much denser and more cellular marrow (Fig. 4B). While the trabecular bony structures, osteoblasts and sinusoid vessels were preserved, the hematopoietic cell components were significantly expanded in AMD3100-treated mice. Furthermore, 3 of 4 PBS- injected- mock control mice had large areas filled with lipid droplets and devoid of cells (Fig. 4B, indicated as stars). These large acellular areas are commonly associated with radiation[33]. In contrast, the large areas (fat droplets) were not found in any of the mice receiving AMD3100 (8 of 8 mice) (Fig. 4B). In addition, we collected one femur and one tibia from each mouse and measured total nucleated marrow cells, total donor-derived CD45.1+ KLS hematopoietic stem cells (c-kit+ Lin2 Sca-1+ cells) and recipient-origin CD45.2+ KLS cells. As shown in Fig. 4C and 4D, AMD3100-treated mice had significantly higher numbers of total marrow nucleated cells and total donor-derived CD45.1+ KLS cells per one femur and one tibia (One femur plus one tibia account for ,7.5% of whole body marrow cells[34]). There was no difference in the recipient- derived CD45.2+ KLS cell (Fig. 4E). These data again suggest that post transplant administration of AMD3100 selectively enhances donor cell marrow engraftment. The total nucleated cells, the donor-derived CD45.1+ KLS cells, and the recipient-origin CD45.2+ KLS cells in the spleen were not significantly different between AMD3100-treated mice and PBS- injected mock mice (data not shown). 5. Post transplant administration of AMD3100 mobilizes recipient’s residual stem cells, increasing marrow niche availability We examined the mechanisms by which AMD3100 selectively enhances donor cell engraftment. We performed additional experiments to investigate the effects of post transplant AMD3100 administration on the mobilization of recipient HSCs and the homing of donor HSCs. Lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (10.5 Gy) were injected via tail vein with sorted KTLS hematopoietic stem cells (2500 cells/mouse) ob- tained from C57BL/Ka CD45.1 Thy1.1 donor mice. At 48 hours after transplantation, recipient mice were injected subcutaneously with a single dose of AMD3100 at 5 mg/kg or PBS control. Twenty-four hours after this injection, the mice were sacrificed, 2 femurs and the spleen harvested, and recipient-originated CD45.2+ KLS and donor-derived CD45.1+ KLS stem cells measured. As shown in Fig. 5A, the mice receiving AMD3100 injection had significantly lower number of recipient CD45.2+ KLS stem cells in the marrow, indicating that AMD3100 mobilizes the recipient stem cells from the marrow. In our 2 separate sets of experiments in which lethally irradiated C57Bl/6 CD45.2 Thy 1.2 mice received sorted CD45.1+ hematopoietic cells (2500 KTLS cells) and 2 femurs harvested at 72 hrs post transplantation, we were not able to measure donor derived CD45.1+ KLS cells in the marrow samples using flow cytometry analysis because of the limited number of KTLS cells infused. Interestingly however, we found that AMD3100-treated mice had significantly higher number of marrow derived CFU-GM and BFU-E, indicating enhanced donor marrow engraftment (Fig. 5B). Furthermore, although the number of recipient-origin KLS stem cells in the spleen was similar in both groups (Fig. 5C), the AMD3100-treated mice had significantly higher number of donor derived CD45.1+ KLS stem cells in the spleen (6.461.5%) compared to PBS control mice (0.9660.36%; p,0.01) (Fig. 5D). These results suggest that AMD3100 administered shortly after transplantation increases donor stem cell homing efficiency by expanding the available marrow niches. The available niches are likely expanded as a consequence of AMD3100-induced mobili- zation of recipient’s residual bone marrow cells from marrow AMD3100 Enhances Engraftment niches, allowing a competitive advantage for the healthy donor cells. To determine if prolonged administration of AMD3100 could still acutely mobilize hematopoietic stem/progenitor cells, we measured colony-forming units and white blood cell counts in peripheral blood 4 hours after giving AMD3100 in transplanted mice that had received AMD3100 every other day beginning at day +2 for 60 days. As shown in Fig. 5E, AMD3100 given every other day for 60 days mobilized hematopoietic progenitor cells at a level comparable to those in na¨ıve mice receiving the first dose of AMD3100 injection. 6. Post transplant administration of AMD3100 increases donor cell division The proliferative status of HSCs affects their ability to engraft[11,14,19] and the speed of hematological recovery[12,13]. Mobilized HSCs are innately more proliferative[35]. Furthermore, binding of SDF-1 to CXCR4 receptor inhibits the division of primitive HSC cells[14,19,20]. To determine whether blocking CXCR4 receptor with AMD3100 could promote in vivo cycling of donor HSCs, we injected 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) - labeled donor KTLS cells (Fig. 6A) and measured their division status at Day +7 post transplant (Fig. 6A). With each cell division, the CFSE is diluted and fluorescence intensity decreases in a step-wise fashion. Compared to control mice, AMD3100- injected mice had reduced percentage of cells in higher intensity peaks (Fig. 6A, lower panels, dot plots and histograms), indicating more rounds of cell division. Additionally, AMD3100-treated mice had much higher number of donor derived CD45.1+ cells compared to PBS-injected mice (Fig. 6A: total CD45.1+ cells). To rule out direct stimulating effects of AMD3100, we measured 3H incorporation in vitro in marrow cells co-cultured with various concentrations of AMD3100 or G-CSF. The proliferating effect of AMD3100 on marrow cells was minimal - approximately 100-1000 fold less than that induced by G-CSF (Fig. 6B). 7. Post transplant administration of AMD3100 maintains hematopoietic stem cell’s long-term repopulating capacity The ability of AMD3100 to increase in vivo donor cell division and proliferation raises the concern that prolonged post transplant administration of AMD3100 may exhaust stem cell pool. To determine the effects of AMD3100 on HSC self-renewing and long-term repopulating capacities, we performed secondary transplant experiments (Fig. 7). We harvested bone marrow cells from transplanted C57BL/6 CD45.2 Thy1.2 recipient mice at day +65 (i.e., primary transplant recipients). These primary transplanted mice received HSCs from donor C57BL/6 CD45.1 mice and were given post transplant administration of AMD3100 at 5 mg/kg body weight or PBS subcutaneously every other day from day +2 to +60. The bone marrow cells from primary transplant recipients were then injected intravenously into lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (i.e., secondary transplant recipients). The whole marrow cells harvested from 1 femur and 2 tibias of one primary mouse were injected into one secondary mouse (one to one matched transplant). At ,7 weeks after transplant, the secondary C57BL/6 CD45.2 Thy1.2 transplanted recipient mice were bled and the CD45.1+ donor cell contribution in peripheral granulocytes determined. In the secondary transplant recipients that received bone marrow from AMD3100-treated primary transplant mice, 79614% blood granulocytes were derived from CD45.1+ donor cells, compared PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11316