenta development.

The paternally expressed imprinted retrotransposon-like 1 (Rtl1) is a retrotransposon-derived gene that has evolved a function in eutherian placentation. Seven miRNAs, including miR-127, are processed from a maternally expressed antisense Rtl1 transcript (Rtl1as) and regulate Rtl1 levels through RNAi-mediated post-transcriptional degradation. To determine the relative functional role of Rtl1as miRNAs in Rtl1 dosage, we generated a mouse specifically deleted for miR-127. The miR-127 knockout mice exhibit placentomegaly with specific defects within the labyrinthine zone involved in maternal-fetal nutrient transfer. Although fetal weight is unaltered, specific Rtl1 transcripts and protein levels are increased in both the fetus and placenta. Phenotypic analysis of single (ΔmiR-127/Rtl1 or miR-127/ΔRtl1) and double (ΔmiR-127/ΔRtl1) heterozygous miR-127- and Rtl1-deficient mice indicate that Rtl1 is the main target gene of miR-127 in placental development. Our results demonstrate that miR-127 is an essential regulator of Rtl1, mediated by a trans-homologue interaction between reciprocally imprinted genes on the maternally and paternally inherited chromosomes.

The basal chorionic trophoblast cell layer: An emerging coordinator of placenta development.

During gestation, fetomaternal exchange occurs in the villous tree (labyrinth) of the placenta. Development of this structure depends on tightly coordinated cellular processes of branching morphogenesis and differentiation of specialized trophoblast cells. The basal chorionic trophoblast (BCT) cell layer that localizes next to the chorioallantoic interface is of critical importance for labyrinth morphogenesis in rodents. Gcm1-positive cell clusters within this layer initiate branching morphogenesis thereby guiding allantoic fetal blood vessels towards maternal blood sinuses. Later these cells differentiate and contribute to the syncytiotrophoblast of the fetomaternal barrier. Additional cells within the BCT layer sustain continued morphogenesis, possibly through a repopulating progenitor population. Several mouse mutants highlight the importance of a structurally intact BCT epithelium, and a growing number of studies addresses its patterning and epithelial architecture. Here, we review and discuss emerging concepts in labyrinth development focussing on the biology of the BCT cell layer.

Generation of Knockout Mouse Trophoblast Stem Cells by CRISPR/Cas9.

The placenta is the organ that dictates the reproductive outcome of mammalian pregnancy by supplying nutrients and oxygen to the developing fetus to sustain its normal growth. During early mammalian development, trophoblast cells are the earliest cell type to differentiate with multipotent capacity to generate the trophoblast components of the placenta. The isolation and use of mouse trophoblast stem cells (mTSCs) to model in vitro trophoblast differentiation, in combination with CRISPR/Cas9 genome editing technology, has provided tremendous insight into the molecular mechanisms governing early mouse placentation. By knocking out a specific gene of interest in mTSCs, researchers are shedding light onto the molecular pathways involved in normal placental development and pregnancy disorders associated with abnormal placentation. In this chapter, we provide a detailed protocol for the genetic modification of mTSCs by using CRISPR/Cas9 genome editing system.

CRISPR Activation in Mouse Trophoblast Stem Cells.

The placenta is a vital organ that regulates nutrient supply to the developing embryo during gestation. In mice, the placenta is composed of trophoblast lineage and mesodermal derivatives, which merge through the chorioallantoic fusion process in a critical event for the progression of placenta development. The trophoblast lineage is derived from self-renewing, multipotent cells known as mouse trophoblast stem cells (mTSCs). These cells are a valuable tool that allows scientists to comprehend the signals regulating major placental cell types' self-renewal and differentiation capacity. Recent advances in CRISPR-Cas9 genome editing applied in mTSCs have provided novel insights into the molecular networks involved in placentation. Here, we present a comprehensive CRISPR activation (CRISPRa) protocol based on the CRISPR/gRNA-directed synergistic activation mediator (SAM) method to overexpress specific target genes in mTSCs.

Titanium Dioxide Nanoparticles Induce Maternal Preeclampsia-like Syndrome and Adverse Birth Outcomes via Disrupting Placental Function in SD Rats.

The escalating utilization of titanium dioxide nanoparticles (TiO2 NPs) in everyday products has sparked concerns regarding their potential hazards to pregnant females and their offspring. To address these concerns and shed light on their undetermined adverse effects and mechanisms, we established a pregnant rat model to investigate the impacts of TiO2 NPs on both maternal and offspring health and to explore the underlying mechanisms of those impacts. Pregnant rats were orally administered TiO2 NPs at a dose of 5 mg/kg body weight per day from GD5 to GD18 during pregnancy. Maternal body weight, organ weight, and birth outcomes were monitored and recorded. Maternal pathological changes were examined by HE staining and TEM observation. Maternal blood pressure was assessed using a non-invasive blood analyzer, and the urinary protein level was determined using spot urine samples. Our findings revealed that TiO2 NPs triggered various pathological alterations in maternal liver, kidney, and spleen, and induced maternal preeclampsia-like syndrome, as well as leading to growth restriction in the offspring. Further examination unveiled that TiO2 NPs hindered trophoblastic cell invasion into the endometrium via the promotion of autophagy. Consistent hypertension and proteinuria resulted from the destroyed the kidney GBM. In total, an exposure to TiO2 NPs during pregnancy might increase the risk of human preeclampsia through increased maternal arterial pressure and urinary albumin levels, as well as causing fetal growth restriction in the offspring.

Oxygen governs Galß1-3GalNAc epitope in human placenta.

It is becoming increasingly apparent that the dynamics of glycans reflect the physiological state of cells involved in several cell functions including growth, response to signal molecules, migration, as well as adhesion to, interaction with, and recognition of other cells. The presence of glycoconjugates in human placenta suggests their major role in maternal-fetal exchanges, intercellular adhesion, cellular metabolism, and villous vessel branching. Although several studies have described glycoconjugate distribution in the human placenta descriptions of their physiological function and control mechanisms during placental development are lacking. In this study we investigated the developmental distribution and regulation of placental core 1 O- and N-glycans focusing on early and late first trimester human pregnancy. To define the control mechanisms of the oligosaccharide chains during early placentation process, chorionic villous explants and human trophoblast cell lines were exposed to various oxygen levels. We found that oxygen tension regulates changes in core-1 O-glycan (the disaccharide Galß1-3GalNAc) epitope expression levels. Moreover, by double affinity chromatography and subsequent analysis with mass spectrometry, we identified in the heat shock protein 90-α (HSP90α) a good candidate as carrier of the Galß1-3GalNAc epitope at low oxygen tension. Our results support a fundamental role of oxygen tension in modulating glycosylation of proteins during placental development.

Fluid shear stress induces a shift from glycolytic to amino acid pathway in human trophoblasts.

BACKGROUND: The human placenta, a tissue with a lifespan limited to the period of pregnancy, is exposed to varying shear rates by maternal blood perfusion depending on the stage of development. In this study, we aimed to investigate the effects of fluidic shear stress on the human trophoblast transcriptome and metabolism. RESULTS: Based on a trophoblast cell line cultured in a fluidic flow system, changes caused by shear stress were analyzed and compared to static conditions. RNA sequencing and bioinformatics analysis revealed an altered transcriptome and enriched gene ontology terms associated with amino acid and mitochondrial metabolism. A decreased GLUT1 expression and reduced glucose uptake, together with downregulated expression of key glycolytic rate-limiting enzymes, hexokinase 2 and phosphofructokinase 1 was observed. Altered mitochondrial ATP levels and mass spectrometry data, suggested a shift in energy production from glycolysis towards mitochondrial oxidative phosphorylation. This shift in energy production could be supported by increased expression of glutamic-oxaloacetic transaminase variants in response to shear stress as well as under low glucose availability or after silencing of GLUT1. The shift towards amino acid metabolic pathways could be supported by significantly altered amino acid levels, like glutamic acid, cysteine and serine. Downregulation of GLUT1 and glycolytic rate-limiting enzymes, with concomitant upregulation of glutamic-oxaloacetic transaminase 2 was confirmed in first trimester placental explants cultured under fluidic flow. In contrast, high fluid shear stress decreased glutamic-oxaloacetic transaminase 2 expression in term placental explants when compared to low flow rates. Placental tissue from pregnancies with intrauterine growth restriction are exposed to high shear rates and showed also decreased glutamic-oxaloacetic transaminase 2, while GLUT1 was unchanged and glycolytic rate-limiting enzymes showed a trend to be upregulated. The results were generated by using qPCR, immunoblots, quantification of immunofluorescent pictures, padlock probe hybridization, mass spectrometry and FRET-based measurement. CONCLUSION: Our study suggests that onset of uteroplacental blood flow is accompanied by a shift from a predominant glycolytic- to an alternative amino acid converting metabolism in the villous trophoblast. Rheological changes with excessive fluidic shear stress at the placental surface, may disrupt this alternative amino acid pathway in the syncytiotrophoblast and could contribute to intrauterine growth restriction.

Folate-depletion alters mouse trophoblast stem cell regulation in vitro.

Maternal folate deficiency increases risk of congenital malformations, yet its effect on placenta development is unclear. Here, we investigated how folate-depleted culture medium affects the developmental potential of mouse trophoblast stem cells (TSCs). When cultured in stem cell conditions, TSC viability was unaffected by folate depletion, but ectopic differentiation of trophoblast cell subtypes occurred. When cultured in conditions that promote differentiation, folate-depleted TSCs were driven towards a syncytiotrophoblast cell fate potentially at the expense of other lineages. Additionally, trophoblast giant cell nuclei were small implicating folate in the regulation of endoreduplication. Therefore, dietary folate intake likely promotes trophoblast development.

Whole genome bisulfite sequencing of sperm reveals differentially methylated regions in male partners of idiopathic recurrent pregnancy loss cases.

OBJECTIVE: To study the genome wide alterations in sperm DNA methylation in male partners of idiopathic recurrent pregnancy loss (iRPL) cases and note regions as potential diagnostic markers. DESIGN: Case-control study and methylome analysis of human sperm. SETTING: Obstetrics and Gynaecology clinics. PATIENT(S): Control group consists of apparently healthy fertile men having fathered a child within the last 2 years (n = 39); and case group consists of male partners of iRPL cases having ≥2 consecutive 1st trimester pregnancy losses (n = 47). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm DNA samples of controls and cases were selected for whole genome bisulfite sequencing analysis based on the previously set thresholds of global methylation levels and methylation levels of imprinted genes (KvDMR and ZAC). Whole genome bisulfite sequencing of selected sperm genomic DNA was performed to identify differentially methylated CpG sites of iRPL cases compared with fertile controls. Pathway analysis of all the differentially methylated genes was done by Database for Annotation, Visualization, and Integrated Discovery annotation tool and Kyoto Encyclopedia of Genes and Genomes tool. Differentially methylated CpGs within genes relevant to embryo and placenta development were selected to further validate their methylation levels in study population by pyrosequencing. RESULT(S): A total of 9497 differentially methylated CpGs with highest enrichment in intronic regions were obtained. In addition, 5352 differentially methylated regions and 2087 differentially methylated genes were noted. Signaling pathways involved in development were enriched on pathway analysis. Select CpGs within genes PPARG, KCNQ1, SETD2, and MAP3K4 showed distinct hypomethylated subpopulations within iRPL study population. CONCLUSION(S): Our study highlights the altered methylation landscape of iRPL sperm, and their possible implications in pathways of embryo and placental development. The CpG sites that are hypomethylated specifically in sperm of iRPL subpopulation can be further assessed as predictive biomarkers.

Animal models of the placenta accreta spectrum: current status and further perspectives.

Placenta accreta spectrum disorder (PAS) is a kind of disease of placentation defined as abnormal trophoblast invasion of part or all of the placenta into the myometrium, even penetrating the uterus. Decidual deficiency, abnormal vascular remodeling in the maternal-fetal interface, and excessive invasion by extravillous trophoblast (EVT) cells contribute to its onset. However, the mechanisms and signaling pathways underlying such phenotypes are not fully understood, partly due to the lack of suitable experimental animal models. Appropriate animal models will facilitate the comprehensive and systematic elucidation of the pathogenesis of PAS. Due to the remarkably similar functional placental villous units and hemochorial placentation to humans, the current animal models of PAS are based on mice. There are various mouse models induced by uterine surgery to simulate different phenotypes of PAS, such as excessive invasion of EVT or immune disturbance at the maternal-fetal interface, which could define the pathological mechanism of PAS from the perspective of the "soil." Additionally, genetically modified mouse models could be used to study PAS, which is helpful to exploring the pathogenesis of PAS from the perspectives of both "soil" and "seed," respectively. This review details early placental development in mice, with a focus on the approaches of PAS modeling. Additionally, the strengths, limitations and the applicability of each strategy and further perspectives are summarized to provide the theoretical foundation for researchers to select appropriate animal models for various research purposes. This will help better determine the pathogenesis of PAS and even promote possible therapy.

Establishment of Trophoblast-Like Tissue Model from Human Pluripotent Stem Cells in Three-Dimensional Culture System.

The placenta has a lifelong impact on the health of both the mother and fetus. Despite its significance, human early placental development is poorly understood due to the limited models. The models that can reflect the key features of early human placental development, especially at early gestation, are still lacking. Here, the authors report the generation of trophoblast-like tissue model from human pluripotent stem cells (hPSCs) in three-dimensional (3D) cultures. hPSCs efficiently self-organize into blastocoel-like cavities under defined conditions, which produce different trophoblast subtypes, including cytotrophoblasts (CTBs), syncytiotrophoblasts (STBs), and invasive extravillous trophoblasts (EVTs). The 3D cultures can exhibit microvilli structure and secrete human placenta-specific hormone. Single-cell RNA sequencing analysis further identifies the presence of major cell types of trophoblast-like tissue as existing in vivo. The results reveal the feasibility to establish 3D trophoblast-like tissue model from hPSCs in vitro, which is not obtained by monolayer culture. This new model system can not only facilitate to dissect the underlying mechanisms of early human placental development, but also imply its potential for study in developmental biology and gestational disorders.