Disruption of polarized membrane domains in cycling normal human HSPCs. (A-C) Analysis of the cell cycling status (A), homing in NSG mice (B), and membrane domain polarity (C) of normal human MPB CD34⁺ cells before ex vivo culture (day 0). (D-F) Analysis of the cell cycling status (D), homing in NSG mice (E), and membrane domain polarity (F) of normal human MPB CD34⁺ cells after ex vivo culture for 4 days in stimulatory conditions in the presence of SCF, FLT3, and TPO. (G-I) Analysis of the cell cycling status (G), homing in NSG mice (H), and membrane domain polarity (I) of normal human MPB CD34⁺ cells after ex vivo culture for 4 days in nonstimulatory conditions in the presence of SCF alone. (J) Percentage of human CD34⁺ cells with polarized domain (± SEM) and percentage of cells in the G₀ phase of the cell cycle (± SEM) for cells at baseline, and cells cultured for 4 to 6 days in stimulatory and/or nonstimulatory conditions (n = 4 donors). (K) Percentage of human cell homing in NSG mice 16 hours after IV injection of uncultured cells or cells cultured for 4 days in stimulatory or nonstimulatory conditions (t test; horizontal bars represent average human cell homing). (L) Immunofluorescence labeling of human CD34⁺ cells with CD82 antibodies after sorting cells in the G₀, G₁, and S/G₂/M phases of the cell cycle. (M) Percentage of cells in G₀, G₁, and S/G₂/M phases of the cell cycle with polarized CD82 (± SEM, n = 3 donors, t test). (N) Percentage of normal CD34⁺ cells in G₀, G₁, and S/G₂/M phases of the cell cycle and percentage of leukemic blast cells from patients with AML, CML, and ALL (Leuk) adhering to osteoblasts in vitro (± SEM, n = 3 donors, t test). Horizontal bars in K represent means. Scale bars in panels C, F, I, and L equal 5 μm (*P ≤ .05; **P < .01; ***P < .001).