Ryan Lahey

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

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 dextran‐coated 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 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.

783. Transient Transduction of Repopulating Human CD34+ Cells Using Nanoparticles in a Clinically Applicable Ex Vivo Protocol

Top of pageAbstract Transient transduction of human hematopoietic stem cells with novel nano-sized iron particles offers a new approach to transient cell modulation in vitro and in vivo. Moreover, nano particles serve a dual purpose by enabling tracking of labeled cells in vivo by means of magnetic resonance imaging. However, defining ex vivo cultivation strategies for the transduction and/or labeling of human HSC using these particles has not yet been well investigated. Here we present in vitro and in vivo data on human cord blood CD34+ HSC subjected to a 24 hr. clinically applicable ex vivo transduction protocol including protamine complexed ferrumoxide nanoparticles conjugated to Alexa 647 dye (FE-PRO[647]). Cell cultivation were carried out in tissue culture-treated 6 well plates coated with the CH-296 fibronectin fragment. We used X-Vivo 15 defined serum free media supplemented with 10 ng/ml rhTPO, rhSCF, and Flt-3-ligand. Initial CFU-GEMM data indicated that the presence of the FE-PRO [647] did not compromise the human HSC ability to generate colonies in vitro. Subsequent transplantation of labeled human cord blood CD34+ into beta2-NOD/SCID mice for in vivo tracking using flow cytometry consistently showed FE-PRO[647] labeled CD34+ cells in the marrow of the recipients three weeks post transplantation. The total human CD45 engraftment as evaluated in total murine marrow was 18.7+/|[minus]|11.3% (N=7), with a CD34+ component of 10.2+/|[minus]|9.0% (N=7). All 7 animals in the cohort were positive for FE-PRO[647] positive engrafted human cells (0.9+/|[minus]| 0.2 %, N=7). Most interestingly, a striking correlation of the total FE-PRO[647] positive cells and the CD34+ HSC was found in the marrow of the recipients (FE-PRO[647]+/CD34+ was 0.8+/|[minus]|0.2, N=7). Though a subset of CD133+ cells was found in all engrafted mice, interestingly enough no correlation of FE-PRO[647] and CD133+ was found. Moreover, correlating the mean fluorescence activity of the initially FE-PRO [647] transduced CB CD34+ cells to the FE-PRO[647]+/ CD34+ cells found in the recipient marrows 3 weeks post transplantation (relative value: 90.5 versus 2.9+/|[minus]|0.7, N=7) indicated that these CD34+ cells had only divided a total of 5 times over the 3 weeks time period. We hypothesize that these CD34+ cells represent long term engrafting human HSC, whereas the majority of the resulting CD34+ engraftment represents committed progenitors, giving rise to short term engraftment. Overall, these data show that nano particles can indeed be used to transiently transduce repopulating human HSC. This has lead us to define conditions for in vivo modulation of human HSC by co-transduction of FE-PRO[647] and the HIVEF1-EGFP lenti viral vector, with the aim of improving the lenti viral transduction efficiency. In vitro and in vivo data will be presented.