Ink4a/Arf−/− Dlk+ cells were transduced with either control enhanced green fluorescent protein (EGFP) or Bmi1 12-18 hours after purification. Enforced expression of Bmi1 was verified by western blot analysis (Fig. 4A). Exogenous Bmi1 in Ink4a/Arf−/− Dlk+ cells did not significantly increase colony number (Fig. 4B). Of note, however, the diameter of Bmi1-overexpressing colonies was significantly larger than that of the control colonies (Fig. 4C).

Furthermore, flow cytometric analyses showed that the percentage of Ink4a/Arf−/− Dlk+ cells labeled with EGFP was higher in Bmi1 cultures than in control cultures (22.6% ± 2.3%, 14.0% ± 1.2%, and 8.8% ± 0.7% versus 8.4% ± 1.1%, 3.4% ± 0.5%, and 2.1% ± 0.2% at days 7, 14, and 28 of culture, respectively) (Fig. Vismodegib in vitro 4D). We next carried out single-cell sorting of Dlk+ cells contained in primary colonies at days 14 and 28 of culture in order to evaluate their self-renewal capacity in terms of replating activity. Dlk+ cells overexpressing Bmi1 gave rise to

3.1-fold to 4.0-fold more secondary colonies than the control ICG-001 (Fig. 5A). Secondary colonies were generated in a similar fashion to the original colonies. Immunocytochemical analyses demonstrated that the frequency of Alb+CK7+ bipotent cells was significantly higher in secondary colonies derived from Dlk+ cells collected from the primary Bmi1-transduced Ink4a/Arf−/− colonies at days 14 and 28 of culture (Fig. 5B,C). In contrast, Bmi1−/−Ink4a/Arf−/− Dlk+ cells behaved like Ink4a/Arf−/− Dlk+ cells (Supporting Fig. 5). Although loss of Bmi1 still affected the function of Ink4a/Arf−/− hepatic stem/progenitor cells to some extent, these findings indicate that Ink4a/Arf is the major target of Bmi1 in hepatic stem medchemexpress cells as in HSCs and NSCs. We then tested whether the loss of both Ink4a and Arf is enough for the transformation of hepatic stem cells. Considering

that a large number of cells were necessary for transplantations assays, these cells were allowed to form colonies in culture for 28 days. Immunocytochemical analyses showed that more than 90% of cells transduced with Bmi1 expressed both EGFP, a marker antigen for retrovirus integration, and Flag-tagged Bmi1 (Supporting Fig. 6). Subsequently, a total of 2 × 106 transduced cells were transplanted into the subcutaneous space of NOD/SCID mice (Fig. 5D). Although all the mice transplanted with Bmi1-transduced Ink4a/Arf−/− Dlk+ cells developed tumors, none of those transplanted with control Ink4a/Arf−/− Dlk+ cells did. Histological analyses revealed that the subcutaneous tumors consisted of both Alb+ parenchymal cells and a CK7+ glandular structure (Fig. 5D). The histological finding is consistent with our previous observation in tumors derived from Bmi1-transduced wild-type hepatic stem cells.3 These findings clearly indicate that repression of the Ink4a and Arf genes is not enough for Bmi1 to achieve its tumorigenic potential in hepatic stem cells.