Cell-cycle regulators expression and flow cytometric cell-cycle analysis. Expression of cell-cycle regulators (A). Eisen tree map computed using the GeneSpring gene tree and the Pearson correlation equation on the modulated transcripts involved in cell-cycle regulation. The normalized signal-based coloring legend is shown at the bottom of the figure. Cell-cycle distribution of normal and CML stem/progenitor cells according to CD34 expression (B). Lin⁻ CD34⁺ and Lin⁻ CD34⁻ cells were isolated from CML patients at diagnosis and healthy donors and analyzed by flow cytometry for their kinetic status. In panel C, we assessed the clonogenic growth in semisolid medium of CML and normal stem/progenitor cells as well as the LTC-IC activity of 2 CML patients (pt). Our data show a significantly higher frequency of CML Lin⁻CD34⁻ and Lin⁻CD34⁺ cell-derived CFU-C compared with normal samples. Specifically, the clonogenic efficiency of leukemic CD34⁻ cells was 0.4% ± 0.08% versus 0.15% ± 0.05% in normal samples. Interestingly, CML Lin⁻CD34⁻ cells also showed LTC-IC activity in 2 of 2 patients studied (the results are expressed as the mean ± SD of quadruplicate cultures of secondary CFU-C × 10⁴ cells). As described in “Methods,” FACS-sorted Lin⁻CD34⁻ cells were also kept in culture for 7 days. After 1 week (t7), cells expressing the CD34 antigen were purified and analyzed for their cell-cycle distribution and BCR-ABL content. As shown in panel D, CML Lin⁻CD34⁻ cells rapidly up-regulated the CD34 antigen on a cell fraction and progressed through the cell cycle. These findings were associated with the higher values of BCR-ABL gene rearrangement as demonstrated by RQ-PCR. The results of panels B and C are expressed as mean ± SD of 3 (B) and 6 (C) different experiments; *P < .05.