Study design.

<p>Experimental design applied to identify sex-biased gene expression in four placental cell types: syncytiotrophoblast (SCT), cytotrophoblasts (CT), arterial (AEC) and venous endothelial cells (VEC), two main placental compartments (trophoblast epithelium and villous vessel endothelium), and in the whole placental villi. The cells were isolated from placentas of male and female fetuses and their mRNA subjected to microarray analysis by hybridization to Affymetrix GeneChip Human 1.0 ST arrays. Genes that reached significance (p<0.05) and with a fold-change >1.3 were considered differentially expressed between the two sexes. Placental villi refers to the combined analysis of all expressed transcripts in syncytiotrophoblast, cytotrophoblasts, arterial and venous endothelial cells comparing gene expression in male vs. female cells. Microarray data were validated by RT-qPCR a) using identical samples as used in the microarray analysis and b) in different isolations cultured independently.</p

Chromosomal distribution of genes showing sex-biased expression in placental endothelium and epithelium.

<p>The number of male- and female-biased genes (p<0.05, fold-change >1.3) is shown for each chromosome separately. The number on top of the bars indicates the number of genes with fold-change >2. The proportion of genes located on autosomal vs. sex chromosomes is given on the top right and left of each graphs, respectively. An ideogram at the right depicts the specific location of upregulated genes in males (blue line) and females (red line) on X and Y chromosomes.</p

Validation of microarray data with RT-qPCR.

<p>FC =  fold-change is the ratio of mean expression for male vs. female cells. N. C. =  no change. N.T. =  not tested. SCT =  syncytiotrophoblasts, CT =  cytotrophoblasts, AEC =  arterial endothelial cells and VEC =  venous endothelial cells. * =  genes validated using samples distinct from those used in microarray analysis.</p

The Human Placental Sexome Differs between Trophoblast Epithelium and Villous Vessel Endothelium

<div><p>Molecular mechanisms underlying sexual dimorphism in mammals, fetal sex influences on intrauterine development, and the sex-biased susceptibility for selected diseases in adulthood are novel areas of current research. As importantly, two decades of multifaceted research has established that susceptibility to many adult disorders originates <i>in utero</i>, commonly secondary to the effects of placental dysfunction. We hypothesized that fetal sex influences gene expression and produces functional differences in human placentas. We thus extended previous studies on sexual dimorphism in mammals, which used RNA isolated from whole tissues, to investigate the effects of sex on four cell-phenotypes within a single key tissue, human placental villi. The cells studied included cytotrophoblasts, syncytiotrophoblast, arterial and venous endothelial cells. The cells were isolated from placentas of male or female fetuses and subjected to microarray analysis. We found that fetal sex differentially affected gene expression in a cell-phenotype dependent manner among all four cell-phenotypes. The markedly enriched pathways in males were identified to be signaling pathways for graft-versus-host disease as well as the immune and inflammatory systems that parallel the reported poorer outcome of male fetuses. Our study is the first to compare global gene expression by microarray analysis in purified, characterized, somatic cells from a single human tissue, i.e. placental villi. Importantly, our findings demonstrate that there are cell-phenotype specific, and tissue-specific, sex-biased responses in the human placenta, suggesting fetal sex should be considered as an independent variable in gene expression analysis of human placental villi.</p></div

Top 10 genes showing higher expression in male and top 10 with higher expression in female placental cell types.

<p>FC =  fold-change is the ratio of mean expression for male vs. female cells. SCT =  syncytiotrophoblasts, CT =  cytotrophoblasts, AEC =  arterial endothelial cells and VEC =  venous endothelial cells.</p

Distribution and the level of sex- biased gene expression across autosomal and sex chromosomes in placental endothelium vs. epithelium.

<p>% of genes is given in relation to the total number of genes showing sex biased expression in villous vessel endothelium and trophoblast epithelium, respectively. The average level of global sex differences is calculated across all genes showing significant change in expression (p<0.05) on autosomal vs. sex chromosomes for each placental compartment separately.</p

Top 5 significantly enriched canonical pathways in Ingenuity Pathway Analysis.

<p>The proportion (%) refers to the amount of sex-biased genes found to play a role in the respective pathway.</p

Number of genes showing sex-biased expression in four distinct placenta cell types.

<p>Number of differentially expressed genes (p<0.05, fold-change >1.3) between male and female syncytiotrophoblasts, cytotrophoblasts, arterial and venous endothelial cells, trophoblast epithelium, villous vessel endothelium, and for placental villi is shown as number of genes upregulated in males vs. the number of genes upregulated in females. Placental villi refers to combined analysis of all expressed transcripts in syncytiotrophoblasts, cytotrophoblasts, arterial and venous endothelial cells.</p

Resolving Tumor Heterogeneity: Genes Involved in Chordoma Cell Development Identified by Low-Template Analysis of Morphologically Distinct Cells

<div><p>The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous) cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. <i>UCHL3</i>, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold) and U-CH1 (3.7-fold) cells. The mannosyltransferase <i>ALG11</i> (695-fold) and the phosphatase subunit <i>PPP2CB</i> (18.6-fold) were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. <i>TMEM144</i>, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology.</p></div

Expression analyses of MUG-Chor1 candidate genes in U-CH1 cells.

<p>RT-qPCR was done on AB7900 TaqMan (Applied Biosystems; Foster City, CA). Normalization (<i>GAPDH</i> and <i>ACTB</i>) and statistical analysis was done with GenEx Professional (MultiD Analysis; Version 5.3.5.6; see also 2.7). Cut-off for multiple testing (<i>ALG11</i>, <i>UCHL3</i>, <i>TMEM144</i> and <i>PPP2CB</i>) was p = 0.01274.</p>a<p>calculated as mean values from quadruplicate or triplicate (in case the Cq value could not be defined) biological samples.</p>b<p>Cq values were normalized to <i>GAPDH</i> and <i>ACTB</i> (ΔCq). Differential expression (ΔΔCq) is given as positive (up-regulated in large cells) or negative (down-regulated in large cells) fold change ( = 2^ΔΔCq).</p