Fluorophore Conjugated Iron Oxide Nanoparticle Labeling and Analysis of Engrafting Human Hematopoietic Stem Cells.
Authors: Maxwell Dustin , Hess David , Bonde Jesper , Hohm Sarah A. , Lahey Ryan , Creer Michael , Piwnica-Worms David , Nolta Jan A. 
Published: October 2019

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. Using the current techniques, flow cytometry analysis gating on CD34+ or CD45+ expression and Fe[750] confirmed the presence of human cells residing in the BM. Using this method, as few as 1 × 105 Fe[750]+CD34+ cells residing in the bone marrow could be detected by fluorescence imaging. Beyond two weeks, the human cell expansion, egress from the marrow, and iron metabolism began to dilute the nanoparticle signal below the limit of detection of both techniques. The current studies provide a method by which investigators can track human stem cells to the marrow vs. different tissues of immune deficient mice. This has been extremely difficult in the past, because stem cells can alter their phenotype after engraftment. The fluorophore-tagged Feridex allows a clean recovery of labeled cells from different tissues by FACS for cell surface phenotype probing and other assays. Due to the fluorophore modification, quantitation of the number of cells that are engrafted in the bone marrow after transplantation is possible, and allows simultaneous probing of cell surface phenotype using flow cytometry, without the requirement for isolating cells based on a pre-determined cell surface marker. The use of fluorophore-labeled Feridex nanoparticles and the clinically relevant incubation procedure described in the current study offers an efficient and safe method to label both cycling and non-cycling human hematopoietic stem and progenitor cells without toxicity as well as to evaluate the homing, localization, phenotype, and short-term engraftment capabilities of defined human HSC subsets. The current data demonstrate that transient labeling of repopulating HSC subsets with fluorescent nanoparticles is a powerful and novel tool for dynamic tracking of human stem cells during the initial weeks after transplantation.