Giant Cell Differentiation Research Articles

Hypoxia-Inducible Factor 1alpha (HIF1A) Mediates Distinct Steps of Rat Trophoblast Differentiation in Gradient Oxygen1

Defective differentiation of invasive, placental trophoblast cells has been associated with several pregnancy-related disorders. This study examines the molecular, functional, and morphological differentiation of lineage-specific, trophoblast giant cells under a gradient of oxygen concentrations. Low oxygen (3%) inhibited differentiation, but this inhibition was relieved in a stepwise fashion with increasing levels of oxygen. The oxygen-sensitive hypoxia-inducible factor 1alpha (HIF1A) is a major transcriptional regulator of the cellular response to low oxygen, and increased HIF1A protein levels and activity corresponded with the maintenance of the stem cell-like state and inhibition of trophoblast differentiation in low oxygen. Furthermore, constitutive expression of an oxygen-insensitive, active form of HIF1A protein mimicked the effects of low oxygen, inhibiting the differentiation of trophoblast giant cells. This study is the first to delineate the stepwise effects of oxygen on the activation of the trophoblast giant cell differentiation process and establishes a new paradigm from which to investigate trophoblast differentiation. In addition, this is the first reported study to demonstrate that constitutive HIF1A activity mediates oxygen's inhibition of differentiation. These results suggest that a dysregulation of HIF1A could contribute to impaired placental development.

Cathepsin proteases have distinct roles in trophoblast function and vascular remodelling

Trophoblast giant cells are instrumental in promoting blood flow towards the mouse embryo by invading the uterine endometrium and remodelling the maternal vasculature. This process involves the degradation of the perivascular smooth muscle layer and the displacement of vascular endothelial cells to form trophoblast-lined blood sinuses. How this vascular remodelling is achieved at the molecular level remains largely elusive. Here, we show that two placenta-specific cathepsins, Cts7 and Cts8, are expressed in distinct but largely overlapping subsets of giant cells that are in direct contact with maternal arteries. We find that Cts8, but not Cts7, has the capacity to mediate loss of smooth muscle alpha-actin and to disintegrate blood vessels. Consequently, conditional ubiquitous overexpression of Cts8 leads to midgestational embryonic lethality caused by severe vascularization defects. In addition, both cathepsins determine trophoblast cell fate by inhibiting the self-renewing capacity of trophoblast stem cells when overexpressed in vitro. Similarly, transgenic overexpression of Cts7 and Cts8 affects trophoblast proliferation and differentiation by prolonging mitotic cell cycle progression and promoting giant cell differentiation, respectively. We also show that the cell cycle effect is directly caused by some proportion of CTS7 localizing to the nucleus, highlighting the emerging functional diversity of these typically lysosomal proteases in distinct intracellular compartments. Our findings provide evidence for the highly specialized functions of closely related cysteine cathepsin proteases in extra-embryonic development, and reinforce their importance for a successful outcome of pregnancy.

Leukemia Inhibitory Factor Regulates Trophoblast Giant Cell Differentiation via Janus Kinase 1-Signal Transducer and Activator of Transcription 3-Suppressor of Cytokine Signaling 3 Pathway

Suppressor of cytokine signaling 3 (SOCS3) inhibits leukemia-inhibitory factor (LIF) signaling and acts as a negative regulator. Deletion of SOCS3 causes embryonic lethality because of placental failure, and genetic reduction of LIF or the LIF receptor (LIFR) in SOCS3-deficient mice rescues placental defects and embryonic lethality; this indicates that SOCS3 is an essential inhibitor of LIFR signaling. However, the downstream signaling molecule that acts as a link between the LIFR and SOCS3 has not been identified. In this study we explored the downstream signaling of LIFR. The administration of LIF to SOCS3-heterozygous pregnant mice promotes trophoblast giant cell differentiation and accelerates placental failure in SOCS3-deficient mice. SOCS3-deficient trophoblast stem cells show enhanced and prolonged signal transducer and activator of transcription 3 (Stat3) activation by LIF stimulation. Further, in the trophoblasts of SOCS3-deficient placenta and differentiating cells from the choriocarcinoma-derived cell line Rcho-1 cells, constitutive activation of Stat3 is observed. The forced expression of SOCS3, dominant-negative Stat3, and dominant-negative Janus kinase 1 (JAK1) in Rcho-1 cells significantly suppressed the trophoblast giant cell differentiation of these cells. In addition, the number of trophoblast giant cells is significantly reduced concomitant with an increased number of precursor trophoblasts in JAK1-deficient placentas. Finally, JAK1 deficiency rescues placental defects and embryonic lethality in SOCS3-deficient mice. These results indicate that the LIFR signaling is finely coordinated by JAK1, Stat3, and SOCS3 and regulates trophoblast giant cell differentiation. In addition, these data establish that LIFR-JAK1-Stat3-SOCS3 signaling is an essential pathway for the regulation of trophoblast giant cell differentiation.

Placental Steroids in Cattle: Hormones, Placental Growth Factors or By-products of Trophoblast Giant Cell Differentiation?

The bovine placenta produces large amounts of steroids, mainly estrone (E1) and progesterone (P4). Specific features of bovine placental steroidogenesis are 1) the expression of all enzymes needed for the production of estrogens from cholesterol in the trophoblast 2) an only marginal and temporal contribution to peripheral maternal P4 levels restricted to a period between approx. days 150 - 240 of gestation 3) the predominance of sulfoconjugated over free E1 and 4) a complementary setting of steroidogenic enzymes in the two morphologically discriminable trophoblast cell types, the uninucleated trophoblast cells (UTC) and the trophoblast giant cells (TGC). In cattle so far no definite information is available on the specific biological roles of placental estrogens and P4. However, the detection of estrogen receptors and progesterone receptors in the placentomes suggests a role primarily as local regulators of caruncular growth, differentiation and functions. Inconsistent with a function as a caruncular growth factor is the strong evidence that in cattle placental estrogens enter the maternal compartment almost completely as estrone sulfate (E1S), which is not active at classical nuclear receptors. On the other hand, E1S may be converted locally to free active estrogens via the action of steroid sulfatase (StS), which has been detected in specific parts of the bovine caruncular epithelium. Alternatively or in addition, StS expression in the caruncular epithelium may serve the utilization of sulfated neutral steroid precursors (e.g. pregnenolone sulfate or cholesterol sulfate) supplied with maternal blood, thus providing free substrates for further metabolization in the adjacent trophoblast. The down-regulation of P450scc and P450c17 and the up-regulation of 3beta-HSD and aromatase during the differentiation of TGC from UTC in parallel with the up-regulation of ER beta and estrogen sulfotransferase in maturing TGC suggests a function of placental estrogens primarily as autoor intracrine regulators during this process and assigns to conjugated placental estrogens a role as inactivated by-products of TGC differentiation intended for excretion. Collectively, despite some evidence from recent studies for putative roles of placental steroids in cattle their exact functions in the bovine species remain still undefined.

Hand it to the nucleolus

Long viewed as merely a biofactory for ribosomes, the nucleolus has recently come to be seen as a multifunctional and dynamic subnuclear organelle. New support for this view comes from a study by David Martindill, Paul Riley (UCL Institute of Child Health, London, UK), and colleagues, who show that a cell fate regulator is held inactive in the nucleolus until phosphorylation releases it to trigger differentiation. Figure 1 The release of Hand1 (green) from the nucleolus coincides with giant cell differentiation (top to bottom). Differentiation of mouse trophoblast stem cells into a specialized cell type called giant cells requires Hand1, a bHLH transcription factor. Hand1 interacts with a wider variety of other bHLH partners than do others of its class. The authors thus wondered whether it might also interact with unrelated partners that help it time giant cell differentiation. Using a yeast two-hybrid approach, they found that Hand1 bound to a nucleolar subunit of a protein called HIC. While Hand1 hung out with HIC in the nucleolus, trophoblast cells did not differentiate. But Hand1 exited the nucleolus at the time of their differentiation to giant cells. This exit required the phosphorylation of Hand1. The kinase that phosphorylated Hand1, called Polo-like kinase 4, is evolutionarily conserved. Phosphorylation-dependent release of transcription factors from the nucleolus may turn out to be a widespread mechanism to control their activity. Reference: Martindill, D.M.J., et al. 2007. Nat. Cell Biol. doi:.10.1038/ncb1633 [PubMed] [Cross Ref]

Metachronus malignant rhabdoid tumor of the ileum and adenocarcinoma of lung: a unique case report

Malignant extrarenal rhabdoid tumors had been described in a variety of anatomic locations including gastrointestinal tract. Carcinomas of small intestine are quite uncommon neoplasms, and those with rhabdoid differentiation are exceedingly rare. These poorly differentiated tumors pose a great deal of difficulty in diagnosis as well as in deciding whether they are primary or metastatic in origin because malignant rhabdoid tumors or carcinomas with rhabdoid differentiation of other sites can metastasize to the small intestine. They behave more aggressively than the typical adenocarcinomas of the same location. Herein, we report a 52-year-old patient with primary small bowel malignant rhabdoid tumor, who was completely disease-free after the initial presentation and management. Five years later, he was found to have a lung mass proved to be adenocarcinoma, exhibiting focal giant cell differentiation without rhabdoid features.

Deletion of the orphan nuclear receptor COUP-TFII in uterus leads to placental deficiency

COUP-TFII (NR2F2), chicken ovalbumin upstream promoter-transcription factor II, is an orphan nuclear receptor of the steroid/thyroid hormone receptor superfamily. The Coup-tfII-null mutant mice die during the early embryonic development because of angiogenesis and heart defects. To analyze the physiological function of COUP-TFII during organogenesis, we used the cre/loxP system to conditionally inactivate COUP-TFII in the ovary and uterus. Homozygous adult female mutants with specific inactivation of the Coup-tfII gene in uterine stromal and smooth muscle cells have severely impaired placental formation, leading to miscarriage at days 10-12 of pregnancy. Deletion of the Coup-tfII gene resulted in an increase in trophoblast giant cell differentiation, a reduction of the spongiotrophoblast layer, and an absence of labyrinth formation causing an improper vascularization of the placenta. This study describes an important maternal role of COUP-TFII in regulating the placentation. The endometrial COUP-TFII might modulate the signaling between the uterus and the extraembryonic tissue for the proper formation of the placenta.

Gradient oxygen differentially regulates trophoblast giant cell differentiation

Preeclampsia is a condition associated with approximately 7–10% of all pregnancies and is known to induce maternal hypertension, increasing the risk for IUGR and premature birth. An important feature of preeclampsia is altered differentiation of invading placental trophoblast cells. While early trophoblast development takes place in severe hypoxia, trophoblast cells encounter an increasing oxygen gradient as they migrate and invade the maternal tissues. In this study, we examined the functional, morphological, and molecular differentiation of Rcho-1 trophoblast cells under a range of oxygen concentrations. Severe hypoxia maintained the expression of trophoblast stem cell marker Id2 and inhibited the induction of palladin and CSH1 protein, and formation of stress fibers. Moderate hypoxia, however, was unable to sustain Id2 expression and low levels of palladin protein induction and stress fiber formation was observed. HIF-1alpha, a potential transcriptional mediator in low oxygen conditions exhibited constitutive mRNA expression under all oxygen conditions. In contrast, HIF-1alpha protein levels and reporter activity decreased with increasing oxygen levels. These results suggest that an oxygen gradient differentially modulates aspects of trophoblast giant cell differentiation in a stepwise fashion and that HIF-1alpha may play a key role in oxygen’s inhibition of trophoblast differentiation. This work was supported by NICHD, National Institute of Health Grant (HD045750, T.L.B).

Hypoxia Represses the Differentiation of Rcho-1 Rat Trophoblast Giant Cells

Background: A hypoxic environment is known to be essential for early placentation. A low oxygen tension induces hypoxia-inducible factor-1 (HIF-1α) which may play an important role as a transcription factor in maintaining the proliferative and undifferentiated phenotype in human trophoblasts. Methods: We analyzed the effect of a low oxygen tension on the rat trophoblast giant cell differentiation pathway using Rcho-1 cells which were derived from rat choriocarcinomas and consist of trophoblast stem cells. Results: We found that a low oxygen tension suppressed the morphological changes and steroidogenesis during differentiation. The anticipated downregulation of the Id-1 transcription factor, a negative regulator of trophoblast giant cell differentiation, was not observed in the hypoxic environment. On the other hand, deferoxamine, which mimics hypoxia and induces HIF-1α, caused downregulation of Id-1 transcription factor and trophoblast giant cell differentiation. Conclusion: These results indicate that hypoxia represses rat trophoblast giant cell differentiation via an HIF-1α-independent pathway.

Endogenous retroviruses regulate periimplantation placental growth and differentiation.

Endogenous retroviruses (ERVs) are fixed and abundant in the genomes of vertebrates. Circumstantial evidence suggests that ERVs play a role in mammalian reproduction, particularly placental morphogenesis, because intact ERV envelope genes were found to be expressed in the syncytiotrophoblasts of human and mouse placenta and to elicit fusion of cells in vitro. We report here in vivo and in vitro experiments finding that the envelope of a particular class of ERVs of sheep, endogenous Jaagsiekte sheep retroviruses (enJSRVs), regulates trophectoderm growth and differentiation in the periimplantation conceptus (embryo/fetus and associated extraembryonic membranes). The enJSRV envelope gene is expressed in the trophectoderm of the elongating ovine conceptus after day 12 of pregnancy. Loss-of-function experiments were conducted in utero by injecting morpholino antisense oligonucleotides on day 8 of pregnancy that blocked enJSRV envelope protein production in the conceptus trophectoderm. This approach retarded trophectoderm outgrowth during conceptus elongation and inhibited trophoblast giant binucleate cell differentiation as observed on day 16. Pregnancy loss was observed by day 20 in sheep receiving morpholino antisense oligonucleotides. In vitro inhibition of the enJSRV envelope reduced the proliferation of mononuclear trophectoderm cells isolated from day 15 conceptuses. Consequently, these results demonstrate that the enJSRV envelope regulates trophectoderm growth and differentiation in the periimplantation ovine conceptus. This work supports the hypothesis that ERVs play fundamental roles in placental morphogenesis and mammalian reproduction.

Sox15 enhances trophoblast giant cell differentiation induced by Hand1 in mouse placenta

Some members of the Sry-type HMG box (Sox) protein family play important roles in embryogenesis as transcription factors. Here, we report that Sox15 transcripts were much more abundant in mouse placenta than in the fetus, the yolk sac, or several adult tissues. In situ hybridization analysis of the mouse E8.0 conceptus indicated that Sox15 mRNA was predominantly expressed in the trophoblast giant cells of the placenta. We also observed that the amount of Sox15 mRNA dramatically increased during the differentiation of mouse trophoblast stem cells. Ectopic expression of Sox15 in Rat choriocarcinoma cells enhanced the giant cell differentiation induced by a bHLH transcription factor, Hand1. Binding experiments in cotransfected 293 T cells and in vitro revealed that Sox15 interacted with Hand1. We next examined the effects of this interaction on the transcriptional activity of Hand1 and Sox15 using the luciferase reporter assay. Overexpression of Hand1 repressed the Sox15-driven reporter expression, but Sox15 enhanced the Hand1-driven transcription. This enhancement required both the Hand1-binding region and the transactivation domain of Sox15. These results may suggest that the increased transcriptional activity of Hand1 caused by Sox15 might promote the transcription of the target gene resulting in the trophoblast giant cell differentiation in the mouse placenta.

Differentiation of Trophoblast Giant Cells and Their Metabolic Functions Are Dependent on Peroxisome Proliferator-Activated Receptor β/δ

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

Hypoxia Inhibits Differentiation of Lineage-Specific Rcho-1 Trophoblast Giant Cells1

Defects in placental development lead to pregnancies at risk for miscarriage and intrauterine growth retardation and are associated with preeclampsia, a leading cause of maternal death and premature birth. In preeclampsia, impaired placental formation has been associated with alterations in a specific trophoblast lineage, the invasive trophoblast cells. In this study, an RT-PCR Trophoblast Gene Expression Profile previously developed by our laboratory was utilized to examine the lineage-specific gene expression of the rat Rcho-1 trophoblast cell line. Our results demonstrated that Rcho-1 cells represent an isolated, trophoblast population committed to the giant cell lineage. RT-PCR analysis revealed that undifferentiated Rcho-1 cells expressed trophoblast stem cell marker, Id2, and trophoblast giant cell markers. On differentiation, Rcho-1 cells downregulated Id2 and upregulated Csh1, a marker of the trophoblast giant cell lineage. Neither undifferentiated nor differentiated Rcho-1 cells expressed spongiotrophoblast marker Tpbpa or labyrinthine markers Esx1 and Tec. Differentiating Rcho-1 cells in hypoxia did not alter the expression of lineage-specific markers; however, hypoxia did inhibit the downregulation of the trophoblast stem cell marker Id2. Differentiation in hypoxia also blocked the induction of CSH1 protein. In addition, hypoxia inhibited stress fiber formation and abolished the induction of palladin, a protein associated with stress fiber formation and focal adhesions. Thus, Rcho-1 cells can be maintained as a proliferative, lineage-specific cell line that is committed to the trophoblast giant cell lineage on differentiation in both normoxic and hypoxic conditions; however, hypoxia does inhibit aspects of trophoblast giant cell differentiation at the molecular, morphological, and functional levels.

Hypoxia-Inducible Factors 1α and 2α Regulate Trophoblast Differentiation

ABSTRACT Placental development initially occurs in a low-oxygen (O 2 ) or hypoxic environment. In this report we show that two hypoxia-inducible factors (HIFs), HIF1α and HIF2α, are essential for determining murine placental cell fates. HIF is a heterodimer composed of HIFα and HIFβ (ARNT) subunits. Placentas from Arnt − / − and Hif1α − / − Hif2α −/− embryos exhibit defective placental vascularization and aberrant cell fate adoption. HIF regulation of Mash2 promotes spongiotrophoblast differentiation, a prerequisite for trophoblast giant cell differentiation. In the absence of Arnt or Hifα , trophoblast stem cells fail to generate these cell types and become labyrinthine trophoblasts instead. Therefore, HIF mediates placental morphogenesis, angiogenesis, and cell fate decisions, demonstrating that O 2 tension is a critical regulator of trophoblast lineage determination. This novel genetic approach provides new insights into the role of O 2 tension in the development of life-threatening pregnancy-related diseases such as preeclampsia.

Modulation of trophoblast stem cell and giant cell phenotypes: analyses using the Rcho-1 cell model

Trophoblast giant cells are located at the maternal–embryonic interface and have fundamental roles in the invasive and endocrine phenotypes of the rodent placenta. In this report, we describe the experimental modulation of trophoblast stem cell and trophoblast giant cell phenotypes using the Rcho-1 trophoblast cell model. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate into trophoblast giant cells. Differentiated Rcho-1 trophoblast cells are invasive and possess an endocrine phenotype, including the production of members of the prolactin (PRL) family. Dimethyl sulfoxide (DMSO), a known differentiation-inducing agent, was found to possess profound effects on the in vitro development of trophoblast cells. Exposure to DMSO, at non-toxic concentrations, inhibited trophoblast giant cell differentiation in a dose-dependent manner. These concentrations of DMSO did not significantly affect trophoblast cell proliferation or survival. Trophoblast cells exposed to DMSO exhibited an altered morphology; they were clustered in tightly packed colonies. Trophoblast giant cell formation was disrupted, as was the expression of members of the PRL gene family. The effects of DMSO were reversible. Removal of DMSO resulted in the formation of trophoblast giant cells and expression of the PRL gene family. The phenotype of the DMSO-treated cells was further determined by examining the expression of a battery of genes characteristic of trophoblast stem cells and differentiated trophoblast cell lineages. DMSO treatment had a striking stimulatory effect on eomesodermin expression and a reciprocal inhibitory effect on Hand1 expression. In summary, DMSO reversibly inhibits trophoblast differentiation and induces a quiescent state, which mimics some but not all aspects of the trophoblast stem cell phenotype.

STAT3-mediated Constitutive Expression of SOCS3 in an Undifferentiated Rat Trophoblast-like Cell Line

In the trophoblast, constitutive expression of SOCS3 is important for the negative regulation of trophoblast giant cell differentiation. In this study, we analyzed the signaling pathway regulating the constitutive SOCS3 expression in undifferentiated Rcho-1 cells, which were derived from rat choriocarcinoma and consist of trophoblast stem cells that are capable of differentiating to trophoblast giant cells in vitro. PD98059, an MEK inhibitor, repressed the SOCS3 expression but AG490, a JAK2 inhibitor, did not. Promoter deletion analysis revealed that the STAT response element (SRE) in the SOCS3 promoter is necessary for the promoter activity. Overexpression of STAT3 increased the SOCS3 promoter activity, whereas expression of dominant-negative STAT3 reduced it. Constitutive STAT3 tyrosine phosphorylation that was not inhibited by either AG490 or PD98059 was demonstrated. Electrophoretic mobility shift assays showed the existence of a protein that bound to SRE and was supershifted with STAT3 antibody. This binding reaction was inhibited by neither AG490 nor PD98059. These findings imply that the ERK/MAPK pathway and STAT3 are involved in the constitutive activation of SOCS3 in undifferentiated Rcho-1 cells. Moreover, they indicate that the constitutive STAT3 tyrosine phosphorylation and the DNA binding activity of STAT3 do not depend on the ERK/MAPK or JAK kinase pathway. These results suggest that a trophoblast-specific STAT3 activation pathway is important for the regulation of giant cell differentiation.

PTHrP promotes murine secondary trophoblast giant cell differentiation through induction of endocycle, upregulation of giant-cell-promoting transcription factors and suppression of other trophoblast cell types

The murine trophoblast cell lineage represents an intriguing experimental cell model as it is composed of four trophoblast stem (TS)-derived cell types: trophoblast giant cells (TGCs), spongiotrophoblast, syncytotrophoblast, and glycogen trophoblast cells. To investigate the role of parathyroid hormone-related protein (PTHrP) in TGC differentiation, we analyzed the effect of exogenous PTHrP on secondary TGCs of day 8.5 p.c. ectoplacental cone explant culture. Secondary TGCs expressed PTHrP and PTHR1 receptor in vivo and in vitro. TGCs treated with PTHrP had reduced proliferation and decreased apoptosis starting from day 2 in culture, and enhanced properties of giant cell differentiation: increased DNA synthesis, number of cells with giant nuclei and expression of placental lactogen-II (PL-II). The induction of TGC formation by PTHrP correlated with downregulation of cyclin B1 and mSNA expression, but upregulation of cyclin D1, thus allowing mitotic-endocycle transition. Moreover, PTHrP treatment influenced TGC differentiation by inducing the expression of transcription factors known to stimulate giant cell formation: Stra13 and AP-2γ, and inhibiting the formation of other trophoblast cell types by suppressing trophoblast progenitors and spongiotrophoblast-promoting factors, Eomes, Mash-2, and mSNA. Taken together with the spatial and temporal patterns of TGC formation and PTHrP synthesis in vivo, these findings indicate an important role for PTHrP in the differentiation of secondary TGCs during placentation.

Trophoblast stem cells differentiate in vitro into invasive trophoblast giant cells

Trophoblast cells are characterized by an invasive behavior into the surrounding uterine tissue. In rodents, an early peri-/endovascular type of invasion exerted by trophoblast giant cells can be distinguished from a late interstitial type carried out by glycogen trophoblast cells. Analysis of the molecular mechanisms of trophoblast invasion has been hampered, however, by the complex temporal and spatial patterns of invasion. We utilized trophoblast stem (TS) cell lines to study trophoblast invasion in vitro and to establish a model that facilitates investigation of this process on the molecular level. Our results showed that trophoblast giant cells that differentiate from TS cells in vitro are capable of penetrating a reconstituted basement membrane matrix. Consequently, invasion rates were increased in various giant cell differentiation-promoting conditions. We also derived TS cell lines that are homozygous for a mutation of the Hand1 transcription factor. The Hand1 −/− TS cells showed reduced levels of giant cell differentiation and exhibited an approximately 50% decrease in invasion rates. In summary, trophoblast giant cells that differentiate from TS cells in vitro recapitulate the invasive capacity of normal trophoblast cells in vivo. The TS cell system is a valuable tool to identify and quantitatively study regulators of trophoblast invasion.

Role of thrombocytosis in diagnosis of giant cell arteritis and differentiation of arteritic from non-arteritic anterior ischemic optic neuropathy.

To investigate the role of thrombocytosis in the diagnosis of giant cell arteritis (GCA), and differentiation of arteritic (A-AION) from non-arteritic (NA-AION) anterior ischemic optic neuropathy; and comparison of the sensitivity and specificity of platelet count to that of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and some other hematologic variables in the diagnosis of GCA. This retrospective study is based on 121 temporal artery biopsy confirmed GCA patients and 287 patients with NA-AION seen in our clinic. For inclusion in this study, all GCA patients, at their initial visit, prior to the initiation of corticosteroid therapy, must have had ESR (Westergren), platelet count and complete blood count, and temporal artery biopsy. From 1985 onwards CRP estimation was done. For inclusion in this study, all NA-AION patients at the initial visit must have undergone evaluation similar to that described above for GCA, except for temporal artery biopsy. Wilcoxon rank-sum test and the two-sample t-test were used to compare hematologic variables between GCA patients with and without visual loss, between those with and without systemic symptoms, and also between GCA and NA-AION patients. Pearson correlation coefficient was computed to measure the association of platelet counts and the other hematologic variables with ESR. Receiver operating characteristic (ROC) curves were constructed for ESR, CRP, platelet count, combinations of ESR and platelet count, and CRP and platelet count, hemoglobin, hematocrit, and white blood cell (WBC) count and the area under the curve (AUC) were compared. Comparison of ESR, CRP, and hematologic variables of GCA patients and of A-AION with the NA-AION group, showed significantly (p <0.0001) higher median levels of ESR, CRP, platelet count, and WBC count and lower levels of hemoglobin and hematocrit in the GCA patients and A-AION than in NA-AION. Comparing AUC of the ROC curve between ESR and platelet count, ESR was a better predictor of GCA compared to platelet count (AUC of 0.946 vs. 0.834). There was a slight improvement in prediction of GCA using the combination of ESR and platelet count (AUC=0.953). The other hematologic variables had an AUC that was smaller than platelet count (0.854 for hemoglobin; 0.841 for hematocrit), with WBC being the least predictive of GCA (AUC=0.666). The AUC of the ROC curve for CRP was 0.978. There was no improvement in prediction of GCA using platelet count in combination with CRP (AUC=0.976). Patients with GCA had significantly (p <0.0001) higher values of platelet count, ESR, CRP and WBC but lower values for hemoglobin and hematocrit compared to the NA-AION group. Predictive ability of an elevated platelet count did not surpass elevated ESR or CRP as a diagnostic marker for GCA. Thrombocytosis may complement ESR. Hemoglobin, hematocrit and WBC were much less predictive of GCA. Elevated CRP had a greater predictive ability for GCA compared to ESR or the other hematologic parameters; thrombocytosis in combination with CRP did not yield an improvement in prediction of GCA.

Trophoblast stem cells differentiate in vitro into invasive trophoblast giant cells

Trophoblast cells are characterized by an invasive behavior into the surrounding uterine tissue. In rodents, an early peri-/endovascular type of invasion exerted by trophoblast giant cells can be distinguished from a late interstitial type carried out by glycogen trophoblast cells. Analysis of the molecular mechanisms of trophoblast invasion has been hampered, however, by the complex temporal and spatial patterns of invasion. We utilized trophoblast stem (TS) cell lines to study trophoblast invasion in vitro and to establish a model that facilitates investigation of this process on the molecular level. Our results showed that trophoblast giant cells that differentiate from TS cells in vitro are capable of penetrating a reconstituted basement membrane matrix. Consequently, invasion rates were increased in various giant cell differentiation-promoting conditions. We also derived TS cell lines that are homozygous for a mutation of the Hand1 transcription factor. The Hand1 −/− TS cells showed reduced levels of giant cell differentiation and exhibited an approximately 50% decrease in invasion rates. In summary, trophoblast giant cells that differentiate from TS cells in vitro recapitulate the invasive capacity of normal trophoblast cells in vivo. The TS cell system is a valuable tool to identify and quantitatively study regulators of trophoblast invasion.