Human Extravillous Trophoblasts Penetrate Decidual Veins and Lymphatics before Remodeling Spiral Arteries during Early Pregnancy

<div><p>In humans, the defective invasion of the maternal endometrium by fetal extravillous trophoblasts (EVTs) can lead to insufficient perfusion of the placenta, resulting in pregnancy complications that can put both mother and baby at risk. To study the invasion of maternal endometrium between (W)5.5–12 weeks of gestation by EVTs, we combined fluorescence in situ hybridization, immunofluorescence and immunohistochemistry to determine the presence of (male) EVTs in the vasculature of the maternal decidua. We observed that interstitial mononuclear EVTs directly entered decidual veins and lymphatics from W5.5. This invasion of decidual veins and lymphatics occurred long before endovascular EVTs remodelled decidual spiral arteries. This unexpected early entrance of interstitial mononuclear EVTs in the maternal circulation does not seem to contribute to the materno-placental vascular connection directly, but rather to establish (and expand) the materno-fetal interface through an alternative vascular route.</p></div

Human EVTs penetrate decidual veins early during first trimester (W5.5-W7.5).

<p>(A) Histological sections of decidua basalis at W5.5 used for Azan staining (left panel), immunostained for pKRT and ACTA2 (left-middle panel) and immunostained for pKRT and PECAM1 (right-middle panel). FISH for chrX/chrY (right) was performed in the pKRT/PECAM1-stained sections. White arrows indicate male EVTs invading veins. The bottom row shows magnifications of the dashed boxes in the top row. (B) Consecutive sections of W5.5 decidua basalis were immunostained for pKRT/PECAM1. (C) FISH for chrX and chrY (right panels) magnifications are shown for the section in dashed box. White arrows point to male EVTs penetrating a decidual vein. (D) Histological sections of decidua basalis at W7.2 used for Azan staining (left panel), immunostained for pKRT and PECAM1 (left-middle panel). FISH for chrX/chrY (right panels) was performed in the pKRT/PECAM1-stained sections. The most right panel shows a magnification of the dashed box. White arrows indicate male EVTs invading veins. (E) Percentage of invaded decidual veins per total veins encountered per histological section (n) between W5.5-W12. Results are sown as mean ± standard deviation. All scale bars are depicted.</p

Human EVTs in the vicinity of arteries early during first trimester (W5.5).

<p>Histological sections of W5.5 decidua basalis showing decidual arteries. Consecutive sections were used for Azan staining (left panel), immunostained for pKRT and ACTA2 (left-middle panel) and immunostained for pKRT and PECAM1 (right-middle panel). FISH for chrX and chrY (right panel) was performed in the pKRT/PECAM1-stained sections. The (middle and) bottom rows show magnifications of the (numbered) dashed boxes in the top rows. White arrows depict male EVTs. All scale bars are depicted.</p

Human extravillous trophoblast cells invade decidual veins and lymphatics early during first trimester.

<p>(A) Histological sections of decidua basalis from a (mosaic) Klinefelter syndrome (47,XXY and 46, XY) pregnancy at W8.4. Left panels show low magnifications of same section immunostained for pKRT and PECAM1 (top) and chrX/chrY FISH (bottom). Middle-left and middle-right panels are magnifications of the (numbered) dashed boxes in the left panels. Right panels are magnifications of the dashed boxes in the middle-right panels. White arrowheads point to XXY fetal EVTs. (B) Histological sections of decidua basalis at W7.2, W8.4 and W10 immunostained for pKRT, PDPN and PECAM1 (left panels). FISH for chrX and chrY (right) was performed in the in the pKRT/PDPN/PECAM1-stained sections. White arrows point to EVTs invading the lymphatic vessels. All scale bars are depicted.</p

Gene expression signature of osteoblastoma.

<p>(A) Unsupervised principal component analysis based on the expression of the 1297 most variable genes (σ/σ_max = 0.3) shows that the five osteoblastomas form a group that has an expression profile separate from the osteosarcomas, mesenchymal stem cells, and osteoblasts differentiated in vitro from mesenchymal stem cells. The first three principal components, representing 34%, 10%, and 9% of the variance, are displayed. Lines connect the three nearest neighbors. By subsequently comparing osteoblastoma and osteosarcoma, 140 genes showed a significantly different expression (<i>p</i> < 0.001, FDR < 0.01; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080725#pone.0080725.s002" target="_blank">Tables S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080725#pone.0080725.s003" target="_blank">S3</a>). (B) The differentially expressed genes are displayed in a heat map. Genes with high and low expression values are labeled in red and green, respectively. (C) Many of the highly expressed genes in osteoblastoma are known to be involved in bone metabolism and at least four of them are induced by the Wnt/beta-catenin signaling pathway; <i>BMP2, BMP4, PTGS2</i> and <i>MMP16</i>. Boxes range from the 25^th to the 75^th percentile. The box whiskers are set at the lowest data point value still within 1.5 times the box range of the lower box limit, and at the highest data point value still within 1.5 times the box range of the upper box limit. The median is displayed as a dotted band. Outliers are defined as data point values falling outside of the box whisker limits.</p

Recurrent deletions in osteoblastoma affect chromosome 22.

<p>By SNP array analyses, hemi-and homozygous deletions affecting chromosome 22 were found in cases 1 and 2. The upper parts of the figure display SNP array plots of the respective case and the lower part show a summary of the losses where blue and green bars represent hemi- and homozygous deletions, respectively. The locations of four homozygously lost genes implicated in bone formation and/or tumorigenesis are shown. </p

Additional file 3: of Mesenchymal stromal cells of osteosarcoma patients do not show evidence of neoplastic changes during long-term culture

Figure S2. Representative example of karyotypic analysis of early passage MSCs (OS patient derived MSC003OS; 46,XX)

Human extravillous trophoblast cells in decidua between W8-W12.

<p>(A) Histological sections of decidua basalis at W8.4 used for Azan staining (left panel), immunostained for pKRT and PECAM1 (left-middle panel). FISH for chrX/chrY (right panels) was performed in the pKRT/PECAM1-stained sections. The most right panel shows a magnification of the dashed box. White arrows indicate male EVTs invading veins. (B) Histological sections of decidua basalis at W10 used for Azan staining (left panel), immunostained for pKRT and ACTA2 (left-middle panel) and immunostained for pKRT and PECAM1 (right-middle panel). FISH for chrX/chrY (right) was performed in the pKRT/PECAM1-stained sections. White arrows indicate male EVTs. Low magnifications are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169849#pone.0169849.s002" target="_blank">S2 Fig</a>. (C) Histological sections of W10 and W12 decidua basalis showing remodeled and unremodelled arteries. Consecutive sections were immunostained for pKRT and ACTA2 (left panels) and immunostained for pKRT and PECAM1 (middle panels). FISH for chrX and chrY (right panels) was performed in the pKRT/PECAM1-stained sections. The bottom rows show magnifications of the dashed boxes in the top rows. All scale bars are depicted.</p

Additional file 2: of Mesenchymal stromal cells of osteosarcoma patients do not show evidence of neoplastic changes during long-term culture

Figure S1. Differentiation capacity and expression of membranous markers of MSCs. Representative examples are shown (40× magnification). A, chondrogenic differentiation was performed using cell pellet cultures and assessed using toluidine blue staining. Adipogenic differentiation was assessed using phase contrast microscopy (B) and Oil-red-O staining (C). Osteogenic differentiation was assessed using phase contrast microscopy (D) and Alizarin red staining (E). F, all samples expressed CD73, CD90 and CD105 (bold histograms) as determined by flow cytometry (isotype controle staining shown for comparison)

The metastable-segregation-unit working model.

<p>(A) Schematic representation of the mitotic consequences of a stable heteroplasmic mtDNA segregation unit. The hypothetical founding cell is 50% heteroplasmic with 72 mtDNA molecules arranged in 9 segregation units each with 8 mtDNA’s, 4 of which are variant. Upon their faithful replication and partitioning to daughter cells for N mitosis, all 2^N descendant cells acquire the same 50% heteroplasmy level. (B) Schematic representation of the mitotic consequences after a metastable event in a cell from (A) such that one of its units has 6 instead of 4 variant mtDNA molecules. Upon random mitotic segregation of the units, 1/9^th of the cells will become fixed at 75% heteroplasmy and 8/9^th at 50%. Other scenarios can be envisaged. For example, a random redistribution of all mtDNAs in a cell from (A) will result in units with 0/8^th, 1/8^th ….7/8^th, 8/8^th mutation loads with binomially distributed frequencies. For simplicity, a uniform heteroplasmic segregation unit with identical number of copies of mtDNA is depicted. Slight variation in mtDNA copy number per unit in a cell will rapidly evolve through random mitotic segregation of these units into multiple cell types with each another uniform heteroplasmic segregation unit (like in B).</p