We recently reported the induction of chimerism and tolerance to renal allografts without GVHD or engraftment syndrome in HLA-mismatched living donor renal transplant recipients through infusion of cryopreserved CD8⁺TCR^– facilitating cells (FC) plus CD34⁺ hematopoietic stem cells (HSC). We hypothesize that one mechanism by which FC enhance HSC engraftment is FC promote homing of HSC to hematopoietic niche. We therefore evaluated whether FC prime the responsiveness of HSC to a low gradient of chemokine SDF-1 in vitro. 500 HSC were placed in the upper chamber of Transwell plates and tested for migration to the lower chamber containing 30ng/ml SDF-1 with or without 30,000 FC for 3 hours. HSC were harvested from the lower chamber and placed in colony-forming cell (CFC) assay. HSC harvested from the lower chamber containing FC formed 6-fold more colonies than the HSC obtained from the lower chamber without FC (p<0.05). We next tested whether FC enhance HSC homing and retention in the hematopoietic niche in vivo using a syngeneic homing model followed by CFC assays. B6 mice were conditioned with a supralethal dose (1200 cGy) of TBI and transplanted with 75,000 B6 FC alone; 25,000 B6 HSC alone; or FC plus HSC 24 after TBI. At 18 hours post-transplantation, bone marrow cells were harvested from femurs and tibias and placed in CFC assay. Bone marrow cells harvested from the mice transplanted with FC without HSC or those receiving conditioning alone did not generate colonies, confirming that FC themselves do not have repopulating capacity in vivo and post-TBI bone marrow was free of recipient HSC under the supralethal dose of TBI. The bone marrow cells harvested from the mice transplanted with HSC and FC formed higher numbers of colonies compared to that of the mice transplanted with HSC alone (p<0.05). When we characterized migration ability of FC, we detected CellTracker Green labeled FC in the bone serial sections of femurs and tibias of the transplanted mice which suggest that FC homed to bone marrow. Further analysis in cell surface markers for putative components of the hematopoietic niche in FC showed that 2% of total FC were CXCL12-producing cells and 5% of total FC were positive for CD169. In summary, our results suggest FC enhance functional HSC homing in vivo and prime HSC migration in vitro. These data indirectly suggest FC may contribute to the hematopoietic niche.

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