Bmi1+ cells and their progeny cells can self-renew in vivo and in vitro. (a) In situ hybridization of Bmi1 in ESCC samples induced by 4NQO for 15 weeks (left) and 22 weeks (right). The brown area is the expression signal of the Bmi1 gene. (b) Bmi1+ cells are labeled in Bmi1CreER;RosatdTomato mice following a single dose of tamoxifen and traced for 7, 14, 21, and 42 days. Red represents Tomato-positive Bmi1-expressing cells and their differentiated progeny cells. Green labels integrin α6, a marker of the epithelial basement membrane. Nuclei are labeled in blue. (c) Three days after tamoxifen injection, EpCAM+Tomato+ double-positive cells sorted by flow cytometry were cultured into tumorspheres in vitro. Scale bars are 100 μm.