Abstract
Interactions between stromal derived factor-1 (SDF-1 or CXCL12), and its receptor CXCR4 regulate hematopoietic stem and progenitor cell retention in the bone marrow. AMD3100, a bicyclam molecule that selectively blocks the interaction between CXCL12 and CXCR4, has recently been used in clinical trials to rapidly mobilize hematopoietic progenitor cells. However, the functional properties of human stem and progenitor cells mobilized with this agent are not well characterized. Here, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized after 5 days of G-CSF treatment. A total of 7 HLA-matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100. After 1 week of drug clearance, the same donor was mobilized with G-CSF, allowing a paired comparison of the repopulating function of cells mobilized by the two agents. Total CD34+ cells mobilized by AMD3100 treatment averaged 1.2±0.4x106 CD34+ cells/kg (range 0.4–2.1x106 CD34+ cells/kg), as compared to G-CSF treatment at 3.2±0.9x106 CD34+ cells/kg (range 1.7–5.7 x106 CD34+ cells/kg). Leukapheresis total mononuclear cell (MNC) fraction or purified CD34+ cells (>90% purity), were isolated and transplanted into sublethally irradiated NOD/SCID mice at varying doses. BM, spleen, and peripheral blood of mice were harvested 7–8 weeks post-transplantation and analyzed by flow cytometry for the presence or absence of engrafting human cells. Low frequency human engraftment events (<0.2% human cells) were confirmed by PCR for P17H8 alpha-satellite human DNA sequences. Injection of 1–40x106 MNC or 0.5–5x105 CD34+ cells produced consistent human engraftment and allowed limiting dilution analysis using Poisson statistics to be performed on paired samples of AMD3100 and G-CSF leukapheresis products from 3 individual patients. The calculated frequencies of NOD/SCID repopulating cells (SRC) were 1 SRC in 11.5x106 AMD3100-mobilized MNC (n=50) compared to 1 SRC in 44.8x106 G-CSF-mobilized MNC (n=55). For purified CD34+ populations, the overall frequency of repopulating cells was 1 SRC in 1.0x105 AMD3100-mobilized CDC34+ cells (n=53) compared to 1 SRC in 3.1x105 G-CSF-mobilized CD34+ cells (n=45). These data correspond to a 3–4-fold increase in overall repopulating function demonstrated by AMD3100 mobilized cells. Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with equivalent production of myelo-monocytic cells (CD33+CD14+), immature B-lymphoid cells (CD19+CD20+), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. These studies indicate that human AMD3100-mobilized MNC and purified CD34+ cells possess enhanced repopulating capacity, as compared to G-CSF mobilized counterparts from the same donor. Thus, AMD3100 mobilized peripheral blood represents a rapidly obtained and highly functional source of repopulating hematopoietic stem cells for clinical transplantation procedures.
Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.
