Hematopoietic stem cell homing to bone injuries observed with Zr-89 oxine positron emission tomographic imaging.

Objectives: Bone marrow is a source for various hematopoietic and non-hematopoietic stem and progenitor cells. Growing evidence suggests that hematopoietic cells possess a surprising degree of differentiation plasticity. Not only can these cells convert from cells of one hematopoietic lineage into another, transplanted bone marrow hematopoietic stem cells (HSCs) can convert into cells of non-hematopoietic organs, such as liver, brain, heart, skeletal muscle, bone and various epithelial tissues, through the process of tissue repair. The use of bone marrow-derived HSCs in therapies for tissue regeneration has been increasingly explored. We investigated HSC migration to injured sites using ⁸⁹Zr-oxine ex vivo labeling of bone marrow cells with PET imaging in soft tissue and bone injury models of mice.

Methods: Bone marrow cells were flashed from femurs and tibias of mice and labeled with ⁸⁹Zr-oxine (9.65-15.7 kBq/10⁶ cells). We first examined if the labeled cells maintain differentiation function as HSCs by culturing them with GM-CFS or IL-15. Cell survival and differentiation were analyzed. Next, we transferred the labeled cells intravenously to syngeneic mice (n=4, 2-2.3x10⁷ cells). Unilateral 1-mm incisions were created either only in the soft-tissue or into the tibia of a distal leg 1 day after ⁸⁹Zr-labeled bone marrow cell transfer, when transferred cells were settled in various organs. Serial microPET/CT imaging was performed and homing of ⁸⁹Zr-labeled cells to the injury was quantified. Because it is suggested that CXCL12 produced in the injured tissue recruit CXCR4⁺ bone marrow cells, we further studied the effects plerixafor, a CXCR4 inhibitor, in bone injured mice. Three daily doses of plerixafor (5 mg/kg) were given intravenously beginning on the day of fracture generation (n=4). The labeled cells localizing to the bone injury were analyzed by flow cytometry and immunohistochemistry.

Results: ⁸⁹Zr-oxine-labeled bone marrow cells differentiated normally to dendritic cells (by GM-CSF) or to NK/NK-T cells (by IL-15). ⁸⁹Zr-labeled bone marrow cells did not show significant accumulation in soft tissue injuries; however, significantly higher donor cell homing was observed in the bone injuries compared to the contra-lateral control side (p<0.0001, 1.7% of transferred cells). Plerixafor treatment significantly reduced bone marrow cell migration to the bone injuries (p<0.01). These results support the previous notion that CXCL12 is produced by bone stroma cells upon injury. Flow cytometry analysis of donor cells collected from the bone injured site revealed a predominance of CD45⁺ hematopoietic stem/progenitor cell populations and subsequent histological analysis demonstrated the presence of donor cells in the sites of bone repair.

Conclusion: ⁸⁹Zr-oxine labeling with PET imaging visualized HSC recruitment to acute bone injuries. These cells seemed to have contributed to the bone regeneration. Research Support: