Transplantation efficacy can be limited by inadequate hematopoietic stem cell (HSC) number, homing, engraftment, or self-renewal. Traditional conditioning prior to HSC transplantation by myeloablative chemotherapy with or without radiation provides therapeutic benefit but also results in immunosuppression and tissue damage that contribute to increased morbidity and mortality, therefore, development of safe myeloablative strategies is a clinical priority. AMD3100 has been shown to be a potent mobilizer of HSCs in multiple animal species and humans and acts by specifically blocking the CXCR4/CXCL12 (SDF-1α) axis that is critical for HSC homing and retention in the niche. Therefore, we hypothesized that rapid mobilization of host HSC out of the bone marrow would result in increased availability of niches for subsequently infused donor HSCs.
C57/BL6 mice were injected subcutaneously with 5 mg/kg AMD3100 and at indicated time points, 40×106 C57/BL6 GFP+ donor bone marrow mononuclear cells were intravenously delivered. The control group received injection of PBS. For additive treatment of the donor cells with PGE2 and Diprotin A, B6 GFP+ HSCs were treated with 1μM PGE2 or 5 mM Diprotin A and then delivered into the recipient mice 60 min post-AMD3100 administration. For combinatorial treatment with both drugs, the cells were treated with PGE2 for 2 hours at 4 °C and then treated with Diprotin A at RT for 15 min prior to transplantation. For homing studies, twelve hours after transplantation, the mononuclear cells from peripheral blood and bone marrow were harvested for flow cytometry and CFU-C assay. Fold increases were based on comparison with control mice that received PBS injection and DMEM-treated donor cells. For long-term engraftment studies, the assays were performed four months after transplantation.
The results demonstrated that a single administration of AMD3100 resulted in significantly increased availability of bone marrow niches by mobilizing the host HSCs into the periphery in a time-dependent manner. The maximal effect of up to 90% vacancy of the niche was observed at 1 hour after AMD3100 administration. Infusion of syngeneic donor bone marrow cells between 45 and 60 min post-AMD3100 administration resulted in maximum homing and engraftment efficiency. Donor CXCR4+CD49d+ cells preferentially homed to the host bone marrow niche. Pretreatment of donor HSCs with PGE2 or Diprotin A (CD26 blockade) further enhanced the homing efficiency following AMD3100 preconditioning. Moreover, AMD3100 preconditioning with or without PGE2 or Diprotin A also resulted in long-term engraftment of donor HSCs. The occupancy of the marrow niche by HSCs was restored to the full capacity in the long term, indicating treatment with these drugs did not result in toxicity. PGE2 and Diprotin A pre-treatment, each of which have been demonstrated to selectively improve donor cell homing and engraftment, further enhances the positive effects of AMD3100 conditioning alone.
Our data support the efficacy of AMD3100 mobilization, alone or in combination with donor cell pre-treatment strategies, as a safe-and-effective preconditioning method for hematopoietic stem cell transplantation.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.