Cell cycle synchronization of CD34⁺ cells and LTC-ICs.

Freshly isolated CD34⁺ cells reside in the G₀/G₁ phase of the cell cycle (A). Cells were prestimulated with SCF, FL, and TPO during 16 hours, after which they were reversibly blocked at the G₁/S transition by a 24-hour treatment with 2 μg/mL aphidicolin (B) or kept in initial conditions (C). Cells were washed extensively and replated in fresh medium and cytokines to allow cell cycle progression. Cells quickly entered S phase after 3 hours (D). The cell cycle progression could be followed at further time points: After 6 and 9 hours, cells progressively reached G₂+M (E-F). After 24 hours, the cells were back to nonsynchronized proliferation (G). Cell cycle status was determined by DNA staining with propidium iodide. A representative experiment is shown. The proportion of LTC-ICs in S phase was measured by HU killing assays (n = 4) at indicated time points.