Fig. 6. Dominant-negative C/EBP (A-C/EBP) inhibits hepatocyte differentiation from stem cells. (A,B) After culturing FACS-sorted EGFP⁺ transduced cells for 10 or 20 days, quantitative PCR was performed. The expression of hepatocyte-differentiation markers ALB, AFP, At, G6P and TO in the stem cell population was significantly suppressed even when cultured with both HGF (H) and OSM (O), which induce hepatocyte differentiation. By contrast, the expression of CK19 and GGT, which are markers of cholangiocyte differentiation, was elevated in cells expressing A-C/EBP. The expression of HNF1, HNF4 and Met was also relatively enhanced, but not significantly. The expression of HNF6, however, was decreased in transduced cells, in a similar fashion to hepatocyte marker genes. All data were normalized to the value of mock controls (day 10; no GF) and fold differences are shown. Representative data from a stem cell clone are shown; three samples were examined for each condition. Data are mean±s.d. (C) Immunocytochemical staining of ALB and PAS-staining were performed on cells expressing EGFP (mock) or A-C/EBP at day 20. In cells expressing A-C/EBP, few cells were positive for ALB and stored glycogen. (counterstain: Hematoxylin). (D) After culture of sorted EGFP (mock) or A-C/EBP-expressing cells for 5 days in clonal density cultures (5x10² cells/well in six-well plates), the number of large (>100 cells in a colony) and small colonies was counted (n=6). The number of large colonies was increased when cells were transduced with A-C/EBP. Data are mean±s.d. (*P<0.005). (E) After transduction of FACS-sorted ALB⁺ or ALB^– cells by A-C/EBP-NGFR and culture of each cell population for 14 days, ALB⁺/NGFR⁺ cells or ALB^–/NGFR⁺ cells were sorted separately and cultured with or without HGF and OSM for 10 days (plating density is 1x10³ cells/cm²). Then, the generation of ALB^– cells from ALB⁺/NGFR⁺ sorted cells and the generation of ALB⁺ cells from ALB^–/NGFR⁺ sorted cells was examined by using FACS. Compared with mock controls, cells expressing A-C/EBP failed to maintain ALB expression in ALB⁺ sorted cells and were unable to generate ALB⁺ cells from ALB^– sorted cells. Representative data from a transfected stem cell clone are shown; three samples were examined for each condition. Data are mean±s.d. Scale bars: 100 µm.