Figure 7. Post transplant administration of AMD3100 main- tains HSC’s long-term repopulating capacity. Bone marrow cells from transplanted C57BL/6 CD45.2 Thy1.2 recipient mice (i.e., primary transplant recipients) were harvested at day +65, and injected intravenously into lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (i.e., secondary transplant recipients) (one to one matched transplant). The primary transplanted mice received HSCs from donor C57BL/6 CD45.1 mice and were given post transplant administration of AMD3100 at 5 mg/kg subcutaneously every other day from day +2 to +60 (AMD3100-primary) or PBS (PBS-primary). 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. (n = 6 in each group). doi:10.1371/journal.pone.0011316.g007 AMD3100 in our current study would suggest its potential use more broadly for any toxic preparatory regimen and for other inflammatory disease processes. Our present studies demonstrate that post transplant adminis- tration of AMD3100 selectively enhances donor hematopoietic cell engraftment in a myeloablative setting. Our data suggest that there are three related mechanisms involved in this selective enhance- ment of donor hematopoietic cell reconstitution by post transplant administration of AMD3100 (Fig. 8). The first is that there is mobilization of residual radioresistant host HSCs and thus further opening of hematopoietic niches and a selective advantage for engraftment of the healthy donor HSCs. Secondly, AMD3100 reverses the proliferation-inhibitory effect of SDF-1. SDF-1 arrests the cycling of primitive HSCs[14,19,20] and thus blocking the SDF-1/CXCR4 interaction results in proliferation/expansion of primitive HSCs. Thirdly, mobilized HSCs are innately more proliferative than HSCs in the niches[35]. Mobilizing the donor HSCs results in more cell division, further increasing the numbers of donor derived marrow cells. Compared to donor HSCs, recipient residual HSCs likely have survival disadvantage and/or impaired engraftment/proliferation capacity due to the insults from total body irradiation, resulting in selective engraftment and expansion of donor hematopoietic cells. AMD3100 Enhances Engraftment The number of available niches determines the degree of donor HSC engraftment. In non-ablated mice, AMD3100 can mobilize recipient HSCs, vacate niches and improve donor HSC engraftment[9]. Our data suggest that even in a myeloablative setting, post transplant administration of AMD3100 could further increase the availability of niches and/or keep the marrow niches open when given repeatedly. The restoration and expansion of niches occurs soon after irradiation and is evident at 48 hours post irradiation[47]. Salter et al recently found that infusion of endothelial progenitor cells alone could rescue mice from total body irradiation, suggesting that some host HSCs could survive total body irradiation[48]. Transplant preparative conditioning such as total body irradiation increases the levels of SDF-1 in the marrow. SDF-1 arrests the cycling of very primitive HSCs[14,19]. In contrast, administration of an SDF-1 antagonist enhances HSC engraftment in vivo[9,14]. Furthermore, Passegue et al have shown that mobilized HSCs are more proliferative than HSCs in the niches[35]. HSC expansion is crucial to hematological recovery following transplantation[12,13]. This would also explain the enhancement of donor cell engraftment and recovery seen with repeated administration of AMD3100 following HCT. We administered AMD3100 at day +2 post transplant and continued the injection every other day. AMD3100 blockade of CXCR4 is reversible and the half-life of AMD3100 is short (terminal half life of ,5 hours). Thus, it is very likely that the 48 hour interval between AMD3100 injections could allow HSCs to mobilize from ‘‘quiescent-storage’’ niche, enter the cell cycle to expand, and then return to their marrow niches and become quiescent again. This repeated, periodic and pulsed release of HSCs followed by expansion and return to quiescent state may be important in the enhanced hematological recovery observed in our study. Although the effects of persistent and continuous blockade of CXCR4 on hematopoiesis following transplant remain to be elucidated, there is a concern that permanent blockade of CXCR4 might actually impair hematopoiesis. Permanent blockage of CXCR4 may result in the failure of proliferating HSCs to return to ‘‘quiescent-storage’’ niches, leading to increased rate of cell death and impaired hematological recovery. In our current study, AMD3100 was given every other day for approximately 60 days. We are currently testing various administration regimens including shorter duration of treatment and the use of AMD3100 as part of conditioning regimens. Our preliminary study indicated that shorter duration of treatment such as every other day for 30 days was as effective as 60 day administration (unpublished data). There were several conflicting articles reporting the effects of SDF-1 and CXCR4 antibody on hematopoiesis after trans- plant[14,17,19,20,49,50]. For example, Peled et al reported reduced homing and impaired engraftment with treatment of graft cells with CXCR4-blocking antibody[17], while Plett et al suggested increased repopulating potential of treated CD34+ cells[50]. One study suggested that in vivo administration of SDF- 1 was accompanied by enhanced transplantability of human CD34+ cells in NOD/SCID mice[20], whereas others reported increased engraftment with SDF-1 antagonist[9,14]. These discrepancies are likely related to the differences in transplant models, the proliferative status of the grafts, the specificities of CXCR4 antibodies/antagonists and even the concentration of SDF-1. SDF-1 exhibits ‘‘concentration-dependent bifunctional effects’’, i.e., SDF-1 can attract or repel cells at different concentrations[50]. Furthermore, AMD3100 may act as CXCR4 partial agonist at high concentration[51]. Our findings are similar to those reported by Plett et al[50], Chen et al[9], Bowie et al[14], and Abraham et al[49] using a novel CXCR4 antagonist (4F- PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e11316