G-CSF drives proliferation and cell-cycle entry within the HSC compartment. (A) Representative time-lapse images of prospectively sorted HSCs on hydrogel microwells at the beginning of acquisition (t = 24 hour) and final time point (t = 80 hour). Proliferation of single HSCs was tracked over time with some cells undergoing no divisions (red) and others dividing (blue). (B) Frequency of proliferation (≥ 1 cell division) for prospectively sorted, single HSCs plated on hydrogel microwells in serum-free, defined media supplemented with FL and SCF (FS) alone or in combination with 40 ng/mL G-CSF. Differences between proliferation frequency for CB (black) and normal BM (gray) at 80 and 92 hours, respectively, were statistically significant (P = .0004, paired, 1-tailed t test). (C) Representative flow cytometric detection of DNA (Hoechst 33342) and RNA (Pyronin Y) in prospectively sorted HSCs cultured on hydrogel substrate for 68 hours with gates corresponding to cell-cycle phase. (D) Frequency of cells in active cycle (ie, G1-S-G2-M) after 60-68 hours of culture with FS or FS+G. Difference in frequency of cells in active cycle is statistically significant (P = .0085, paired, 1-tailed t test).