Effects of proteinase K treatment on the inhibitory activity of CM-hAMTC and CM-hAM.

<p>(<b>A</b>) Representative SDS-PAGE of untreated and proteinase K-treated (for 2 and 6 hrs) UltraCulture medium, CM-hAMTC and CM-hAM. Digestion level was calculated as the reduction of the density of the BSA band (arrow). (<b>B</b>) Inhibitory effect on lymphocyte proliferation, induced by anti-CD3 stimulation, of 100 μl of untreated (NT) and proteinase K-treated UltraCulture medium, CM-hAMTC and CM-hAM. Similar results were also obtained using 50 μl of CMs (data not shown). Data represent the mean and SD of at least five independent experiments. * = p<0.05, ** = p<0.01 versus UltraCulture.</p

Cytokine quantification in UltraCulture medium, CM-hAMTC and CM-hAM.

<p>MAD: Median Absolute Deviation.</p><p>Values are expressed in pg/ml.</p>*<p>quantified by ELISA.</p

Study of the potential molecular weight of the inhibitory factor(s).

<p>(<b>A</b>) Lymphocyte proliferation, stimulated by anti-CD3, in presence of 100 μl of CM-hAMTC (white bar), CM-hAMTC-fraction-mix (grey bars) or mix of fractions with different molecular weights (white-grey- and black-grey-striped bars). (<b>B</b>) Lymphocyte proliferation, stimulated by anti-CD3, in presence of 100 μl of complete UltraCulture medium (black bar) or its fractions (grey and black-grey-striped bars). Data represent the mean and SD from at least six independent experiments. ** = p<0.01, *** = p<0.001 versus PBMC + αCD3; # = p<0.05.</p

Inhibition of production of NO, kynurenine, and prostaglandins (PGs) in PBMC culture.

<p>Lymphocyte proliferation, stimulated with anti-CD3, in presence of 100 μl of control medium or CM-hAMTC and CM-hAM with (white bars) or without (black bars) the addition of indomethacin (<b>A</b>), L-NAME (<b>B</b>) or DL-methyl-tryptophan (<b>C</b>). Data represent the mean and SD of at least three independent experiments. * = p<0.05, ** = p<0.01, *** = p<0.001 versus PBMC + αCD3.</p

Inhibitory effect of CM-hAMTC and CM-hAM in presence of neutralizing antibodies.

<p>Lymphocyte proliferation, stimulated with anti-CD3 (PBMC + αCD3), in presence of 100 μl of control medium or CM with (white bars) or without (black bars) the addition of neutralizing antibodies against IL-10 (<b>A</b>), TGF-β (<b>B</b>), HGF (<b>C</b>), IL-6 (<b>D</b>), mix of antibodies anti-IL-10, -TGF-β, -HGF and -IL-6 (<b>E</b>). Data represent the mean and SD of at least four independent experiments. * = p<0.05, ** = p<0.01 versus PBMC + αCD3.</p

Effect of PGs on the proliferation of stimulated PBMC in presence or absence of CMs obtained from hAMTC or hAM treated or not with indometahcin.

<p>(<b>A</b>) Lymphocyte proliferation, stimulated with anti-CD3, in presence of different amount of prostaglandin (PG) PGD2 (⧫), PGE2 (□), PGF2α (<b>Δ</b>) or PGI2 (X). (<b>B</b>) Lymphocyte proliferation, stimulated with anti-CD3, in presence of 100 μl of control medium (UltraCulture) or CM derived from untreated (CM-hAMTC) or indomethacin treated (CM-hAMTC Indo) hAMTC with (white bars) or without (black bars) the addition of a mix of PGs (PGE2 500 ng/ml + PGD2 100 ng/ml + PGF2α 25 ng/ml). (<b>C</b>) Lymphocyte proliferation, stimulated with anti-CD3, in presence of 100 μl of control medium (UltraCulture) or CM derived from untreated (CM-hAM) or indomethacin treated (CM-hAM Indo) hAM with (white bars) or without (black bars) the addition of a mix of PGs (PGE2 25 ng/ml + PGD2 6 ng/ml + PGF2α 10 ng/ml). * = p<0.05, ** = p<0.01, *** = p<0.001.</p

NO, kynurenine, and prostaglandins (PGs) in CM-hAMTC and CM-hAM.

<p>(<b>A</b>) NO quantification (total nitrate quantification) in UltraCulture, CM-hAMTC, and CM-hAM performed by means of the Griess test. (<b>B</b>) Lymphocyte proliferation (expressed in cpm) after stimulation with anti-CD3 and in presence of 100 μl of complete UltraCulture medium, CM derived from untreated (black bar), or L-NAME-treated (white bar) hAMTC and hAM cultures. (<b>C</b>) IDO activity, shown as the ratio between the concentration of kynurenine and tryptophan, in the control medium, CM-hAMTC, CM-MLR or CM-MLR-hAMTC, and in the presence of 0.5 mM DL-methyl-tryptophan (CM-MLR-hAMTC+MT). (<b>D</b>) Lymphocyte proliferation (expressed in cpm), induced by MLR, in the presence of hAMTC cultured with or without the addition of DL-methyl-tryptophan. (<b>E</b>) Lymphocyte proliferation (expressed in cpm), stimulated with anti-CD3, in presence of 100 μl of control medium or CM derived from untreated (black bar) or 0.5 mM DL-methyl-tryptophan-treated (white bar) hAMTC and hAM cultures. (<b>F–I</b>) Lymphocyte proliferation (expressed in cpm) after stimulation with anti-CD3, in presence of 100 μl of control medium or CM derived from untreated (black bar) or indomethacin- (<b>F</b>), ketoprofen- (<b>G</b>), niflumic acid- (<b>H</b>) or inhibitors-mix- (<b>I</b>) treated (white bars) hAMTC and hAM cultures. Data represent the mean and the SD of at least three independent experiments. * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Prostanoids quantification in UltraCulture medium and CM derived from untreated or indomethacin treated hAMTC and hAM.

<p>Values are expressed in ng/ml.</p

Analysis of inhibitory effect of hAMTC, BM-MSC and of the respective CMs.

<p>Effects of the following on the proliferation of anti-CD3-stimulated lymphocytes (PBMC + αCD3): (<b>A</b>) hAMTC at passage 0 (white-grey striped bar), different amounts (50–75–100 μl) of CM obtained from the culture of hAMTC at p0 when plated at density of 1×10⁶ cells/ml (CM-hAMTC p0 1*×10⁶/ml) or 0.25×10⁶ cells/0.5 ml (CM-hAMTC p0 0.25*×10⁶/0.5 ml) and different amounts (50–75–100 μl) of CM-hAM; (<b>B</b>) hAMTC at passage 3 (vertical black line bar), BM-MSC at passage 3 (horizontal black line bar) and different amounts (50–100 μl) of CM derived from the culture of these cells. Data represent the mean and standard deviation (SD) of at least four independent experiments. * = p<0.05, ** = p<0.01, *** = p<0.001 versus PBMC + αCD3; ## = p<0.01.</p

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