Novel stem cell-based therapies require new imaging techniques to enable the visualization and tracking of transplanted cells in vivo for evaluation of homing and engraftment parameters. Here we present in vitro and in vivo data on nanoparticle labeling of umbilical cord blood (UCB) CD34+ and lineage depleted HSC subsets, in addition to labeling of CD34+ human bone marrow, G-CSF and AMD-3100 M-PBSC. For these studies, we used a 24 hr. clinically applicable ex vivo labeling protocol including protamine complexed ferrumoxide nanoparticles conjugated to Alexa 647 dye or Alexa 750 (FE-PRO[647] or Fe-Pro[750]). Cell cultivation was carried out using serum free X-Vivo 15 defined medium with 10 ng/ml rhTPO, rhSCF, and Flt-3-ligand on retronectin. Transplantation of FACS sorted 97.5% pure FE-PRO [647] labeled human UCB-derived CD34+ cells into NOD/SCID/B2M null mice resulted in mean engraftment levels of 66.7%+/−1.0% CD45+ human cells, after 8 weeks, as compared to 41.8%+/−20.4% in control mice that received non-loaded cells. These data indicate that the FE-PRO [647] did not compromise the engraftment capacity of the human HSC (p>0.05). Moreover, transplantation of labeled human UCB-derived CD34+ cells into NOD/SCID/B2M null mice for in vivo tracking using flow cytometry and magnetic resonance imaging allowed visualization of the FE-PRO[647] labeled CD34+ cells in the spleen and marrow of the recipients, up to three weeks post transplantation. In spleens, human CD34+ FE-PRO [647]+ levels decreased from 20.6.0+/−13.4% (N=5) one week post transplantation to undetectable levels after three weeks (N=7). The total human CD45+ engraftment as evaluated in total murine marrow was 18.7+/−11.3% (N=7) after three weeks. All animals in the cohort were positive for CD34+ FE-PRO[647]+ engrafted human cells (0.8+/− 0.2 %, N=7). In vivo imaging of animals transplanted with 2–5 x 105 human CD34+ cells (16.8% Fe-Pro[750]+ labeled) was performed using the Kodak 4000 MM multimodal imaging unit in which the luminescence signal arising from the nano-labeled human cells can be precisely localized by overlaying the images with x-ray pictures of the animals. Surprisingly, asymmetric engraftment was repeatedly observed between right leg tibia-femur and left-leg tibia-femur in a cohort of 8 NOD/SCID mice at various timepoints over a total of 20 days after intravenous transplantation. Human engraftment was subsequently confirmed and correlated to the luminescence signal by flow cytometry of the bones and spleens of the imaged animals, at the same timepoints. These data demonstrate that nano-particles can be used to label repopulating human HSC for subsequent in vivo tracking, without toxicity to the engrafting cells. This technique offers new methods to dynamically image the homing and engraftment of purified human hematopoietic stem cells over the initial three weeks post-transplantation, in live animals.

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