Human CD34+ Cells Rapidly Mobilized by AMD3100 Repopulate NOD/SCID Mice with Equivalent Efficiency as CD34+ Cells Mobilized by G-CSF.
Authors: Hess David A. , Bonde Jesper , Craft Timothy P. , Wirthlin Louisa , DiPersio John F. , Devine Steven M. , Nolta Jan A. 
Published: April 2020

Abstract
Interactions between the chemokine receptor CXCR4 and its ligand, stromal derived factor-1, regulate hematopoietic stem cell migration. The CXCR4 antagonist, AMD3100, has recently been shown to rapidly mobilize primitive hematopoietic cells. However, the functional properties of stem and progenitor cells mobilized with this agent are not well characterized. Thus, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized by 5 days of G-CSF treatment. Healthy, matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100, and produced an enrichment of circulating CD34+ cells to 0.6% of the total mononuclear cells (MNC). After 2 weeks of drug clearance, the same donor was mobilized with G-CSF (0.4% CD34+ cells), allowing a paired comparison of the repopulating function of cells mobilized by these two regimens. Total MNC, CD34+ cells (&gt;95% purity), and lineage depleted (Lin−) cells (48–55% CD34+) were isolated and transplanted into NOD/SCID mice at various doses. Injection of 106–107 (approximately 5x103–5x104 CD34+ cells) AMD3100-mobilized MNC resulted in bone marrow (BM) engraftment in 6 of 10 mice, whereas equal doses of G-CSF mobilized MNC engrafted 3 of 10 mice. Higher cell doses (2x107 MNC, approximately 1x105 CD34+ cells) consistently produced engraftment in the BM, spleen, and peripheral blood of all mice, with higher levels of engraftment with AMD3100-mobilized cells (1.8±0.5%) compared to G-CSF-mobilized cells (0.4±0.05%, p&lt;0.05). Similar analyses performed using purified CD34+ cells revealed similar engraftment frequencies for both leukapheresis products. Transplantation of 5x104–105 AMD3100-mobilized CD34+ cells engrafted 4 of 7 mice and G-CSF-mobilized CD34+ cells engrafted 5 of 10 mice. However, transplantation of 5x105 AMD3100-mobilized CD34+ cells consistently resulted in higher engraftment levels compared to G-CSF-mobilized CD34+ cells (3.4±1.1% versus 0.8±0.4% human cells, p&lt;0.05). Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with production of myeloid cells (CD33+, CD14−CD13+CD66abce+/−), monocytes (CD14+), immature B-lymphoid cells (CD19+/−CD20+/−), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. Similarly, AMD3100 and G-CSF-mobilized Lin− cells produced consistent engraftment in all transplanted mice (n=20), and demonstrated equivalent engraftment levels and multilineage differentiation when directly comparing AMD3100 versus G-CSF-mobilization. Ongoing analysis of additional patient samples will allow direct comparison of these repopulating cells by limiting dilution analysis using Poisson statistics. These preliminary studies indicate that human AMD3100-mobilized CD34+ cells possess at least equivalent repopulating capacity compared to G-CSF mobilized cells, and therefore represent a more rapidly obtainable source of hematopoietic stem cells for clinical transplantation.