Trophoblast cells play an essential role in the interactions between the fetus and mother. Mouse trophoblast stem (TS) cells have been derived and used as the best model for molecular and functional analysis of mouse trophoblast lineages, but attempts to derive human TS cells have so far been unsuccessful. Here we show that activation of Wingless/Integrated (Wnt) and EGF and inhibition of TGF-β, histone deacetylase (HDAC), and Rho-associated protein kinase (ROCK) enable long-term culture of human villous cytotrophoblast (CT) cells. The resulting cell lines have the capacity to give rise to the three major trophoblast lineages, which show transcriptomes similar to those of the corresponding primary trophoblast cells. Importantly, equivalent cell lines can be derived from human blastocysts. Our data strongly suggest that the CT- and blastocyst-derived cell lines are human TS cells, which will provide a powerful tool to study human trophoblast development and function. Trophoblast cells are specialized cells in the placenta that mediate the interactions between the fetus and mother. Okae et al. report the derivation of human trophoblast stem cells from blastocysts and early placentas, which will provide a powerful tool to study human placental development and function.
The trophoblast cell lineage is essential for the survival of the mammalian embryo in utero. This lineage is specified before implantation into the uterus and is restricted to form the fetal portion of the placenta. A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permitted the isolation of permanent trophoblast stem cell lines. These cell lines differentiated to other trophoblast subtypes in vitro in the absence of FGF4 and exclusively contributed to the trophoblast lineage in vivo in chimeras.
Both paracrine and autocrine factors are involved in the regulation of trophoblast invasion. One of these factors is human chorionic gonadotropin (hCG), which stimulates trophoblast invasion. The stimulatory activity has especially been ascribed to a hyperglycosylated form of hCG (hCG-h) that is expressed in early pregnancy. We compared the stimulatory activities of different forms of hCG and its free β-subunit (hCGβ) on trophoblast invasion. hCG, hCG-h, hCGβ, and its hyperglycosylated form (hCGβ-h) stimulated the invasion of JEG-3 choriocarcinoma cells. The stimulatory effect of hCGβ was also confirmed with primary human trophoblasts. Down-regulation of the LH/hCG receptor by RNA-interference did not significantly reduce the effect of hCGβ and hCG on cell invasion. Increased invasion was associated with increased levels of MMP-2, MMP-9 and activity of uPA. Our findings suggest that hCG, hCGβ and their hyperglycosylated forms stimulate the invasion of trophoblast cells independent of the classical LH/hCG-receptor.
Objective To use trophoblast cells accumulating in the endocervical canal at the beginning of pregnancy for noninvasive prenatal testing. Design Prospective, double-blinded test for fetal gender. Setting Academic medical center. Patient(s) Fifty-six women with singleton pregnancies at gestational age 5–20 weeks. Intervention(s) Isolation of fetal cells from resident maternal cells in endocervical specimens using anti-human leukocyte antigen G coupled to magnetic nanoparticles; cell phenotyping immunofluorescently with a panel of trophoblast subtype-specific proteins; DNA integrity assessment with terminal dUTP nick-end labeling (TUNEL); and polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) to detect sex chromosomes in individual cells. Main Outcome Measure(s) Trophoblast phenotype, TUNEL index, and percentage male cells. Result(s) The women were given a routine Papanicolaou test; fetal genders were verified from medical records. Recovery after immunomagnetic isolation averaged 746 ± 59 cells across gestational age, with 99% expressing chorionic gonadotropin, whereas the depleted cell fraction expressed none. The isolated cells had an extravillous trophoblast phenotype and intact nuclear DNA (>95%). Fetal gender was determined in 20 specimens without error by PCR. The FISH analysis of isolated cells from male specimens validated their fetal origin. Conclusion(s) Noninvasive prenatal testing is feasible beginning at a gestational age of 5 weeks.
The mammalian fetus represents a semiallograft within the maternal uterus yet is not rejected. This situation is particularly pronounced in species with a hemochorial type of placentation, such as humans and rodents, where maternal tissues and blood are in direct contact with fetal trophoblast and thus potentially with paternal antigens. The main polymorphic antigens responsible for graft rejection are MHC antigens. In humans the trophoblast cells invading into the decidua have a unique pattern of MHC class I expression characterized by both classical (HLA-C) and nonclassical (HLA-G and HLA-E) molecules. Whether such an unusual MHC repertoire on the surface of trophoblast is a conserved feature between species with hemochorial placentation has not been resolved. Here we demonstrate, using a range of methods, that C57BL/6 mouse trophoblast predominantly expresses only one MHC class I antigen, H2-K, at the cell surface of giant cells but lacks expression of nonclassical MHC molecules. Antigenic disparity between parental MHCs affects trophoblast-induced transformation of the uterine vasculature and, consequently, placental and fetal gowth. Maternal uterine blood vessels were more dilated, allowing for increased blood supply, in certain combinations of maternal and paternal MHC haplotypes, and these allogeneic fetuses and placentas were heavier at term compared with syngeneic controls. Thus, maternal-fetal immune interactions are instrumental to optimize reproductive success. This cross-talk has important implications for human disorders of pregnancy, such as preeclampsia and fetal growth restriction.
Problem The extravillous trophoblasts (EVT) express HLA‐C and HLA‐G, but HLA‐E and HLA‐F are the subject of conflicting reports. In this study, we define the HLA expression profile during active EVT placental implantation, pregnancy development, and parturition. Method of study Immunohistochemistry, q‐PCR, and Western blot were used to investigate HLA‐C, HLA‐E, and HLA‐F placental expression across gestation from the early first trimester, late first trimester, second trimester (n=10 in each), preterm gestation (n=6) to elective term cesarean section and term vaginal deliveries (n=12, 38‐41 weeks). EVT explants and Swan71 cells were used to assess HLA‐C and HLA‐F during active EVT migration. Results HLA‐G, HLA‐C, and HLA‐F were expressed by 1st‐trimester EVT and became intracellular and weaker as gestation progressed. HLA‐E was only expressed in 1st‐trimester placenta. HLA‐F and HLA‐C mRNA and protein expression levels showed a significant increase in the fetal villous mesenchyme across gestation. HLA‐C levels increased with labor. We detected a 100‐kDa HLA‐F band in early pregnancy suggesting dimer formation on the EVT surface. These results were confirmed in EVT outgrowths and Swan71 trophoblast which showed that HLA‐F and HLA‐G are increased on the cell surface of migrating EVT, while HLA‐C was internalized. Conclusion Expression of HLA‐F and HLA‐G on the cell surface of actively migrating EVT supports their specific role in early EVT invasion and interactions with uterine natural killer cells. HLA‐C's limited expression to the proliferative EVT suggests a protective role in the earliest events of implantation but not in active EVT invasion. We also show for the first time that HLA‐C may be involved in parturition.
The HELLP syndrome is a pregnancy-associated disease inducing hemolysis, elevated liver, enzymes, and low platelets in the mother. Although the HELLP symptoms occur in the third trimester in the mother, the origin of the disease can be found in the first trimester fetal placenta. A locus for the HELLP syndrome is present on chromosome 12q23 near PAH. Here, by multipoint nonparametric linkage, pedigree structure allele sharing, and haplotype association analysis of affected sisters and cousins, we demonstrate that the HELLP locus is in an intergenic region on 12q23.2 between PMCH and IGF1. We identified a novel long intergenic noncoding RNA (lincRNA) transcript of 205,012 bases with (peri)nuclear expression in the extravillous trophoblast using strand-specific RT-PCR complemented with RACE and FISH. siRNA-mediated knockdown followed by RNA-sequencing, revealed that the HELLP lincRNA activated a large set of genes that are involved in the cell cycle. Furthermore, blocking potential mutation sites identified in HELLP families decreased the invasion capacity of extravillous trophoblasts. This is the first large noncoding gene to be linked to a Mendelian disorder with autosomal-recessive inheritance.
The placenta forms the interface between the maternal and fetal circulation and is critical for the establishment of a healthy pregnancy. Trophoblast cell proliferation, migration and invasion into the endometrium are fundamental events in the initiation of placentation. Leukemia inhibitory factor (LIF) has been shown to promote trophoblast invasion in vitro, however its precise role in trophoblast invasion in vivo is unknown. We hypothesized that LIF would be required for normal trophoblast invasion and spiral artery remodeling in mice. Both LIF and its receptor (LIFR alpha) co-localized with cytokeratin-positive invasive endovascular extravillous trophoblasts (EVT) in mouse implantation sites during mid-gestation. Temporally blocking LIF action during specific periods of placental development via administration of our unique LIFR alpha antagonist, PEGLA, resulted in abnormal trophoblast invasion and impaired spiral artery remodeling compared to PEG control. PEGLA-treated mouse decidual vessels were characterized by retention of alpha-smooth muscle actin (alpha SMA)-positive vascular smooth muscle cells (VSMCs), while PEG control decidual vessels were remodelled by cytokeratin-positive trophoblasts. LIF blockade did not alter F4/80-positive decidual macrophage numbers between treatment groups, but resulted in down-regulation of decidual transcript levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10), which are important immune cell activation factors that promote spiral artery remodeling during pregnancy. Our data suggest that LIF plays an important role in trophoblast invasion in vivo and may facilitate trophoblast-decidual-immune cell cross talk to enable adequate spiral artery remodeling.
Abstract STUDY QUESTION Is it possible to improve vascular remodeling by inhibiting the excessive expression of protease-activated receptor 1 (PAR-1) in trophoblast of abnormal placenta? SUMMARY ANSWER Inhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, offering an alternative therapeutic approach for preeclampsia. WHAT IS KNOWN ALREADY PAR-1 is high-affinity receptor of thrombin. Thrombin increases sFlt-1 secretion in trophoblast via the activation of PAR-1. It is reported that the expression of both thrombin and PAR-1 expression are increased in placentas of preeclampsia patients compared with normal placentas. STUDY DESIGN, SIZE, DURATION Trophoblast cells were transfected with PAR-1 short hairpin RNA (shRNA) or PAR-1 overexpression plasmids in vitro. Tube formation assays and a villus-decidua co-culture system were used to study the effect of PAR-1 inhibition on placental angiogenesis and vascular remodeling, respectively. Placentas from rats with preeclampsia were transfected with PAR-1 shRNA to confirm the effect of inhibiting PAR-1 overexpression in placenta. PARTICIPANTS/MATERIALS, SETTING, METHODS The trophoblast cell line HTR-8/SVneo was transfected with PAR-1 shRNA or PAR-1 overexpression plasmids. After 48 h, supernatant was collected and the level of sFlt-1 secretion was measured by ELISA. Human umbilical cord epithelial cells and a villus-decidua co-culture system were treated with conditioned media to study the effect of PAR-1 inhibition on tube formation and villi vascular remodeling. A preeclampsia rat model was established by intraperitoneal injection of L-NAME. Plasmids were injected into the placenta of the preeclampsia rats and systolic blood pressure was measured on Days 15 and 19. The effect of different treatments was evaluated by proteinuria, placental weights, fetal weights and fetal numbers in study and control groups. The level of serum sFlt-1 in rats with preeclampsia was also measured. Changes in the placenta microvessels were studied by histopathological staining. MAIN RESULTS AND THE ROLE OF CHANCE PAR-1 shRNA inhibited PAR-1 expression and significantly suppressed sFlt-1 expression in trophoblasts. Soluble Flt-1 level in the supernatant was suppressed by PAR-1 inhibition plasmid transfection and increased by PAR-1 overexpression plasmids (46.93 ± 5.22 vs. 25.21 ± 4.18 vs. 67.84 ± 3.58 ng/ml, P < 0.01). Tube formation assays showed that conditioned media from shPAR-1 transfected cells resulted in an increase in the total number of branching points compared with that of blank controls (P < 0.05). The villus-decidua co-culture system confirmed down-regulation of PAR-1 was conducive to angiogenesis and vascular remodeling. Transfecting placenta with PAR-1 shRNA plasmids improved placental vascular development and ameliorated the symptoms of preeclampsia in rats. After treatment with shRNA, blood pressure was controlled (140.83 ± 1.08 vs. 123.6 ± 1.47 mmHg, P < 0.001) and proteinuria levels were decreased (4.48 ± 0.36 vs. 2.64 ± 0.25 μg/μl, P < 0.01). sFlt-1 protein levels were significantly higher in preeclampsia group than in the control group (1.44 ± 0.33 vs. 2.92 ± 0.85 ng/ml, P < 0.001), but was reduced (0.92 ± 0.06 ng/ml, vs. PE, P < 0.001) in the treatment group. The histopathological changes of the placental microvessels showed that in the preeclampsia group, the number of blood vessels was reduced, while in treatment group, the placental microvasculature was improved (P < 0.001). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Despite our promising results, the evaluation of kidney damage was studied only by proteinuria measurement. Histochemistry of kidney damage will be supplemented in a further study. WIDER IMPLICATIONS OF THE FINDINGS The data showed that inhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, potentially offering an alternative therapeutic approach for preeclampsia. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81100442 and 81771605 for Y.Z. and 81179584 for L.Z.) and the Hubei Province Health and Family Planning Scientific Research Project (Grant No. WJ2017 M093 for Y.Z.). The authors declare that there is no conflict of interest.
We aimed to determine the effect of expression on the expression profile of long noncoding RNAs (lncRNAs) in trophoblasts, and we studied the involvement of certain lncRNAs and in the pathogenesis of recurrent miscarriage (RM). RT2 lncRNA PCR arrays revealed that YY1 overexpression in trophoblasts significantly promoted the expression of the HOX transcript antisense RNA HOTAIR and demonstrated that HOTAIR expression was significantly lower in the RM trophoblasts than in control trophoblasts. Ectopic HOTAIR overexpression and knockdown experiments revealed that it was a novel target of . Bioinformatics analysis identified two YY1-binding sites in the HOTAIR promoter region, and chromatin immunoprecipitation (ChIP) analysis verified that YY1 binds directly to its promoter region. Interestingly, HOTAIR overexpression enhanced trophoblast invasion in an ex vivo explant culture model, while its knockdown repressed these effects. Furthermore, liquid chromatography-tandem mass spectrometry (LC-MS/MS) label-free quantitative proteomics screening revealed that HOTAIR overexpression activated phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling in trophoblasts. In an ex vivo explant culture model, HOTAIR overexpression effectively elevated matrix metalloproteinase 2 (MMP2) expression via the PI3K-AKT signaling pathway, enhancing trophoblast migration and invasion. These findings reveal a new regulatory pathway in which activates PI3K-AKT signaling via HOTAIR, promoting MMP2 expression, suggesting that HOTAIR is a potential therapeutic target for RM. Zhang et al. found that HOTAIR plays a key role in trophoblast invasion and migration and further identified a new regulatory pathway in which YY1 activates PI3K-AKT signaling via HOTAIR, promoting MMP2 expression, suggesting that HOTAIR is a potential therapeutic target for recurrent miscarriage.