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

Abstract

The use of nanometer-sized iron oxide particles combined with molecular imaging techniques enables 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 dextrancoated iron oxide nanoparticles (Feridex) to characterize human HSC labeling to monitor the engraftment process. Conjugating fluorophores to the dextran coat for fluorescence-activated cell sorting purification eliminated spurious signals from nonsequestered nanoparticle contaminants. A short-term defined incubation strategy was developed that 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 fluorophorelabeled iron oxide nanoparticles for fluorescence imaging in combination with flow cytometry allows evaluation of labeling efficiencies and homing capabilities of defined human HSC subsets. STEM CELLS 2008;26:517-524 Disclosure of potential conflicts of interest is found at the end of this article.

Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity

“…30,31 Purified ALDH lo and ALDH hi cells were incubated with Fe[750] nanoparticles in fibronectin (CH-296 25 g/cm 2 )-coated plates (Takara Shuzo, Otsu, Japan) as described, 31 and cultured in serum free X-Vivo 15 media (Lonza Walkersville) supplemented with 10 ng/mL recombinant human thrombopoietin, recombinant human stem cell factor, and Flt-3 ligand (R&D Systems, Minneapolis, MN). Fe[750] nanoparticles were prepared with protamine sulfate (American Pharmaceuticals, Schaumberg, IL) and added to 2 ϫ 10 5 cells/well at a concentration of 10 g/mL.…”

“…31 Excess unbound Fe[750] nanoparticles were removed from Fe-labeled cells by extensive washes in PBS and by the selection of the cells based on forward and side scatter properties (R1, Figure 3A) Figure 3B). Our previous studies have shown that potentially phagocytic myeloid progenitors and monocytes label efficiently in vitro.…”

Transient loading of CD34⁺ hematopoietic progenitor cells with polystyrene nanoparticles

“…29 Other studies in which iron oxide and Gd 2 O 3 NPs by HPCs were used for MRI applications have reported solely their uptake and little effect on cellular function. [9][10][11]30,31 None of these preceding studies have reported the short-time kinetics of NPs in HPCs. Transient loading of cells requires the release of the NPs, as we have described using the physics model.…”

Gd2O3 nanoparticles in hematopoietic cells for MRI contrast enhancement

“…According to the literature, the particle uptake in cells varies also at approximately similar experimental conditions. 15,20,23,24 It is therefore concluded that different uptake might depend on the natural and biological difference in the cells in addition to different labeling methods and incubation times. To increase the internalization of particles, in particular Gd 2 O 3 nanoparticles, other transfection agents, other incubation times, and higher incubation concentrations as well as surface modification of the particles can be considered.…”