Primers used for quantitative RT-PCR analyses.

<p>Primers used for quantitative RT-PCR analyses.</p

Differential stem cells markers in undifferentiated and differentiated human mesenchymal stem cells.

<p>Levels of CD13, CD49e, CD166, CD133 and VEGFR2 in undifferentiated cells (UC), CM1 and CM2-treated cells after 21 days of culture. (Conditioned medium: CM). Values are expressed as mean of percentage ± standard deviation. (a p<0.001 and b p<0.01 vs. CM1-treated cells; +++ p<0,001 and + p<0,05 vs. undifferentiated cells).</p

The activation of Wnt/β-catenin during hepatocyte differentiation is associated with the presence of related proteins to tumoral phenotype.

<p>Relative abundance of specific proteins (DIGE analysis) in human mesenchymal stem cells undifferentiated after 21 days of culture (UC21d) and in mesenchymal stem cells differentiated into hepatocytes with conditioned medium 1 (CM1) or 2 (CM2). B) Western blot confirmation of the changes observed by DIGE analysis in the abundance of some proteins in CM1 and CM2 hepatocytes: Adenine phosphoriobosyl transferase (APT), cathepsin B precursor (CATB), L-lactate dehydrogenase β chain (LDHB), transgelin (TGL2), tropomyosin β chain (TPM2) and nuclear β-catenin. Tubulin and TFIIB were used as cytoplasm and nuclear loading control respectively.</p

The treatment of human mesenchymal stem cells with CM2 induces nuclear translocation ofβ-catenin and Wnt signaling activation.

<p>A) To determine β-catenin subcellular localization, human mesenchymal stem cells undifferentiated (UC) and treated with conditioned medium 1 (CM1) or 2 (CM2) after 21 days of culture were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue). Merged image of β-catenin-FITC and DAPI staining is also shown. Original magnification: 40×. B) mRNA expression of Lrp5/6, Frizzled- 3 (FZD3) and c-myc was evaluated in undifferentiated cells and cells treated with conditioned medium 1 (CM1) or 2 (CM2). Fold of undifferentiated cells at 21 days of culture. ^a p<0.001 vs. CM1-treated cells. C) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034656#pone-0034656-g004" target="_blank">Figure 4</a> c shows western blot of p53 and α-tubulin as loading control. Image is representative of three independent experiments.</p

Comparative analysis by DIGE of proteins differentially expressed in hepatocytes obtained with CM1 or CM2 differentiation protocols.

<p>Cyt: Cytoplasm; Nuc: Nucleus; Secr: Secreted protein; ER: Endoplasmic reticulum; Mit: Mitochondria; Lys: Lysosome.</p

Stem cells/cancer stem cells markers expression in undifferentiated cells at time 0 days.

<p>Data are included as mean ± standard deviation.</p

The treatments with CM1 or CM2 increase the expression of hepatospecific genes in human mesenchymal stem cells.

<p>Relative levels of mRNA expression of A) albumin (ALB), B) α-fetoprotein (αFP), C) α1-antitrypsin (α-1-AT), D) CCAAT/enhancer-binding protein beta (C/EBP) and E) cytochrome P450 (CYP3A5) were determined in human undifferentiated mesenchymal stem cells before and after differentiation with conditioned medium 1 (CM1) or 2 (CM2) after 7, 14 and 21 days of culture; Gene expression is shown as fold-changes compared to undifferentiated cells at each time. Values are expressed as mean ± standard deviation. All genes were increased significantly respect to undifferentiated cells (UC). ^a p<0.001, ^b p<0.01 vs. CM1 or CM2.</p