When human MSCs were cultured during 21 days for hepatocytes differentiation, marked differences in www.selleckchem.com/products/Nilotinib.html the levels of CD13, CD49e, CD133, CD166 and VEGFR2 were observed in CM1 and CM2-treated cells. The levels of these markers were significant higher in CM2-treated cells as compared to undifferentiated cells or CM1-treated cells (Figure 1). Figure 1 Differential stem cells markers in undifferentiated and differentiated human mesenchymal stem cells. Table 1 Stem cells/cancer stem cells markers expression in undifferentiated cells at time 0 days. Expression of hepatospecific markers in human mesenchymal stem cells during their differentiation into hepatocytes To compare the potential for hepatic differentiation of both protocols, the expression of hepatospecific genes was evaluated measuring mRNA levels by RT-PCR and protein expression by immunocytochemistry.
The mRNA levels of albumin (ALB), ��-fetoprotein (��FP), ��1-antitrypsin (��1-AT), C/EBP�� and CYP3A5 were strongly induced in human MSCs treated with CM1 or CM2 at 7, 14 and 21 days, compared to undifferentiated cells (UC). Comparatively, at 21 days of differentiation, there are no significant differences between both differentiation protocols. In CM2-treated cells there were not significant differences in the expression of these genes between cells to time 0 and cells after 48 h of treatment. The expressions of albumin and C/EBP were bigger in CM1 than CM2-treated cells while the expressions of ��1-AT and CYP3A5 were increased in CM2 vs. CM1-tretaed cells. The expression of ��FP was similar with both protocols (Figure 2).
Figure 2 The treatments with CM1 or CM2 increase the expression of hepatospecific genes in human mesenchymal stem cells. Expression of hepatospecific proteins, such as albumin, ��1-antitrypsin, ��-fetoprotein and cytokeratin 19 were also immunodetected in cells after 21 days of differentiation with both protocols CM1 or CM2 (Figure 3). Both protocols expressed with similar intensity hepatospecific proteins. Undifferentiated cells at 21 days were negative for the expression of these proteins. PAS stain was positive after treatment with CM1 and CM2 although a higher intensity was observed in cells cultured with CM2. Figure 3 The treatment with CM1 or CM2 induces the presence of hepatospecific proteins in human mesenchymal stem cells.
Role of Wnt/��-catenin pathway and p53 during human mesenchymal stem cells differentiation Cilengitide into hepatocytes Since Wnt/��-catenin pathway plays a main role in the control of differentiation of adult stem cells, confocal microscopy was used to study the subcellular localization of ��-catenin during differentiation into hepatocytes in both CM1 and CM2 (Figure 4A). Immunofluorescence staining demonstrated that ��-catenin localized to the cell membrane or to the peri-membrane region in undifferentiated and CM1 cells after 21 days, while there was no evidence of nuclear translocation.