Figure 1.
Differential stem cells markers in undifferentiated and differentiated human mesenchymal stem cells.
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).
Table 1.
Stem cells/cancer stem cells markers expression in undifferentiated cells at time 0 days.
Figure 2.
The treatments with CM1 or CM2 increase the expression of hepatospecific genes in human mesenchymal stem cells.
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.
Figure 3.
The treatment with CM1 or CM2 induces the presence of hepatospecific proteins in human mesenchymal stem cells.
The presence of hepatospecific proteins such as albumin, α 1-antitrypsin, α-fetoprotein, cytokeratin-19 and PAS stain were evaluated by immunohistochemistry after 21 days of culture with conditioned medium CM1 or CM2. Arrows show positive staining area.
Figure 4.
The treatment of human mesenchymal stem cells with CM2 induces nuclear translocation ofβ-catenin and Wnt signaling activation.
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) Figure 4 c shows western blot of p53 and α-tubulin as loading control. Image is representative of three independent experiments.
Figure 5.
A) Number of cells after 7, 14 and 21 days of culture in undifferentiated, CM1 and CM2-treated cells. Values expressed as mean ± standard deviation. a p<0.001 vs. CM1-treated cells and undifferentiated cells. B) The presence of nuclear PCNA (brown nucleus) was evaluated by immunohistochemistry after 21 days of culture in undifferentiated cells, CM and CM2-treated cells. Image is representative of three experiments. Original magnification: 20×. C) Cell cycle was analyzed at 21 days of hepatocyte differentiation in undifferentiated, CM1 and CM2-tretaed cells. Data are showed as mean of percentage plus standard deviation. a p<0.001 vs. CM1-treated cells, ++ p<0.01 and +++ p<0.001 vs. undifferentiated cells. D) Primary spheroid assay with count of number of cells after 4 days of culture with conditioned medium for spheroid formation. Data are showed that mean ± standard deviation (a p<0.001 vs. CM1-treated cells and +++ p<0.001 vs. undifferentiated cells). E) Secondary spheroid formation assay. Number of secondary spheroids was counted in an inverted microscope. Three experiments were carried out and data are expressed as mean ± standard deviation (a p<0.001 vs. CM1-treated cells and +++ p<0.001 vs. undifferentiated cells). F) Detail of secondary spheroids is showed in the microphotographs of undifferentiated cells, CM1 and CM2-treated cells.
Figure 6.
The activation of Wnt/β-catenin during hepatocyte differentiation is associated with the presence of related proteins to tumoral phenotype.
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.
Table 2.
Comparative analysis by DIGE of proteins differentially expressed in hepatocytes obtained with CM1 or CM2 differentiation protocols.
Table 3.
Primers used for quantitative RT-PCR analyses.