AMD3100 Enhances Engraftment administration of AMD3100 led to a significant reduction in the levels of 9 pro-inflammatory cytokines/chemokines (IL-1, IL-3, IL- 12, IL-17, tissue necrosis factor-a, GM-CSF, interferon-l, macrophage-inhibitory protein-1 and eotaxin) (Fig. 3C). We next examined if AMD3100 could protect mice from lethal irradiation without the need for HCT. Lethally irradiated C57BL/ 6 CD45.2 Thy1.2 mice (10.5 Gy) were given AMD3100 at 5 mg/ kg body weight either at day 2 post irradiation and then every other day until the death of mice or within one hour post irradiation and then daily for 5 days. As shown in Fig. 3D, without HSC transplant, all the irradiated mice died by day 17 and there were no significant differences in animal survival between AMD3100-treated mice and PBS-injected mice. These data imply that the effects of AMD3100 on HSCs may indeed play a critical role in improving animal survival. 4. Post transplant administration of AMD3100 enhances donor marrow cell engraftment To further investigate the effects of AMD3100 on marrow hematopoietic progenitor/stem cell recovery, we administered the last AMD3100 injection at day +56 and sacrificed the mice at day +65 and harvested bone marrow for CFU-GM assay, histopath- ological examination and flow cytometry analyses. Compared to PBS-injected- mock control mice, mice receiving AMD3100 injection had increased frequencies of CFUs-GM (represented as colony numbers/105 marrow nucleated cells, Fig. 4A) and had a Figure 4. Post transplant administration of AMD3100 enhances donor cell marrow engraftment. The mice as described in Fig. 1 were sacrificed at Day +65 post transplantation, and 2 femurs and 2 tibias were harvested and processed and analyzed as described in Methods. A: CFU-GM assay. CFU-GM colonies were measured in individual mice using MethoCultH GF 3434 mouse colony-forming cell assay. B: Histology. Bone marrow from individual mouse was examined by microscopy (4X: top panel and 100X: bottom panel) after decalcification and H/E staining. Trabecular bony structure, osteoblasts, hematopoietic cells, sinusoid vessels and acellular areas filled with lipid droplets were indicated. Normal mice did not receive transplant or AMD3100 injection. C: Total nucleated cell number from 1 femur and 1 tibia. D: Donor-derived CD45.1+ KLS stem cell number from 1 femur and 1 tibia of individual mouse. E: Recipient-origin CD45.2+ KLS stem cell number from 1 femur and 1 tibia of individual mouse. *: p,0.05. doi:10.1371/journal.pone.0011316.g004 PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11316