Identification of HTRA4 as a Transcriptional Target of p63 in Trophoblast.

The placenta plays a crucial role in pregnancy success. ΔNp63α (p63), a transcription factor from the TP53 family, is highly expressed in villous cytotrophoblasts (CTBs), the epithelial stem cells of the human placenta, and is involved in CTB maintenance and differentiation. We examined the mechanisms of action of p63 by identifying its downstream targets. Gene expression changes were evaluated following overexpression and knockdown of p63 in the JEG3 choriocarcinoma cell line, using microarray-based RNA profiling. High-temperature requirement A4 (HTRA4), a placenta-specific serine protease involved in trophoblast differentiation and altered in preeclampsia, was identified as a gene reciprocally regulated by p63, and its expression was characterized in primary human placental tissues by RNA-sequencing and in situ hybridization. Potential p63 DNA-binding motifs were identified in the HTRA4 promoter, and p63 occupancy at some of these sites was confirmed using chromatin immunoprecipitation, followed by quantitative PCR in both JEG3 and trophoblast stem cells. These data begin to identify members of the transcriptional network downstream of p63, thus laying the groundwork for probing mechanisms by which this important transcription factor regulates trophoblast stemness and differentiation.

Placental pathology and fetal growth outcomes in pregnancies complicated by maternal obesity.

BACKGROUND: The rising prevalence of maternal obesity presents a significant health concern because of the possible implications for obstetric complications and neonatal outcomes. Understanding the impact of obesity on placental structure and function as well as fetal growth and infant outcomes is important to improve the care of these potentially high-risk pregnancies. This study aimed to determine the effect of elevated maternal BMI on histopathologic patterns of placental injury and its consequences on fetal growth. METHODS: Data were collected from an ongoing cohort of maternal-infant dyads in the UCSD Obstetric Registry spanning 2011-2020. Maternal characteristics, including BMI, hypertensive disease and diabetes, placental gross and histopathology, and infant characteristics, including sex and birthweight, were recorded and analyzed. ANOVA and chi-square tests were used in initial analyses, followed by log-binomial and linear regression models adjusted for relevant confounders to determine associations between maternal BMI, specific patterns of placental injury, and infant birthweight percentiles. RESULTS: Among 1366 maternal-infant dyads, placentas from mothers with overweight and obesity were heavier and demonstrated higher adjusted relative risks of chronic villitis (CV), decidual vasculopathy, intervillous thrombosis, and normoblastemia. Placental efficiency, determined by fetal-placental weight ratio, was decreased with increasing BMI. Maternal obesity was associated with higher rates of preterm birth and higher birthweight percentiles. Multiple placental lesions, including maternal (MVM) and fetal vascular malperfusion (FVM), exhibited significant effects on birthweight percentiles; however, only MVM showed a differential effect based on maternal obesity. CONCLUSIONS: Presence of obesity in pregnancy is associated with increased rates of placental patterns of injury, decreased placental efficiency, and increased birthweight percentiles. While placental lesions, such as CV, have the potential to negatively impact fetal growth, the resulting birthweight percentiles demonstrate a more complex relationship between maternal obesity and fetal growth, that likely involves placental and fetal adaptation to the altered in utero environment.

Chronic villitis as a distinctive feature of placental injury in maternal SARS-CoV-2 infection.

BACKGROUND: SARS-CoV-2 infection during pregnancy is associated with an increased risk for stillbirth, preeclampsia, and preterm birth. However, this does not seem to be caused by intrauterine fetal infection because vertical transmission is rarely reported. There is a paucity of data regarding the associated placental SARS-CoV-2 histopathology and their relationship with the timing and severity of infection. OBJECTIVE: This study aimed to determine if maternal SARS-CoV-2 infection was associated with specific patterns of placental injury and if these findings differed by gestational age at time of infection or disease severity. STUDY DESIGN: A retrospective cohort study was performed at the University of California San Diego between March 2020 and February 2021. Placentas from pregnancies with a positive SARS-CoV-2 test were matched with 2 sets of controls; 1 set was time-matched by delivery date and sent to pathology for routine clinical indications, and the other was chosen from a cohort of placentas previously collected for research purposes without clinical indications for pathologic examination before the SARS-CoV-2 outbreak. Placental pathologic lesions were defined based on standard criteria and included maternal and fetal vascular malperfusion and acute and chronic inflammatory lesions. A bivariate analysis was performed using the independent Student t test and Pearson chi-square test. A logistic regression was used to control for relevant covariates. Regions of SARS-CoV-2-associated villitis were further investigated using protein-based digital spatial profiling assays on the GeoMx platform, validated by immunohistochemistry, and compared with cases of infectious villitis and villitis of unknown etiology. Differential expression analysis was performed to identify protein expression differences between these groups of villitis. RESULTS: We included 272 SARS-CoV-2 positive cases, 272 time-matched controls, and 272 historic controls. The mean age of SARS-CoV-2 affected subjects was 30.1±5.5 years and the majority were Hispanic (53.7%) and parous (65.7%). SARS-CoV-2 placentas demonstrated a higher frequency of the 4 major patterns of placental injury (all P<.001) than the historic controls. SARS-CoV-2 placentas also showed a higher frequency of chronic villitis and severe chronic villitis (P=.03 for both) than the time-matched controls, which remained significant after controlling for gestational age at delivery (adjusted odds ratio, 1.52; 95% confidence interval, 1.01-2.28; adjusted odds ratio, 2.12; 95% confidence interval, 1.16-3.88, respectively). Digital spatial profiling revealed that programmed death-ligand 1 was increased in villitis-positive regions of the SARS-CoV-2 (logFC, 0.47; adjusted P value =.002) and villitis of unknown etiology (logFC, 0.58; adjusted P value =.003) cases, but it was conversely decreased in villitis-positive regions of the infectious villitis group (log FC, -1.40; adjusted P value <.001). CONCLUSION: Chronic villitis seems to be the most specific histopathologic finding associated with SARS-CoV-2 maternal infection. Chronic villitis involves damage to the vasculosyncytial membrane of the chorionic villi, which are involved in gas and nutrient exchange, suggesting potential mechanisms of placental (and perhaps neonatal) injury, even in the absence of vertical transmission. Surprisingly, changes in protein expression in SARS-CoV-2-associated villitis seem to be more similar to villitis of unknown etiology than to infectious villitis.

Iron nitrosyl complexes are formed from nitrite in the human placenta.

Placental nitric oxide (NO) is critical for maintaining perfusion in the maternal-fetal-placental circulation during normal pregnancy. NO and its many metabolites are also increased in pregnancies complicated by maternal inflammation such as preeclampsia, fetal growth restriction, gestational diabetes, and bacterial infection. However, it is unclear how increased levels of NO or its metabolites affect placental function or how the placenta deals with excessive levels of NO or its metabolites. Since there is uncertainty over the direction of change in plasma levels of NO metabolites in preeclampsia, we measured the levels of these metabolites at the placental tissue level. We found that NO metabolites are increased in placentas from patients with preeclampsia compared to healthy controls. We also discovered by ozone-based chemiluminescence and electron paramagnetic resonance that nitrite is efficiently converted into iron nitrosyl complexes (FeNOs) within the human placenta and also observed the existence of endogenous FeNOs within placentas from sheep and rats. We show these nitrite-derived FeNOs are relatively short-lived, predominantly protein-bound, heme-FeNOs. The efficient formation of FeNOs from nitrite in the human placenta hints toward the importance of both nitrite and FeNOs in placental physiology or pathology. As iron nitrosylation is an important posttranslational modification that affects the activity of multiple iron-containing proteins such as those in the electron transport chain, or those involved in epigenetic regulation, we conclude that FeNOs merit increased study in pregnancy complications.

Histopathologic and Transcriptomic Profiling Identifies Novel Trophoblast Defects in Patients With Preeclampsia and Maternal Vascular Malperfusion.

Preeclampsia (PE) is a heterogeneous disease for which the current clinical classification system is based on the presence or absence of specific clinical features. PE-associated placentas also show heterogeneous findings on pathologic examination, suggesting that further subclassification is possible. We combined clinical, pathologic, immunohistochemical, and transcriptomic profiling of placentas to develop integrated signatures for multiple subclasses of PE. In total, 303 PE and 1388 nonhypertensive control placentas were included. We found that maternal vascular malperfusion (MVM) in the placenta was associated with preterm PE with severe features and with small-for-gestational-age neonates. Interestingly, PE placentas with either MVM or no histologic pattern of injury showed a linear decrease in proliferative (p63+) cytotrophoblast per villous area with increasing gestational age, similar to placentas obtained from the nonhypertensive patient cohort; however, PE placentas with fetal vascular malperfusion or villitis of unknown etiology lost this phenotype. This is mainly because of cases of fetal vascular malperfusion in placentas of patients with preterm PE and villitis of unknown etiology in placentas of patients with term PE, which are associated with a decrease or increase, respectively, in the cytotrophoblast per villous area. Finally, a transcriptomic analysis identified pathways associated with hypoxia, inflammation, and reduced cell proliferation in PE-MVM placentas and further subclassified this group into extravillous trophoblast-high and extravillous trophoblast-low PE, confirmed using an immunohistochemical analysis of trophoblast lineage-specific markers. Our findings suggest that within specific histopathologic patterns of placental injury, PE can be subclassified based on specific cellular and molecular defects, allowing the identification of pathways that may be targeted for diagnostic and therapeutic purposes.

Placental pathology is necessary to understand common pregnancy complications and achieve an improved taxonomy of obstetrical disease.

The importance of a fully functioning placenta for a good pregnancy outcome is unquestioned. Loss of function can lead to pregnancy complications and is often detected by a thorough placental pathologic examination. Placental pathology has advanced the science and practice of obstetrics and neonatal-perinatal medicine by classifying diseases according to underlying biology and specific patterns of injury. Many past obstacles have limited the incorporation of placental findings into both clinical studies and day-to-day practice. Limitations have included variability in the nomenclature used to describe placental lesions, a shortage of perinatal pathologists fully competent to analyze placental specimens, and a troubling lack of understanding of placental diagnoses by clinicians. However, the potential use of placental pathology for phenotypic classification, improved understanding of the biology of adverse pregnancy outcomes, the development of treatment and prevention, and patient counseling has never been greater. This review, written partly in response to a recent critique published in a major obstetrics-gynecology journal, reexamines the role of placental pathology by reviewing current concepts of biology; explaining the most recent terminology; emphasizing the usefulness of specific diagnoses for obstetrician-gynecologists, neonatologists, and patients; previewing upcoming changes in recommendations for placental submission; and suggesting future improvements. These improvements should include further consideration of overall healthcare costs, cost-effectiveness, the clinical value added of placental assessment, improvements in placental pathology education and practice, and leveraging of placental pathology to identify new biomarkers of disease and evaluate novel therapies tailored to specific clinicopathologic phenotypes of both women and infants.

The role of BMP4 signaling in trophoblast emergence from pluripotency.

The Bone Morphogenetic Protein (BMP) signaling pathway has established roles in early embryonic morphogenesis, particularly in the epiblast. More recently, however, it has also been implicated in development of extraembryonic lineages, including trophectoderm (TE), in both mouse and human. In this review, we will provide an overview of this signaling pathway, with a focus on BMP4, and its role in emergence and development of TE in both early mouse and human embryogenesis. Subsequently, we will build on these in vivo data and discuss the utility of BMP4-based protocols for in vitro conversion of primed vs. naïve pluripotent stem cells (PSC) into trophoblast, and specifically into trophoblast stem cells (TSC). PSC-derived TSC could provide an abundant, reproducible, and ethically acceptable source of cells for modeling placental development.

FundAl Retroflexion (FAR) Angle is a Novel Sonographic Marker Associated With Cesarean Scar Pregnancies in the First Trimester: A Case-Control Study.

OBJECTIVES: Early diagnosis of Cesarean scar pregnancies (CSP) remains difficult. This study describes a novel sonographic marker, the FundAl Retroflexion (FAR) angle, that may be used in the first trimester. The objective of the study is to compare the FAR angle between CSP and normal pregnancies. METHODS: For this case-control study, we reviewed images from our institution's database that were acquired from January 2016 to December 2019. All cases of CSP and randomly selected controls, defined as patients with history of Cesarean delivery and normal implantation, that underwent ultrasound evaluation at <14 weeks were included. The FAR angle, defined as the acute angle created between the endometrial echo and cervical canal, was measured. The mean FAR angle was then compared between the two groups and a receiver operating characteristic (ROC) curve was generated. RESULTS: We identified 15 cases of CSP during the study period and were able to measure the FAR angle in 14 of the cases. The mean FAR angle was larger in CSP than in normal control pregnancies (45° versus 27°, respectively, P < 0.001). Using an ROC curve, a FAR angle cut off of 40° maximizes the ability to distinguish between CSP from normal pregnancies. CONCLUSIONS: The FAR angle provides an easily obtainable and numerical measurement. CSP have larger FAR angle compared to normal controls with a distinguishing cut off of 40°. Larger studies are needed to determine if using the FAR angle can improve first trimester diagnosis for CSP.

Comparative analysis of mouse and human placentae across gestation reveals species-specific regulators of placental development.

An increasing body of evidence points to significant spatio-temporal differences in early placental development between mouse and human, but a detailed comparison of placentae in these two species is missing. We set out to compare placentae from both species across gestation, with a focus on trophoblast progenitor markers. We found that CDX2 and ELF5, but not EOMES, are expressed in early post-implantation trophoblast subpopulations in both species. Genome-wide expression profiling of mouse and human placentae revealed clusters of genes with distinct co-expression patterns across gestation. Overall, there was a closer fit between patterns observed in the placentae when the inter-species comparison was restricted to human placentae through gestational week 16 (thus, excluding full-term samples), suggesting that the developmental timeline in mouse runs parallel to the first half of human placental development. In addition, we identified VGLL1 as a human-specific marker of proliferative cytotrophoblast, where it is co-expressed with the transcription factor TEAD4. As TEAD4 is involved in trophectoderm specification in the mouse, we posit a regulatory role for VGLL1 in early events during human placental development.

Nitric oxide metabolism in the human placenta during aberrant maternal inflammation.

KEY POINTS: Nitric oxide (NO) is a gasotransmitter with important physiological and pathophysiological roles in pregnancy. There is limited information available about the sources and metabolism of NO and its bioactive metabolites (NOx) in both normal and complicated pregnancies. The present study characterized and quantified endogenous NOx in human and mouse placenta following determination of the stability of exogenous NOx in placental homogenates. NOx have differential stability in placental homogenates. NO and iron nitrosyl species (FeNOs), are relatively unstable in placental homogenates from normal placentas. Exogenous NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. FeNOs were also detected endogenously in mouse and human placenta. NOx levels in placental villous tissue are increased in fetal growth restriction vs. placentas from women with normal pregnancies, particularly in fetal growth restriction associated with pre-eclampsia. Villitis was not associated, however, with an increase in NOx levels in either normotensive or pre-eclamptic placentas. The results call for further investigation of FeNOs in normal and complicated pregnancies. ABSTRACT: Nitric oxide (NO) is a gasotransmitter with important roles in pregnancy under both physiological and pathophysiological conditions. Although products of NO metabolism (NOx) also have significant bioactivity, little is known about the role of NO and NOx under conditions of aberrant placental inflammation during pregnancy. An ozone-based chemiluminescence approach was used to investigate the stability and metabolic fate of NOx in human placental homogenates from uncomplicated pregnancies in healthy mothers compared to that in placental tissue from normotensive and pre-eclamptic pregnancies complicated with fetal growth restriction (FGR) with and without villitis of unknown aetiology. We hypothesized that placental NOx would be increased in FGR vs. normal tissue, and be further increased in villitis vs. non-villitis placentas. Findings indicate that nitrate, nitrite and nitrosothiols, but not NO or iron nitrosyl species (FeNOs), are relatively stable in placental homogenates from normal placentas, and that NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. Furthermore, NOx levels in placental villous tissue are increased in FGR vs. placentas from women with normal pregnancies, particularly in FGR associated with pre-eclampsia. However, in contrast to our hypothesis, villitis was not associated with an increase in NOx levels in either normotensive or pre-eclamptic placentas. Our results also strongly support the involvement of FeNOs in both mouse and human placenta, and call for their further study as a critical mechanistic link between pre-eclampsia and fetal growth restriction.

Modeling human trophoblast, the placental epithelium at the maternal fetal interface.

Appropriate human trophoblast lineage specification and differentiation is crucial for the establishment of normal placentation and maintenance of pregnancy. However, due to the lack of proper modeling systems, the molecular mechanisms of these processes are still largely unknown. Much of the early studies in this area have been based on animal models and tumor-derived trophoblast cell lines, both of which are suboptimal for modeling this unique human organ. Recent advances in regenerative and stem cell biology methods have led to development of novel in vitro model systems for studying human trophoblast. These include derivation of human embryonic and induced pluripotent stem cells and establishment of methods for the differentiation of these cells into trophoblast, as well as the more recent derivation of human trophoblast stem cells. In addition, advances in culture conditions, from traditional two-dimensional monolayer culture to 3D culturing systems, have led to development of trophoblast organoid and placenta-on-a-chip model, enabling us to study human trophoblast function in context of more physiologically accurate environment. In this review, we will discuss these various model systems, with a focus on human trophoblast, and their ability to help elucidate the key mechanisms underlying placental development and function. This review focuses on model systems of human trophoblast differentiation, including advantages and limitations of stem cell-based culture, trophoblast organoid, and organ-on-a-chip methods and their applications in understanding placental development and disease.

Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease.

Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6-8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2(+)/p63(+) CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2(+)/p63(+) CTB stem-like cells. These cells can be replated and further differentiated into STB- and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G(+) EVTs in an hypoxia-inducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies.

Identification of Patients with Preeclampsia by Measuring Fluorescence of an Amyloid-Binding Aryl Cyano Amide in Human Urine Samples.

Preeclampsia (PE) is a hypertensive disorder of pregnancy and one of the leading contributors to both maternal and perinatal morbidity and mortality. Reliable diagnostic parameters unique to the disorder that accurately define and diagnose PE are currently unavailable. Recent studies have revealed that PE is accompanied by the accumulation of amyloidogenic deposits in the placenta and the presence of congophilic amyloid-like protein aggregates in the urine. Here, we evaluate the capability of an amyloid-targeting aryl cyano amide (ARCAM-1) fluorophore to identify PE patients from analysis of urine samples. Our results reveal that this probe can distinguish patients with PE from gestationally healthy patients and patients suffering from non-PE hypertension, highlighting the potential for amyloid-targeting fluorophores to help identify PE patients during pregnancy.

Hypoxia Directs Human Extravillous Trophoblast Differentiation in a Hypoxia-Inducible Factor-Dependent Manner.

Villous cytotrophoblasts are epithelial stem cells of the early human placenta, able to differentiate either into syncytiotrophoblasts in floating chorionic villi or extravillous trophoblasts (EVTs) at the anchoring villi. The signaling pathways regulating differentiation into these two lineages are incompletely understood. The bulk of placental growth and development in the first trimester occurs under low oxygen tension. One major mechanism by which oxygen regulates cellular function is through the hypoxia-inducible factor (HIF), a transcription factor complex stabilized under low oxygen tension to mediate cellular responses, including cell fate decisions. HIF is known to play a role in trophoblast differentiation in rodents; however, its role in human trophoblast differentiation is poorly understood. Using RNA profiling of sorted populations of primary first-trimester trophoblasts, we evaluated the first stage of EVT differentiation, the transition from epidermal growth factor receptor+ villous cytotrophoblasts into human leukocyte antigen-G+ proximal column EVT (pcEVT) and identified hypoxia as a major pcEVT-associated pathway. Using primary cytotrophoblasts, we determined that culture in low oxygen directs differentiation preferentially toward human leukocyte antigen-G+ pcEVT, and that an intact HIF complex is required for this process. Finally, using global RNA profiling, we identified integrin-linked kinase and associated cytoskeletal remodeling and adhesion to be among HIF-dependent pcEVT-associated signaling pathways. Taken together, we propose that oxygen regulates EVT differentiation through HIF-dependent modulation of various cell adhesion and morphology-related pathways.

Knockout maternal adiponectin increases fetal growth in mice: potential role for trophoblast IGFBP-1.

AIMS/HYPOTHESIS: The main objective of this study was to investigate whether maternal adiponectin regulates fetal growth through the endocrine system in the fetal compartment. METHODS: Adiponectin knockout (Adipoq (-/-) ) mice and in vivo adenovirus-mediated reconstitution were used to study the regulatory effect of maternal adiponectin on fetal growth. Primary human trophoblast cells were treated with adiponectin and a specific peroxisome proliferator-activated receptor α (PPARα) agonist or antagonist to study the underlying mechanism through which adiponectin regulates fetal growth. RESULTS: The body weight of fetuses from Adipoq (-/-) dams was significantly greater than that of wild-type dams at both embryonic day (E)14.5 and E18.5. Adenoviral vector-mediated maternal adiponectin reconstitution attenuated the increased fetal body weight induced by maternal adiponectin deficiency. Significantly increased blood glucose, triacylglycerol and NEFA levels were observed in Adipoq (-/-) dams, suggesting that nutrient supply contributes to maternal adiponectin-regulated fetal growth. Although fetal blood IGF-1 concentrations were comparable in fetuses from Adipoq (-/-) and wild-type dams, remarkably low levels of IGF-binding protein 1 (IGFBP-1) were observed in the serum of fetuses from Adipoq (-/-) dams. IGFBP-1 was identified in the trophoblast cells of human and mouse placentas. Maternal fasting robustly increased IGFBP-1 levels in mouse placentas, while reducing fetal weight. Significantly low IGFBP-1 levels were found in placentas of Adipoq (-/-) dams. Adiponectin treatment increased IGFBP-1 levels in primary cultured human trophoblast cells, while the PPARα antagonist, MK886, abolished this stimulatory effect. CONCLUSIONS/INTERPRETATION: These results indicate that, in addition to nutrient supply, maternal adiponectin inhibits fetal growth by increasing IGFBP-1 expression in trophoblast cells.

Gene Expression Profiling Reveals a Novel Regulatory Role for Sox21 Protein in Mouse Trophoblast Stem Cell Differentiation.

Appropriate self-renewal and differentiation of trophoblast stem cells (TSCs) are key factors for proper placental development and function and, in turn, for appropriate in utero fetal growth. To identify novel TSC-specific genes, we performed genome-wide expression profiling of TSCs, embryonic stem cells, epiblast stem cells, and mouse embryo fibroblasts, derived from mice of the same genetic background. Our analysis revealed a high expression of Sox21 in TSCs compared with other cell types. Sox21 levels were high in undifferentiated TSCs and were dramatically reduced upon differentiation. In addition, modulation of Sox21 expression in TSCs affected lineage-specific differentiation, based on both marker analysis and functional assessment. Our results implicate Sox21 specifically in the promotion of spongiotrophoblast and giant cell differentiation and establish a new mechanism through which trophoblast sublineages are specified.

BMP4-directed trophoblast differentiation of human embryonic stem cells is mediated through a ΔNp63+ cytotrophoblast stem cell state.

The placenta is a transient organ that is necessary for proper fetal development. Its main functional component is the trophoblast, which is derived from extra-embryonic ectoderm. Little is known about early trophoblast differentiation in the human embryo, owing to lack of a proper in vitro model system. Human embryonic stem cells (hESCs) differentiate into functional trophoblast following BMP4 treatment in the presence of feeder-conditioned media; however, this model has not been widely accepted, in part owing to a lack of proof for a trophoblast progenitor population. We have previously shown that p63, a member of the p53 family of nuclear proteins, is expressed in proliferative cytotrophoblast (CTB), precursors to terminally differentiated syncytiotrophoblast (STB) in chorionic villi and extravillous trophoblast (EVT) at the implantation site. Here, we show that BMP4-treated hESCs differentiate into bona fide CTB by direct comparison with primary human placental tissues and isolated CTB through gene expression profiling. We show that, in primary CTB, p63 levels are reduced as cells differentiate into STB, and that forced expression of p63 maintains cyclin B1 and inhibits STB differentiation. We also establish that, similar to in vivo events, hESC differentiation into trophoblast is characterized by a p63(+)/KRT7(+) CTB stem cell state, followed by formation of functional KLF4(+) STB and HLA-G(+) EVT. Finally, we illustrate that downregulation of p63 by shRNA inhibits differentiation of hESCs into functional trophoblast. Taken together, our results establish that BMP4-treated hESCs are an excellent model of human trophoblast differentiation, closely mimicking the in vivo progression from p63(+) CTB stem cells to terminally differentiated trophoblast subtypes.

Signaling pathways in mouse and human trophoblast differentiation: a comparative review.

The mouse is often used as a model for understanding human placentation and offers multiple advantages, including the ability to manipulate gene expression in specific compartments and to derive trophoblast stem cells, which can be maintained or differentiated in vitro. Nevertheless, there are numerous differences between the mouse and human placentas, only the least of which are structural. This review aims to compare mouse and human placentation, with a focus on signaling pathways involved in trophoblast lineage-specific differentiation.

p63 inhibits extravillous trophoblast migration and maintains cells in a cytotrophoblast stem cell-like state.

Proper differentiation of placental epithelial cells, called trophoblast, is required for implantation. Early during placentation, trophoblast cell columns help anchor the developing embryo in the uterine wall. Although proximally continuous with villous cytotrophoblast (CTB) distally, these cells differentiate into invasive extravillous trophoblast. We previously reported that p63, a p53 family member, is highly expressed in proliferative villous CTB and required for induction of the trophoblast lineage in human pluripotent stem cells. We now further explore its function in human trophoblast by using both primary CTB from the early placenta and established trophoblast cell lines. We show that p63 is expressed in epidermal growth factor receptor-positive CTB and that its expression decreases with differentiation into HLA-G(+) extravillous trophoblast. In trophoblast cell lines, p63 is expressed in JEG3 cells but absent from HTR8 cells. Overexpression of p63 in both cell lines enhances cell proliferation and significantly reduces cell migration; conversely, down-regulation of p63 in JEG3 cells reduces cell proliferation and restores cell migration. Analysis of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways shows that p63 blocks epithelial-to-mesenchymal transition, promotes a CTB-specific cell adhesion profile, and inhibits expression of matrix metalloproteinases. Taken together, these data show that p63 maintains the proliferative CTB state, at least partially through regulation of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways.