extracellular matrix. CXCR4 is expressed on HSCs, osteoblasts, T cells and other inflammatory cells. SDF-1 is the only known ligand for the CXCR4 receptor. Following transplantation conditioning, the level of SDF-1 in the marrow microenviron- ment significantly increases. The gradient difference of SDF-1 between the niche microenvironment and blood stream promotes HSC transendothelial migration and homing to the niches[17]. Additionally, SDF-1 in the marrow extracellular matrix binds to the CXCR4 receptors on HSCs and has dual effects: tethering HSCs in the niches, and arresting the cycling of very primitive HSCs[14,19,20]. AMD3100 is a highly specific and reversible antagonist of the CXCR4 receptor[21]. AMD3100 is a bicyclam molecule with an aromatic bridge[21], and binds with extreme specificity to CXCR4, with contacts at amino acid positions 171, 182, 193 and 262 of CXCR4. AMD3100 was originally studied as an agent for the treatment of patients infected with human immunodeficiency virus (HIV) since some strains of HIV require CXCR4 as a co-receptor for cell entry. However, during initial clinical trials, rapid and reversible leukocytosis was noted in human volunteers and in HIV patients treated with AMD3100 and led the focus of AMD3100 development towards hematopoietic stem cell mobilization[22,23]. AMD3100 blockade of the SDF-1/CXCR4 interaction results in rapid release of HSCs from marrow niches and mobilization of HSCs into the peripheral blood[17,24,25,26,27,28]. AMD3100 was recently approved by the Food and Drug Administration (FDA) to be used in combination with G-CSF for stem cell mobilization in patients with non-Hodgkin’s lymphoma and multiple myeloma. Most of the work with AMD3100 has been exclusively on its use as a mobilization agent[24,27,28,29,30]. We hypothesized that post transplant administration of AMD3100 would enhance donor cell reconstitution in myeloablative recipients. We reasoned that AMD3100 would induce in vivo proliferation of transplanted donor stem cells and increase additional availability of marrow niches for donor stem cell engraftment. We report here the effects of post transplant administration of AMD3100 on animal survival and on donor cell engraftment in a mouse transplantation model. AMD3100 Enhances Engraftment Results 1. Post transplant administration of AMD3100 significantly improves animal survival We first examined the effects of post transplant administration of AMD3100 on animal survival following transplantation. We injected sorted c-Kit+ Thy1low Lin2 Sca-1+ hematopoietic stem cells (KTLS cells) from C57BL/Ka CD45.1 Thy1.1 mice via tail vein into lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (250 KTLS cells/recipient mouse). Transplanted mice were then injected subcutaneously with PBS control buffer or AMD3100 (5 mg/kg body weight) every Monday, Wednesday and Friday beginning at day +2 post transplant for a total of 56 days. Animal survival was monitored daily. We performed 3 separate sets of experiments with 10 mice per group in each experiment. The mice received 9.5–10.5 Gy total body irradiation. At the 10.5 Gy dose, 60% of transplanted mice receiving PBS control died within 2 weeks following transplantation. In contrast, only 13.363% (mean 6 SEM) of transplanted mice treated with AMD3100 died. The animal survival results from our 3 separate sets of experiments are shown in Fig. 1. These data indicate that post transplant administration of a CXCR4 antagonist (AMD3100) significantly improves animal survival following HCT. 2. Post transplant administration of AMD3100 selectively enhances donor cell recovery To determine the effects of AMD3100 on hematological recovery, we performed blood counts weekly beginning at Day +7. The total nucleated cells, the donor-derived cells (CD45.1+ cells), and the recipient-origin cells (CD45.2+ cells) in the peripheral blood were measured using multi-color flow cytometry analyses. As demonstrated in Fig. 2A, mice given AMD3100 had faster nucleated cell recovery. The difference in blood nucleated cell counts between AMD3100-treated mice and PBS-injected control mice became statistically significant beginning at Day +36, and was completely attributed to faster donor-derived cell recovery (Fig. 2B). Contribution of recipient-derived cells (CD45.2+) was minimal (,10%) in all transplanted mice and there was no difference in the number of recipient-derived CD45.2+ cells Figure 1. Post transplant administration of AMD3100 improves animal survival in a murine transplantation model. KTLS cells from C57BL/Ka CD45.1 Thy1.1 mice were injected via tail vein into lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (250 KTLS cells per mouse). Beginning 2 days after transplant (day +2), the mice were injected subcutaneously with PBS control or AMD3100 at 5 mg/kg body weight every Monday, Wednesday, and Friday until day +56. Animal survival was monitored daily. In experiment #1 and #2, the mice received 10.5 Gy total body irradiation using a Cesium irradiator. In experiment #3, the mice received 9.5 Gy total body irradiation. (n = 10 mice in each group in each experiment). doi:10.1371/journal.pone.0011316.g001 PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11316