Supplementary Material for: Detection of Fetal Sex, Aneuploidy and a Microdeletion from Single Placental Syncytial Nuclear Aggregates

<b><i>Objectives:</i></b> A key problem in prenatal screening using extra-embryonic cells is the feasibility of extracting usable DNA from a small number of cells. Syncytial nuclear aggregates (SNAs) are multinucleated structures shed from the placenta. This study assesses the potential of SNAs as a source of fetal DNA for the detection of genetic abnormalities. <b><i>Methods:</i></b> SNAs were collected in vitro. Whole-genome amplification was used to amplify DNA from single SNAs, and DNA quality and quantity was assessed by spectrophotometry and PCR. Confocal microscopy was used to count nuclei within SNAs, determine metabolic activity and investigate DNA damage. Fetal sex and chromosomal/genetic abnormalities were investigated with array-comparative genomic hybridization (aCGH). <b><i>Results:</i></b> DNA was amplified from 81% of the individual SNAs. A mean of 61 ± 43 nuclei were found per SNA. DNA strand breaks were found in 76% of the SNAs. Seventy-five percent of SNAs yielded whole-genome-amplified DNA of sufficient quality for aCGH after storage and shipping. Individual SNAs from the same pregnancy reliably gave the same chromosomal profile, and fetal sex and trisomies could be detected. A microdeletion was detected in one pregnancy. <b><i>Conclusion:</i></b> SNAs could provide a source of extra-embryonic DNA for the prenatal screening/diagnosis of fetal sex and chromosomal and sub-chromosomal genetic abnormalities

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

Array-CGH profiles of MUG-Chor1 phenotypes.

<p>Comparative genome hybridization of 100 large cells (red line) and small cells (blue line) each yielded identical chromosomal profiles. Both populations show gains at chromosomes 2q, 5q, 7, 17q and losses at 2q, 6p, 9p, 10p, 10q, 12p, 17p and 22. Small gains at chromosomes 2q as well as a small loss at chromosome 17q were detected in the large cell phenotype. This indicates that both morphologically different cell types evolved from a common clonal origin. Bars on the left of the moving average indicate losses of DNA. Bars on the right of the moving average indicate gains of DNA. Both profiles are in line with previously published data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087663#pone.0087663-Rinner1" target="_blank">[8]</a>.</p

Expression analyses of chordoma specific and candidate genes in MUG-Chor1 cells.

<p>RT-qPCR was done on AB7900 TaqMan (Applied Biosystems; Foster City, CA). <i>GAPDH</i> and <i>ACTB</i> were used for normalization. Normalization and statistical analysis was done with GenEx Professional (MultiD Analysis; Version 5.3.5.6). All non-template controls were undetermined (Cq>45) except for <i>GAPDH</i> showing two replicates with Cq values >37 and <i>VIM</i> yielding one replicate at Cq = 27. 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>c<p>Cq values w/o outlier. Outliers were identified by means of Grubbs’ outlier test.</p

Ultrastructural analysis of small and intermediate cells.

<p>A) Small MUG-Chor1 cells show typical ultrastructural features of a diploid cell including nucleus (N), mitochondria (M), and endoplasmic reticulum (ER) in a dense cytoplasm. B) Stitched image of two intermediate cells tightly connected alongside their cell membranes (arrow) present with nucleus (N, only visible in one cell), ER, and mitochondria. Both cells already contain prominent vacuoles (V₁, V₂) and show highly organized cytoskeleton in close proximity to the nucleus and vacuoles (asterisk). Scale bars: 1 µm (A), 5 µm (B).</p

Morphological and immunohistochemical characterization of the chordoma tumor giving rise to MUG-Chor1 cell line

<p>. A) Hematoxylin/eosin stained section show lobulated myxoid tumor tissue with cords, strands and nests of tumor cells with pale/eosinophilic to vacuolated cytoplasm. B, C) In detail, the tumor is composed of small cells with eosinophilic cytoplasm and partly spindle cell morphology and large vacuolated/physaliferous tumor cells including “signet ring” shaped cells. D) All cell phenotypes yield the chordoma-specific nuclear staining for brachyury. Arrowheads: small cells; asterisks: large vacuolated/physaliferous cells; arrows: “signet ring” cells. Scale bars: 500 µm (A), 50 µm (B, C); D: original magnification X20.</p

Morphological characteristics of vacuoles in large cells.

<p>Large physaliferous cells consisting of equal vacuole content may present with either few very large (A, C, E) or numerous smaller vacuoles (B, D, F). Both morphological phenotypes were detected throughout all samples ranging from tumor tissue (A, B), cell culture (C, D) to sample preparations just before being micromanipulated (E) or microdissected (F) indicating stable characteristics of this chordoma tumor. C, D: original magnification X10; F: original magnification X40.</p

HPF fixed vacuoles within the chordoma cells.

<p>All micrographs indicate the connection of the vacuoles. A) Overview of the vacuoles (V); arrows indicate the network within the MUG-Chor1 cells. B) Cells show a high number of small vacuoles connected to each other as well as endosomes and Golgi apparatus, surrounded by dense cytoplasm. C) Higher magnification enables the visualization of the linkages between either small vacuoles or endosomes and vacuoles; arrows indicate the exchange of material within the vacuoles.</p

Intracellular calcium response to four different agonists in MUG-Chor1 cells was measured.

<p>A) Histamine (His), acetylcholine (ACh), serotonin (5HT), and P2Y purinoceptor agonist mes-ATP were applied at a concentration of 10 µM for a period of 60 seconds as indicated by the grey bars; the ratio between 340 nm and 380 nm is given. After stimulation, the respective agonist was washed out for another 60 seconds. B) The first two peaks of ratio values of MUG-Chor1 cells indicate ACh-application in the presence of extracellular calcium (NT+[Ca²⁺]_ex; upper, open bar); peak reduction was induced by removing external calcium (NT w/o [Ca²⁺]_ex; upper, grey bar). Small bars represent the time of perfusion with ACh at the two different concentrations (10 µM: lower, grey bar; 100 µM: lower, black bar). C) Within the bar chart a summary of ACh-induced changes is given. The first two bars represent effects in [Ca²⁺]_i induced by ACh (10 µM; 100 µM) given as average value (± standard deviation) of the change in intracellular calcium concentration [Ca²⁺]_i (nM). The usage of calcium within the perfusion solution as well as the different concentrations of ACh is indicated below the x-axis. Significant changes evaluated by the students t-test are given (***, p<0.001; **, p<0.01).</p