Cy/G-CSF treatment induces changes in the osteoblastic niche. (A) Experimental strategy. For HSC mobilization, mice were injected intraperitoneally with Cy (200 mg/kg) and then on successive days with human G-CSF (250 μg/kg/day) administered as a single daily subcutaneous injection. The day of Cy treatment was considered day −1 and the first day of G-CSF treatment as day 0. The timeline of Cy and G treatments for each experimental group is shown. For in vivo BrdU labeling experiments, mice in every experimental group (untreated, D0, D2, and D4) received 9 successive injections of BrdU (1 mg/mouse in 0.9% saline, intraperitoneally, indicated by green arrows), over a 4.5-day period. All experimental groups began BrdU treatment 12 hours after Cy injection in the first experimental group (D4) and received continued BrdU injections every 12 hours thereafter (indicated by green arrows). Thus, all animals were sacrificed (SAC) after the same time period of BrdU exposure. (B) Osteoblast (Opn⁺CD45⁻Ter119⁻) frequencies are increased in Cy/G-treated mice. Representative FACS plots are shown (n = 10), with the frequency of Opn⁺CD45⁻Ter119⁻ shown in each gate. (C) Total numbers of osteoblasts are increased in response to Cy/G (D2) treatment but return to normal numbers by D4. Data are plotted as means plus or minus SD. Differences were significant for untreated versus D0 (*P < .05) and untreated vs D2 (*P < .05). Differences were not significant for untreated versus D4 (*P > .05). (D) Osteoblasts proliferate in response to Cy/G treatment as demonstrated by increased in vivo uptake of BrdU in osteoblasts from mobilized (D0, D2, and D4) as opposed to untreated (untreated, far left) mice. Histograms indicate the percentage BrdU⁺ cells (determined by control staining of osteoblasts from mice that did not receive BrdU) among Opn⁺CD45⁻Ter119⁻ cells. This is representative of 2 independent experiments.