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. 2015 Oct 13;23(12):1843–1853. doi: 10.1038/mt.2015.166

Figure 2.

Figure 2

Cancer upregulated drug-resistant (CUDR) enhances the embryonic stem cells (ESC) diffentiation into the hepatocyte-like cells. (a) Nuclear run-on (upper) and reverse-transcription polymerase chain reaction (RT-PCR) (lower) analysis of CUDR in human ESC line MEL-2 transfected with pCMV-A-GFP-CUDR and pCMA6-A-GFP respectively. β-actin as internal control. (b) Human ESC line MEL-2 transfected with pCMV-A-GFP-CUDR or pCMA6-A-GFP could efficiently generate definitive endoderm (DE) tissue by treating the the modified cultures with high concentrations of the TGFβ family ligand activin A (100 ng/ml) for 5 days. Western blot analysis with anti-Foxa2, anti-Sox17 in these induced cells (the 2nd, 3rd, and 5th day). β-actin as internal control. (c) A number of groups have generated hepatoblasts using this DE tissue as a starting material, plating the DE on matrix to mimic the hepatic ECM and then added FGF4 (100 ng/ml) and BMP (100 ng/ml) to mimic hepatic induction for 5 days. Western blotting with anti-AFP, anti-Albumin, and anti-HNF4α in the induced cells (the 6th and 10th day). β-actin as internal control. (d) Some combination of insulin-transferrinselenite (ITS, 5 μg/ml), hepatocyte growth factor (HGF) (20 ng/ml), oncostatin M (OSM) (10 ng/ml), acid fibroblast growth factor (aFGF) (50 ng/ml), and dexamethasone (10−7M) to expand the hepatoblast population and to promote hepatic maturation for 10 days. Western blotting with anti-Albumin in the induced cells (the 11th, 14th, 17th, and 20th day). β-actin as internal control. (e) Albumin promoter luciferase activity assay in derived hepatocyte-like cells. Each value was presented as mean ± standard error of the mean (SEM). **P < 0.01. (f) Nuclear run-on (upper) and RT-PCR (lower) analysis of CUDR in human ESC line MEL-2 transfected with pGFP-V-RS-CUDR and pGFP-V-RS, respectively, β-actin as internal control. (g) Western blot analysis with anti-Foxa2, anti-Sox17 in these derived cells (the 2nd, 3rd, and 5th day). β-actin as internal control. (h) Western blotting with anti-alpha fetoprotein (AFP), anti-Albumin, and anti-HNF4α in the derived cells (the 6th and 10th day), β-actin as internal control. (i) Western blotting with anti-Albumin in the induced cells (the 11th, 14th, 17th, and 20th day), β-actin as internal control. (j) Albumin promoter luciferase activity assay in derived hepatocyte-like cells (CUDR knockdown or control). Each value was presented as mean ± SEM. **P < 0.01).