Fig. 2. Separation of differentiating ALB⁺ cells from the stem cell population. (A) After gene transfer of the ALB enhancer/promoter-EGFP construct into expanding stem cell populations, ALB⁺ and ALB^– cells were sorted restrictively using FACS. Immediate re-analysis of sorted cells showed purification of both ALB⁺ and ALB^– cells. After the culture of each sorted cell subpopulation for 16 days on type IV collagen-coated dishes, FACS analysis demonstrated that ALB^– cells emerged from ALB⁺ cells, and that ALB⁺ cells emerged from ALB^– cells. Percentages of fractionated cells are shown at the top of each panel. Establishment of the gate was based on the profile of the negative control. (B) Semi-quantitative RT-PCR analysis of sorted EGFP⁺ (ALB⁺) and EGFP^– (ALB^–) cells. Note that ALB⁺ sorted cells expressed hepatocyte-lineage markers, such as ALB, At, G6P and TO, at much higher levels than did ALB^– cells. (C) Quantitative analysis of sorted ALB⁺ cells using real-time quantitative PCR. All data were normalized to the value of ALB^– sorted cells and fold-differences are shown. Representative data from a transfected stem cell clone are shown; three samples were examined for each protein. Data are mean±s.d. (D) (a-f) Several sorted ALB^– cells could form clonal colonies including both ALB⁺ and ALB^– cells at day 20. (g-i) Moreover, ALB⁺ sorted cells gave rise to ALB^– cells even 1 day after the initiation of culture, and finally formed mosaic colonies similar to those from ALB^– cells (black arrowhead in g, an original EGFP⁺ cell; white arrowhead in g, a daughter cell). (a,d,g) Phase contrast. (b,e,h) Enhanced green fluorescence protein (EGFP) imaging. (c,f,i) Merge. d-f are magnifications of a-c, respectively. Scale bars: 100 µm.