Correction: Human Adipose Tissue-Derived Mesenchymal Stem Cells Target Brain Tumor-Initiating Cells

<p>Correction: Human Adipose Tissue-Derived Mesenchymal Stem Cells Target Brain Tumor-Initiating Cells</p

mRNA expression of cyto-chemokine receptors in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) after co-culture with brain tumor-initiating cells (BTICs).

<p>Real-time Quantitative Polymerase Chain Reaction (RT-qPCR) analysis of cyto-chemokine receptors after co-culture of (A) medulloblastoma-BTICs, (B) atypical teratoid/rhabdoid tumors (AT/RT)-BTICs and (C) glioblastoma-BTICs. The mRNA level of each cytokine receptor was normalized to the level of GAPDH. All data are representative of three independent experiments, and each value represents the mean ± SD. *Significant difference from control (P < 0.05).</p

Induced cytokine levels in the BTICs after co-cultured with HFF1 or hAT-MSCs (pg/ml)^*.

<p>BTICs: brain tumor initiating cells, HFF1: human foreskin fibroblast, hAT-MSCs: human adipose-derived mesenchymal stem cells, AT/RT: atypical teratoid rhabdoid tumor, MCP-1: monocyte chemoattractant protein 1, SDF-1: stromal cell-derived factor 1, RANTES: regulated on activation, normal T cell expressed and secreted, IL-6: interleukin-6 ligand, IL-8: interleukin-8, IGF-1: insulin-like growth factor 1ligand, PDGF-bb: platelet-derived growth factor, VEGF: vascular endothelial growth factor, Ang-1: angiopoietin1</p><p>*[induced cytokine level in the BTICs with HFF1] = [total cytokine level in the BTICs with HFF1]-[cytokine level in the HFF1 only]</p><p>[induced cytokine level in the BTICs with hAT-MSCs] = [total cytokine level in the BTICs with hAT-MSCs]-[cytokine level in the hAT-MSCs only]</p><p>Induced cytokine levels in the BTICs after co-cultured with HFF1 or hAT-MSCs (pg/ml)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129292#t001fn002" target="_blank">*</a>}.</p

Migratory ability of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) toward brain tumor-initiating cells (BTICs) <i>in vivo</i>.

<p>Fluorescence images of the brains were taken at the indicated times. (A) In the absence of tumor, the fluorescent signals of hAT-MSCs (blue) are weakened at 2 weeks and are not detectable at 3 weeks. (B) <i>In vivo</i> sequential tracking was performed by injecting both NEO-LIVE 675-labeled BTICs (red) and NEO-LIVE 797-labeled hAT-MSCs (blue). hAT-MSCs gradually migrate toward the tumor site, and strong fluorescent signals are observed at 4 weeks post-injection at the tumor site. (C) Mice were injected the NEO-LIVE 797-labeled hAT-MSCs or HFF1. Representative <i>in vivo</i> fluorescence images show that the HFF1 signals are decreased or fade out. (D) On the contrary, the hAT-MSC signals are widened at all BTIC-derived mouse brain tumor sites at 3 weeks post-injection when compared with HFF1 cells. The color bar indicates radiant efficiency.</p

Knock down of cytokine receptors on hAT-MSCs.

<p>After siRNAs treatment to each cytokine receptors on hAT-MSCs, (A) the protein expressions were confirmed in hAT-MSCs by western blot and (B) the migratory ability toward BTICs is assessed using trans-well assay. (C) The quantified results show inhibition of migration by selective knock down of cytokine receptors. ×50 magnification. All data are representative of three independent experiments, and each value represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.</p

Vliv mechanizované sklizně hroznů na kvalitu vína

The thesis deals with the mechanized harvesting of the vine and its influence on the quality and composition of the harvested berries. He compares the partially mechanized harvest with a fully mechanized harvest and harvest impact on health status and composition of the product to be harvested. The quality of the berries, their damage and health affect the quality of the produced wines. For fully mechanized harvesters during the harvest time has undergone development, the influence of which was to eliminate the undesirable effects associated with harvesting in this way, the minimum. While in the 1970s. the years of the last century, meant the mechanized harvest rather additional and enhanced way of harvesting, nowadays many of the larger vine growers, due to the growing costs of labor, fails without the cultivation of the vine ever imagined. Yet they are not the views of the mechanized harvesting of the single, the answer to the question, to what extent does the quality of the wine harvest reduced mechanical, corresponds to a comparison of several vine growers

Recombinant PEDF promotes stemness and self-renewal of GSCs.

<p>(A) Sphere formation assay was performed in X02 cells treated with recombinant PEDF (rPEDF) (100 ng/ml) or control vehicle. Images are representative of three independent experiments. The graph represents average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (<i>n</i> = 3). * <i>p</i> < 0.05. (B) IB analysis of Sox2, Nestin, and GFAP in X02 cells treated or not treated with rPEDF (100 ng/ml). β-actin was used as a loading control. (C) Sphere formation assay was performed in X01 cells cultured in serum-free GSC medium (containing EGF and bFGF) or serum-free GSC medium without EGF and bFGF. X01 cells cultured in serum-free GSC medium without EGF and bFGF were treated with rPEDF (100 ng/ml) or control vehicle. Images are representative of three independent experiments. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (<i>n</i> = 3). ** <i>p</i> < 0.01. (D) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in X01 cells cultured in three different conditions (C). α-tubulin was used as a loading control. All images were taken at 5x magnification.</p

PEDF expression maintains stemness and self-renewal of GSCs.

<p>(A, C) LDA was performed in GSCs (CSC2 and X01) infected with shPEDF1-expressing lentiviral, shPEDF1 with PEDF-overexpressing lentiviral, or control construct. CSC2-Con or CSC-shPEDF1; <i>p</i> = 0.000201, CSC2-Con or CSC-shPEDF1-PEDF; <i>p</i> = 0.576, CSC2-shPEDF1 or CSC-shPEDF1-PEDF; <i>p</i> = 2.23e-05 (A) and X01-Con or X01-shPEDF1; <i>p</i> = 0.000265, X01-Con or X01-shPEDF1-PEDF; <i>p</i> = 0.589, X01-shPEDF1 or X01-shPEDF1-PEDF; <i>p</i> = 2.99e-05 (C). (B, D) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in CSC2-Con, CSC2-shPEDF1, or CSC2-shPEDF1-PEDF (B) and X01-Con, X01-shPEDF1, or X01-shPEDF1-PEDF (D). β-actin was used as a loading control. (E) LDA was performed in X02 infected with PEDF-expressing lentiviral or control construct. X02-Con or X02-PEDF; <i>p</i> = 0.00936. (F) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in X02-Con or X02-PEDF cells. β-actin was used as a loading control. (G) IB analysis of PEDF (in medium), p-EGFR, EGFR, p-STAT3, STAT3, Nestin, Sox2, and GFAP in X02-Con, X02-EGFRvIII, X02-shPEDF2, or X02-EGFRvIII coinfected with shPEDF2-expressing lentiviral construct. (H) Sphere formation assay was performed in X02-Con, X02-EGFRvIII, X02-shPEDF2, or X02-EGFRvIII coinfected with shPEDF2-expressing lentiviral construct. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (<i>n</i> = 3). * <i>p</i> < 0.05.</p

Identification of PEDF as a novel autocrine factor regulated by EGFRvIII through STAT3 signaling.

<p>(A) Sphere formation assay of CSC2 cell cultured in control medium (serum-free GSC medium), serum-free GSC-conditioned medium (CM), or serum-CM. Prior to the harvest of CMs, the cells were washed with phosphate buffered saline (PBS) and changed with F12 medium without serum or other supplement for 24 h. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± standard error of the mean (SEM) (<i>n</i> = 3). * <i>p</i> < 0.05; ** <i>p</i> < 0.01. (B) Schematic representation of mass spectrometry analysis. CMs from CSC2 cultured in serum-free GSC or serum-cultured medium were respectively harvested after 2 wk. Also, Serum-free GSC CM from CSC2 (EGFRvIII⁺ GSC) or Ex Vivo (EGFRvIII^- GSC) were respectively harvested after 2 wk. Prior to the harvest of CMs, the cells were washed with PBS and changed with F12 medium without serum or other supplement for 24 h. All of them were analyzed by liquid chromatography–mass spectrometry–mass spectrometry (LC-MS/MS). (C) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in GSCs (CSC2, X01, and X02 cells) incubated in serum-free GSC or serum-cultured medium. α-tubulin was used as a loading control. (D) Sphere formation assay was performed in CSC2 cells incubated in serum-free CSC2-Con or CSC2-shPEDF2 CM. Two CMs respectively obtained from CSC2 cells infected with shPEDF2-expressing lentiviral or control construct, cultured in serum-free GSC medium. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (<i>n</i> = 3). ** <i>p</i> < 0.01. (E) IB analysis of p-EGFR, EGFR, p-STAT3, STAT3, and PEDF (in medium) in GSCs (CSC2 and X01cells) transfected with EGFRvIII siRNA or its control. β-actin was used as a loading control. (F) IB analysis of p-EGFR, EGFR, p-STAT3, STAT3, PEDF (in medium), Sox2, Nestin, and GFAP in GSCs (CSC2 and X01) transfected with siEGFR-WT or siControl. α-tubulin was used as a loading control. (G) IB analysis of p-EGFR, EGFR, p-STAT3, STAT3, PEDF (in medium), Sox2, Nestin, and GFAP in GSCs (CSC2 and X01) infected with EGFR WT-expressing lentiviral or control construct. α-tubulin was used as a loading control. (H) IB analysis of p-EGFR, EGFR, p-STAT3, STAT3, PEDF (in medium), Sox2, Nestin, and GFAP in X02 cells infected with EGFR-WT, EGFRvIII-expressing lentiviral or their control construct. β-actin was used as a loading control. (I) IB analysis of PEDF (in medium), Nestin, Sox2, GFAP, p-STAT3, and STAT3 in CSC2 and X01 cells treated with a small-molecule inhibitor of STAT3 (Stattic, 5 uM) or vehicle for 6 h. α-tubulin was used as a loading control. (J) IB analysis of PEDF (in medium), Nestin, Sox2, GFAP, p-STAT3, and STAT3 in CSC2 and X01 cells transfected with siSTAT3 or its control. α-tubulin was used as a loading control. (K) IB analysis of PEDF (in medium), Nestin, Sox2, GFAP, p-STAT3, and STAT3 in X02 cells infected with EGFRvIII-expressing lentiviral or their control construct. Also, these cells were transfected with siSTAT3 or its control. GAPDH was used as a loading control.</p

EGFRvIII/STAT3/PEDF signaling in GSCs.

<p>(A) IB analysis of EGFR, EGFRvIII, p-EGFR, p-STAT3, STAT3, and PEDF (in medium) in 13 GSCs. α-tubulin was used as a loading control. EGFRvIII⁺/PEDF^high GSCs are CSC2, X01, X03, X04, X06, X08, and X09 cells. EGFRvIII⁺/PEDF^low GSCs are MD30, 1123NS, and 83NS cells. EGFRvIII^-/PEDF^low GSCs are X02, Ex Vivo, and 528NS cells. (B) Histopathology of Balb-c/nu mouse brain tissue was orthotopically injected with three representative types of GSCs (A). Upper panel is hematoxylin and eosin (H&E) staining of the whole brain. Red box indicates a site of corpus callosum far from the injection site. This site was stained by H&E, Nestin, EGFRvIII, p-STAT3, and Sox2.</p