A Quantitative System for Discriminating Induced Pluripotent Stem Cells, Embryonic Stem Cells and Somatic Cells

<div><p>Induced pluripotent stem cells (iPSCs) derived from somatic cells (SCs) and embryonic stem cells (ESCs) provide promising resources for regenerative medicine and medical research, leading to a daily identification of new cell lines. However, an efficient system to discriminate the different types of cell lines is lacking. Here, we develop a quantitative system to discriminate the three cell types, iPSCs, ESCs, and SCs. The system consists of DNA-methylation biomarkers and mathematical models, including an artificial neural network and support vector machines. All biomarkers were unbiasedly selected by calculating an eigengene score derived from analysis of genome-wide DNA methylations. With 30 biomarkers, or even with as few as 3 top biomarkers, this system can discriminate SCs from pluripotent cells (PCs, including ESCs and iPSCs) with almost 100% accuracy. With approximately 100 biomarkers, the system can distinguish ESCs from iPSCs with an accuracy of 95%. This robust system performs precisely with raw data without normalization as well as with converted data in which the continuous methylation levels are accounted. Strikingly, this system can even accurately predict new samples generated from different microarray platforms and the next-generation sequencing. The subtypes of cells, such as female and male iPSCs and fetal and adult SCs, can also be discriminated with this method. Thus, this novel quantitative system works as an accurate framework for discriminating the three cell types, iPSCs, ESCs, and SCs. This strategy also supports the notion that DNA-methylation generally varies among the three cell types.</p> </div

The discriminating system performs precisely on converted data.

<p>A, a high correction relationship exists between methylation percentage measured from sequencing and the beta value measured from Illumina microarray. B, Our system discriminates the three cell types with high accuracy with converted data. For visualization purposes, only percentage of NNET was shown here due to its similarity with SVM and the high correlation between accuracy percentage and kappa. This practice was also applied to following figures in this study.</p

Overall methylation profiling of three cell types.

<p>All methylation sites measured by microarray were used to profile the overall methylation patterns of the three cell types, iPSCs, ESCs, and SCs. A, unsupervised clustering analysis revealed that SCs were separated from PCs (iPSCs and ESCs). In the PCs subgroup, most ESCs were separated from iPSCs. B, Correspondence analysis classified three cell types, SCs, iPSCs, and ESCs. SCs and PCs were separated in first component while most of ESCs and iPSCs were separated in second component. C, iPSCs and ESCs were further classified by correspondence analysis in 3D space. For visualization purposes, only one subset of data was shown here.</p

Top biomarker list.

<p>Left panel, top 10 biomarkers for discriminating SCs from PCs. Right panel, top 10 biomarkers for discriminating iPSCs from ESCs. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056095#pone.0056095.s002" target="_blank">Table S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056095#pone.0056095.s003" target="_blank">S3</a>, and S4 for complete list used in this study.</p

Our system works accurately with raw data.

<p>Our system reaches the similar discriminating power as that with normalized data.</p

Carbohydrate metabolism is highly related to fiber initiation.

<p>(A) Sucrose synthase (w58) differentially accumulated in the wt and <i>fl</i> mutant cotton ovules. The bars indicate the log-transformed values of the fold-change ratios of differentially accumulated proteins at five stages. (B) Carbohydrate contents as determined by an enzymatic method. The results are the mean values from three independent experiments. Statistical analyses were performed using student’s T test. Abbreviations: *, p<0.05; **, p<0.01.</p

Additional file 1 of Comparing the effects of pulsed and radiofrequency catheter ablation on quality of life, anxiety, and depression of patients with paroxysmal supraventricular tachycardia: a single-center, randomized, single-blind, standard-controlled trial

Additional file 1. Supplementary Material 1

Synergistic Antitumor Activities of Docetaxel and Octreotide Associated with Apoptotic-Upregulation in Castration-Resistant Prostate Cancer

<div><p>Androgen deprivation therapy has become the fist-line treatment of metastatic prostate cancer; however, progression to castrate resistance disease occurs in the majority of patients. Thus, there is an urgent need for improvements in therapy for castration-resistant prostate cancer. The aims of the present study were to determine the efficacy somatostatin analogue octreotide (OCT) combined with a low dose of docetaxel (DTX) using castration resistant prostate cancer cells and to investigate the involved molecular mechanisms in vitro. The anti-proliferative and synergism potential effects were determined by MTT assay. Induction of apoptosis was analyzed employing annexing V and propidium iodide staining and flow cytometry. VEGFA, CASP9, CASP3 and ABCB1 gene expression was evaluated by RT-PCR and Q-RT-PCR analysis. OCT in combination with DTX treatments on DU145 cell migration was also evaluated. Investigation revealed that combined administration of DTX and OCT had significant, synergistically greater cytotoxicity than DTX or OCT treatment alone. The combination of the two drugs caused a more marked increase in apoptosis and resulted in greater suppression of invasive potential than either individual agent. There was obvious increase in caspase 3 expression in the OCT alone and two-drug combined treatment groups, however, VEGFA expression was markedly suppressed in them. These results support the conclusion that somatostatin analogues combined with docetaxel may enhance the chemotherapy efficacies through multiple mechanisms in castration-resistant PCa cell line. This work provides a preclinical rationale for the therapeutic strategies to improve the treatment in castrate resistance disease.</p></div

Effects of DTX, OCT alone and the combination of DTX/OCT on the mRNA expression of VEGFA, caspase 9, caspase 3 and ABCB1 in DU145 cells.

<p>M: Marker, The expression levels of objective genes were examined by RT-PCR (A, B, C and D) and quantitative real-time -PCR (B, D, F and H) respectively, using cells treated with the test drugs for 72 hours. The expression level of each mRNA was normalized to the level of β-actin mRNA. Values represent the means±SD of triplicate analyses (*<i>p</i><0.05, **<i>p</i><0.01).</p

Effect of DTX, OCT alone and two-drug combination treatment on DU145 cells apoptosis.

<p>Data are mean ± SEM of two independent experiments performed in triplicate. ***<i>p</i><0.000 vs. control.</p