The CXCR4 Antagonist BL-8040 Induces a Robust Mobilization of CD34+CD38−CD45RA−CD90+ CD49f+ HSCs with Long-Term and Secondary Myeloid and Lymphoid Repopulating Activity

Introduction: Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates.

The bulk of HSCs are CD34+, however, most CD34+ cells are lineage-restricted progenitors and HSCs remain rare. HSCs can be enriched further based on CD45RA, Thy1 (CD90), and CD38 expression. Loss of CD90 expression was proposed to be sufficient to separate CD34+CD38−CD45RA− CD90+ HSCs from CD34+CD38−CD45RA− CD90- multipotent progenitors (MPPs). Recently, it was demonstrated that the expression CD49f is a specific HSC marker. Single CD49f(+) cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting MPPs.

Results: CD34+ cells were purified from BL-8040 and G-CSF mobilized grafts and stained for CD38, CD45RA, CD90, and CD49f. The percentage of CD34+CD38- hematopoietic stem and progenitors was similar in both grafts (Figure 1A). However, whereas 23.2 % of BL-8040 mobilized CD34+ CD38- cells did not express CD45RA; only 1.6% of G-CSF mobilized CD34+ CD38- cells did not express CD45RA (Figure 1B). The percentages of CD34+CD38−CD45RA−CD49f+CD90+/-, CD34+CD38−CD45RA−CD49f+ CD90+, and CD34+CD38−CD45RA− CD90+ HPCS were increased significantly by 45, 25 and 12 -fold in the BL-8040 graft compared to G-CSF graft derived CD34+CD38- cells (Figure 1C). To assess the long-term engraftment potential of the BL-8040 mobilized CD34+ cells, engraftment was allowed for 4, 8, and 22 weeks after transplantation. Successful and robust long-term human engraftment of CD45+ and CD45+CD34+ cells was observed at week 22 (Figure 2A, 2B). The % of human CD45 cells remained stable in the BM whereas the percentage of CD45 cells in the blood and spleen increased at week 22 (Figure 2C). At 4 weeks, human CD3+CD4+ T cells were only observed at a low percentage in the spleen but not in the BM, whereas no significant percentage of CD3+CD8+ cells were found neither in the BM nor in the spleen (Figure 2D, 2E). 22 weeks after transplantation, the percentage of human CD3+CD4+ and CD3+CD8+ T cells was significantly increased in the spleen (30% vs. 5%, respectively) and to much lower levels in the BM (Figure 2D, 2E). Furthermore, successful and robust long-term human engraftment of secondary recipient was observed 14 weeks following the second transplantation (Figure 2A and 2B).

Conclusion: In association with the high percentage of HPCs in the BL-8040-derived graft, we found a robust myeloid and lymphoid long-term engraftment (week 22) of BL-8040 mobilized human CD34+ cells in NSG mice. The ability of BL-8040 to collect high numbers of HPCs may be beneficial for a variety of HPCs dependent therapeutics.

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