CD8⁺TCR^- Bone Marrow Facilitating Cells Prime Migration and Enhance Homing of Hematopoietic Stem Cells

CD8⁺TCR^- bone marrow facilitating cells (FCs) facilitate engraftment of hematopoietic stem cells (HSCs) in both allogeneic and syngeneic recipients. We recently reported that co-administration of FCs with HSCs established chimerism and induced tolerance to renal allografts without graft-versus-host disease and engraftment syndrome in HLA-mismatched living donor renal transplant recipients. Homing of HSCs to the bone marrow niche is believed to be a crucial prerequisite for engraftment. Therefore, we evaluated whether FCs enhance functional HSC homing and retention in the hematopoietic niche using the in vivo syngeneic homing model followed by colony-forming cell (CFC) assay. B6 mice were conditioned with a supralethal dose (1200 cGy) of total body irradiation (TBI) and transplanted with 75,000 B6 FCs, 25,000 B6 HSCs or FCs plus HSCs 24 hours after TBI. At 18 hours after transplantation, bone marrow cells were harvested from the recipient’s femurs and tibias and placed in CFC assay using methylcellulose-based media. Bone marrow cells harvested from mice transplanted with FCs without HSCs or those receiving conditioning alone did not generate colonies, confirming that FCs themselves do not have repopulating capacity in vivo and post-TBI bone marrow was free of recipient HSCs under the supralethal dose of TBI. FCs significantly enhanced the efficiency of HSC homing as reflected by the bone marrow cells harvested from the mice transplanted with HSCs and FCs formed significantly higher numbers of colonies compared to that of the mice transplanted with HSCs alone (24.7 ± 2.9 vs. 15.6 ± 2.7; p < 0.05). The chemokine receptor CXCR4 plays a pivotal role in HSC homing. We therefore determined whether the effect of FCs on HSC homing was mediated by FCs increasing CXCR4 expression in HSC. CXCR4 expression in HSCs was measured by flow cytometry 18 hours after co-culture of HSCs and FCs in vitro. Incubation of FCs with HSCs did not lead to increased CXCR4 expression on HSCs compared with HSCs cultured alone. We next tested whether FCs enhance the responsiveness of HSCs to a low concentration of stromal cell-derived factor 1 (SDF-1, also known as CXCL12) gradient in Transwell chemotaxis assays in vitro. 500 HSCs were placed in the upper chamber and tested for migration of cells to the lower chamber of the Transwell in response to a 30 ng/ml SDF-1 gradient for 3 hours. The cells harvested from the lower chamber were placed in CFC assay. The migrated cells harvested from the lower chamber containing FCs formed 6-fold more colonies than HSCs obtained from the lower chamber that did not contain FC (22.3 ± 3.9 vs. 4.0 ± 1.2; p < 0.05). Furthermore, we found that small factions of total FCs were CXCL12-producing cells (2% of total FCs) and CD169⁺ cells (5% of total FCs). The migration ability of FCs was characterized by following patterns of homing after transplantation of 300,000 CellTracker Green labeled FCs or 300,000 FCs bearing different congenic marker. In 8,000,000 bone marrow cells harvested from femurs and tibias of the transplanted mice 18 hours post-transplantation, we detected 100 to 300 of transplanted CellTracker Green labeled FCs. On the 10 serial bone sections from femurs and tibias of the transplanted mice, we enumerated 15 to 20 of CellTracker Green labeled FCs. These data suggested that FCs might home to bone marrow within 18 hours post-transplantation. In summary, our results suggest that FCs enhance functional HSC homing and retention in the hematopoietic niche in vivo. This is not mediated by increased CXCR4 expression in HSCs. FCs also prime HSC migration to a low concentration SDF-1 gradient in vitro, possibly through production of priming factors.