Time and dose response effects of AMD3100 as a mobilizer of HPC (CFU-GM, BFU-E and CFU-GEMM) to the blood of mice. (A) Time course of mobilization in response to a single s.c. injection of 5 mg/kg AMD3100 into C3H/HeJ mice; results are for one full experiment (5 mice/group and time 0 control numbers of CFU-GM, BFU-E, and CFU-GEMM per ml of blood were 78 ± 18, 17 ± 2, and 10 ± 2, respectively). (B) Dose response analysis at 1 h post s.c. injections of 0.625, 1.25, 2.5, 5.0, or 10 mg/kg AMD3100 into C3H/HeJ mice; results are average of 7 mice/group from a total of two separate experiments. (C) Influence of multiple injections of 5 mg/kg AMD3100 or saline given on day 1; days 1 and 2; or days 1, 2, and 3 given 24 h apart, and analyzed 1 h after the last injection into C3H/HeJ mice; results are average of 14 mice for control group for different timed saline injections and 8 mice for each test group from a total of two complete experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

AMD3100 mobilizes competitive repopulating mouse HSCs with self-renewal capacity (A) and G-CSF synergizes this effect (B). (A, left) 18 C57Bl/6 mice (CD45.2) serving as donors were injected s.c. with 0.1 ml saline, and 36 C57Bl/6 mice were injected with 0.1 ml AMD3100 at 5 mg/kg (=∼100 μg/mouse). Blood was collected 1 h later and LDMNCs isolated. BM from nonirradiated B6.BoyJ (CD45.1) mice served as competitor cells. The ratio of donor (CD45.2) blood cells to competitor (CD45.1) BM cells was set as the number of LDMNC blood cells in 3 donor mice to a constant number of 0.5 × 10⁶ competitor BM cells equaling 3:1. A 2:1 ratio was the number of LDMNCs in blood of 2 donor mice to 0.5 × 10⁶ competitor cells, and a 1:1 ratio was LDMNC in 1 donor mouse to 0.5 × 10⁶ competitor cells. Results are based on analysis of transplantation of 6 lethally irradiated recipients per test group of i.v. infused cells. Right panel: BM cells were removed from femurs of primary mice 4 mo after injection of 3:1 saline or 3:1 AMD3100:competitor cell mixture and 2.5 × 10⁶ marrow cells from 3 mice of each group injected separately into 3 lethally secondary irradiated B6.BoyJ mice in a noncompetitive assay. Results are shown as percent CD45.2 C57Bl/6 donor cell chimerism in CD45.1 B6.BoyJ recipients. *P < 0.05; **P < 0.01; ***P < 0.001 compared with group 1 at each time point. (B) G-CSF and AMD3100 were administered as in Figure 2, part A, with G-CSF given twice a day for 2 d. Left panel: competitive repopulation in primary recipients 6 mo post transplant. The results are an average of mouse cells from five donors into six recipients each. Right panel: secondary repopulation in a noncompetitive assay 6 mo after transplant. Results are an average of 3 donor mice with cells transplanted into three recipients each. *P < 0.01 or **P < 0.001 compared with control.

Influence of AMD3100, G-CSF, and the combination of G-CSF plus AMD3100 on mobilization of NOD-SCID SRCs from normal human volunteers (apheresis samples), surface expression of adhesion molecules and chemotaxis of CD34⁺ cells, and homing of mobilized murine Sca1⁺Lin⁻ cells. (A) SRCs per kg in apheresis samples from G-CSF– (n = 3), G-CSF + AMD3100- (160 μg/kg; n = 3), and AMD3100- (240 μg/kg; n = 4) mobilized circulating blood. Each set of test samples was assayed simultaneously in limiting dilutions in conditioned NOD-SCID mice. For every sample, four different cell concentrations were used and four mice were transplanted with each cell concentration. Mice were assayed for chimerism 8 wk later; those that demonstrated >0.2% chimerism (total CD45⁺ cells in BM) were considered to be positive. Percentage of negative mice were used to calculate SRC frequencies. (B, i) Expression of CD49d (VLA-4), CD49e (VLA-5), CD26L (L-selectin), and CXCR4 on mobilized CD34⁺ cells (mean ± 1SEM of percent positive cells of five to seven different G-CSF samples, three AMD3100 plus G-CSF samples, and four BM samples). (B, ii) Mean fluorescent intensity (MFI) of positive samples. Same number of samples evaluated as in B, i except G-CSF group has a different number (n =7). (C) CD34⁺ cells isolated from G-CSF (n = 6) and G-CSF plus AMD3100 (n = 3) mobilized peripheral blood were assessed for chemotaxis to SDF-1/CXCL12 (100 ng/ml) and results expressed as percentage of migrated cells. Data for each sample were collected in duplicate. (D, i) Homing of CD45.2⁺ Sca1⁺Lin⁻ BM cells from C57Bl/6 mice into lethally irradiated CD45.1⁺ B6.BoyJ BM from samples mobilized with G-CSF (2 times/d for 4 d as in Fig. 2B; n = 3), G-CSF + AMD3100 (5 mg/kg; n = 4), and AMD3100 (5 mg/kg; n = 3). Homing was assessed as in Materials and methods. (D, ii) Competitive repopulation of CD45.2⁺ BM cells recovered after homing shown in D, i. Results of chimerism are after 4 mo in 2° irradiated B6.BoyJ recipients. For A–D, significant differences compared with G-CSF: *P < 0.001; **P < 0.01; ***P < 0.05; all other values are P > 0.05. NA, data not depicted.

Comparative AMD3100 enhancement of G-CSF–induced mobilization of CFU-GM, BFU-E, and CFU-GEMM in: (A) 2 d G-CSF: C57Bl/6, C3H/HeJ, DBA/2, (B) 4 d G-CSF: C57Bl/6, and (C) 4 d of G-CSF: Fancc^−/− and ^+/+ mice. The results shown in A are an average from a total of three experiments of 15 C57Bl/6, 15 DBA/2, and 11 C3H/HeJ mice. Results in B are for one experiment of 5 mice/group. Results shown in C are the averages of 15 wild-type and 8 Fancc^−/− mice per group averaged from a total of two experiments. For parts A–C, all experimental points within a group are at least P < 0.001 compared with the control for the specific progenitor cell of each mouse strain.

Influence of AMD3100, alone and with G-CSF, on circulating HPCs in healthy human volunteers. (A) Influence of multiple injections of AMD3100 alone. Three volunteers were injected i.v. with AMD3100 (80 μg/kg) on the first day (day 1) and again at 24 h. Progenitors/ml blood were assessed preinjection (day 1, 0 h) and at the noted time intervals. Fold changes are calculated based on the following day 1, 0 h progenitors/ml for three donors: CFU-GM (387, 78, 201), BFU-E (712, 78, 217), and CFU-GEMM (77, 29, 93) grown in methylcellulose cultures in the presence of Epo, stem cell factor, and interleukin-3. The fold changes for CFU-GM also include progenitor cells/ml as calculated in agar culture medium with GM-CSF and stem cell factor (control numbers for three donors: 108, 18, 54). *P < 0.001 compared with 0 h counts of that particular day. (B) Influence of AMD3100 on mobilization of circulating HPCs in healthy human volunteers receiving G-CSF or G-CSF plus AMD3100 (160 μg/kg). The results shown are the mean plus range of fold increases of the absolute numbers of CFU-GM and CFU-GEMM per ml of blood for two healthy volunteers each without apheresis. Fold changes are based on the following numbers of G-CSF day 5, 0 h control progenitor cells/ml: group I: G-CSF (CFU-GM: 34403 and 27194; CFU-GEMM: 14363 and 10083); group II: G-CSF (4 d) plus AMD3100 (day 5; CFU-GM: 11968 and 2675; CFU-GEMM: 3366 and 510), and group III: G-CSF (5 d) plus AMD3100 (day 5; CFU-GM: 2698 and 2380; CFU-GEMM: 1028 and 1238). (C) Results of apheresis after mobilization of circulating HPCs. Results are expressed as total progenitors collected (in thousands) for donors receiving G-CSF (n = 3), G-CSF + AMD3100 (160 μg/kg; n = 3), or AMD3100 (240 μg/kg; n = 4). *P < 0.01 for G-CSF– or AMD3100 versus G-CSF + AMD3100-mobilized cells. (D) Results of apheresis shown in C but expressed as progenitors mobilized per kg donor. *P < 0.05 for same comparisons as in C.