Fluorophore Conjugated Iron Oxide Nanoparticle Labeling and Analysis of Engrafting Human Hematopoietic Stem Cells

¹ Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO.
² Division of Oncology, Hematopoietic Development and Malignancy Program, Washington University School of Medicine, St. Louis, MO.
³ Vascular Biology Program, Robarts Research Institute, London, Ontario, Canada
⁴ Department of Internal Medicine, Stem Cell Program, University of California, Davis, CA
⁵ Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO., Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO

^* To whom correspondence should be addressed. E-mail: Jan.nolta{at}ucdmc.ucdavis.edu.

	Abstract

The use of nanometer-sized iron oxide particles combined with molecular imaging techniques enable dynamic studies of homing and trafficking of human hematopoietic stem cells (HSC). Identifying clinically applicable strategies for loading nanoparticles into primitive HSC requires strictly defined culture conditions to maintain viability without inducing terminal differentiation. In the current study, fluorescent molecules were covalently linked to dextran-coated iron oxide nanoparticles (Feridex) to characterize human HSC labeling to monitor the engraftment process. Conjugating fluorophores to the dextran coat for FACS purification eliminated spurious signals from non-sequestered nanoparticle contaminants. A short-term defined incubation strategy was developed which allowed efficient labeling of both quiescent and cycling HSC, with no discernable toxicity in vitro or in vivo. Transplantation of purified primary human cord blood lineage-depleted and CD34⁺ cells into immunodeficient mice allowed detection of labeled human HSC in the recipient bones. Flow cytometry was used to precisely quantitate the cell populations that had sequestered the nanoparticles, and to follow their fate post-transplantation. Flow cytometry endpoint analysis confirmed the presence of nanoparticle-labeled human stem cells in the marrow. The use of fluorophore-labeled iron oxide nanoparticles for fluorescence imaging in combination with flow cytometry allows evaluation of labeling efficiencies and homing capabilities of defined human HSC subsets.