CD41⁺ Evi1 leukemia cells have a higher LIC than CD41⁻ cells. (A) The surface expression of CD41 on Evi1 leukemia cells. BM- and SP-MNCs were harvested from Evi1 leukemia mice and stained with an allophycocyanin (APC)-conjugated anti-CD41 antibody. Representative FACS data are shown. The graph shows frequencies of CD41⁺ cells in BM and SP derived from 9 individual leukemia mice. (B) Surface-marker profiles of Evi1 leukemia BM cells. Cells were stained with a PE-conjugated anti-CD41 antibody and APC-conjugated antibodies (c-kit, CD150, Gr-1, or Mac-1). Data for GFP⁺ cells are shown. Bar graphs show frequencies of c-kit⁺, CD150⁺, Gr-1⁺, and Mac-1⁺ cells in CD41⁺ and CD41⁻ fractions. Error bars indicate SD (n = 5; **P < .01, Student t test). (C) CD41⁺ and CD41⁻ Evi1 leukemia cells within a GFP⁺ fraction were sorted and subjected to further analysis. A representative FACS plot is shown. (D) The morphologic feature of CD41⁺ and CD41⁻ cells was examined by Wright-Giemsa staining, and the proportion of myeloblasts with a high nucleus/cytoplasm ratio was compared between these fractions. Pictures were captured by a BH-2 microscope equipped with an NC SPlan objective lens and a DP20 camera module (both from Olympus, Tokyo, Japan). Scale bars represent 10 μm. Error bars indicate SD (n = 5; **P < .01, Student t test). (E) CD41⁺ and CD41⁻ cells were cultured in MethoCult M3434 medium and examined their colony-forming activities. Representative pictures of colonies and a bar graph showing colony numbers from each fraction are presented. Scale bars represent 500 μm. Error bars indicate SD (n = 8; **P < .01, Student t test). (F) The cell-cycle status of CD41⁺ and CD41⁻ cells was analyzed by propidium iodide staining. Error bars indicate SD (n = 3). (G) Apoptosis analysis of CD41⁺ and CD41⁻ cells. Freshly isolated BM-MNCs were stained with a PE-conjugated anti-CD41 antibody, followed by staining with APC-conjugated Annexin V. Apoptotic rates in CD41⁺ and CD41⁻ fractions were determined by FACS. Error bars indicate SD (n = 4; *P < .05, Student t test). (H) CD41⁺ and CD41⁻ fractions were sorted from primary Evi1 leukemia BM cells and IV injected into sublethally irradiated (5.25 Gy) mice (1 × 10⁴ cells per mouse). Survival curves of mice transplanted with CD41⁺ (n = 9; red line) or CD41⁻ (n = 10; blue line) Evi1 leukemia cells are shown (P = .0016, log-rank test). (I-J) LIC frequencies in the 4 subfractions of Evi1 leukemia cells. (I) The GFP⁺/c-kit⁺ fraction in Evi1 leukemia BM cells was divided into 4 subfractions: Fr.1 (CD41⁺/CD150⁺), Fr.2 (CD41⁺/CD150⁻), Fr.3 (CD41⁻/CD150⁺), and Fr.4 (CD41⁻/CD150⁻). (J) LIC frequencies in each fraction as determined by a limiting dilution transplantation assay are shown. See supplemental Table 5 for detailed transplantation results.