The Combination of AMD3100 Plus GROβ Rapidly Mobilizes Hematopoietic Stem Cells with Enhanced Homing, Adhesion and Survival Properties

Abstract

Mobilized peripheral blood stem cells (PBSC) are the primary cell source for hematopoietic transplantation. In mice, combined single administration of AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4 with GROβ, a CXCR2 agonist, rapidly mobilizes hematopoietic stem and progenitor cells (HSPC) within 15 minutes of administration that is equal or greater in magnitude to a multiday regimen of G-CSF and demonstrates significant synergy compared to either compound used alone. In non-competitive transplant models, AMD3100 plus GROβ mobilized 6.2±0.3 CRU/105 PBSC compared to 1.8±0.2, 1.9±0.2 and 2.0±0.3 CRU, respectively, for G-CSF, AMD3100 or GROβ used alone. Furthermore, AMD3100 plus GROβ mobilized long-term repopulating cells that show superior hematopoietic engrafting capacity in lethally irradiated mice. Analysis of chimerism at 6 months post competitive transplant of mobilized PBSC compared to normal bone marrow at a ratio of 1:1, showed 15±2%, 10±2% and 28±2% donor chimerism for G-CSF, AMD3100 and GROβ mobilized PBSC, respectively, whereas the equivalent number of AMD3100 plus GROβ mobilized PBSC produced 55±6% donor chimerism. In order to define the mechanism(s) responsible for increased engraftment of AMD3100 plus GROβ mobilized PBSC, we performed extensive phenotypic and functional analysis of combination AMD3100 plus GROβ mobilized HSPC and compared them to HSPC mobilized by G-CSF, AMD3100 or GROβ used alone. Flow cytometry analysis of highly enriched HSC using SLAM family receptor markers demonstrated that the AMD3100 plus GROβ mobilized PBSC population contained 1.8±0.1, 3.2±0.15 and 1.6±0.18 fold more CD150+ CD48− SKL cells, respectively, than the PBSC products mobilized by G-CSF, AMD3100 or GROβ used alone. In addition, a higher proportion of AMD3100 plus GROβ mobilized CD150+ CD48− SKL cells expressed higher levels of the adhesion receptors CD11a, CD49e and CD49d. Cell cycle analysis demonstrated that AMD3100 plus GROβ mobilized PBSC contained more cells (92±4 %) in G0/G1 phase of the cell cycle than PBSC mobilized by G-CSF (82±6%), AMD3100 (76±4%) and GROβ (85±2%) alone. In homing studies using CFSE labeled mobilized mononuclear cells injected into lethally irradiated mice, ~2-fold more SKL cells mobilized by AMD3100 plus GROβ homed to bone-marrow compared to SKL cells mobilized by G-CSF, AMD3100 or GROβ used alone. In addition, analysis of apoptosis in homed CFSE+ SKL cells indicated that 2.92±0.4% of homed SKL cells mobilized by AMD3100 plus GROβ were Annexin-Vpositive vs. 6.4±1.5%, 10.4±2.6% and 4.5±1.2%, respectively, for homed SKL cells mobilized by G-CSF, AMD3100 or GROβ alone, suggesting that mobilization by AMD3100 and GROβ in combination results in enhanced survival of mobilized HSC. Our results suggest that the enhanced engraftment observed upon transplantation of PBSC mobilized by combination treatment with AMD3100 plus GROβ in results from mobilization of a greater number of more primitive quiescent HSC that have enhanced intrinsic homing, adhesion and survival properties.