Nestinhighbreast CSC have potent tumorigenicity. The isolated breast CD44+CD24− CSC from 12 freshly resected breast cancer tissues were stained with anti-Nestin, and the Nestin+ and Nestin− breast CSC were purified as Nestinhigh or Nestinlow CSC by flow cytometry, respectively. The levels of Nestin expression in Nestinhigh, Nestinlow, and unstained control CSC were determined by western blotting, and these CSC were tested for their capacity to form mammospheres in vitro and to induce solid tumors in vivo. Finally, the levels of Nestin expression in the solid tumors induced by Nestinhigh, Nestinlow, or control CSC were determined by western blotting. Data are representative charts and images, and expressed as the mean ± SD of each group of samples (n = 12 per group). (A) Flow cytometric analysis of CSC. (B) Western blot analysis of the levels of Nestin expression. (C) Mammosphere formation. (D) The rates of tumor formation in SCID mice (n = 10 animals per group) from three separate experiments. (E) Western blot analysis of Nestin expression in the formed tumors (n = 2 to 10 per group). CSC, cancer stem cell; SCID, severe combined immunodeficiency; SD, standard deviation.