Hematopoietic stem cell (CD34D) uptake of superparamagnetic iron oxide is enhanced by but not dependent on a transfection agent TIMOTHY J. ENGLAND 1 , PHILIP M.W. BATH 1 , MARYAM ABAEI 2 , DOROTHEE AUER 2 & D. RHODRI E. JONES 3 1 Stroke Trials Unit, 2 Academic Radiology, University of Nottingham, and 3 Clinical Tissue Laboratory, Department of Pathology, Nottingham University Hospitals NHS Trust, Nottingham, UK Abstract Background aims. Tracking the fate of cells after infusion would be a valuable asset for many stem cell therapies, but very few (cell) labels are approved for human therapeutic use. Superparamagnetic iron oxide particles (SPIO) can be inter- nalized into stem cells in vitro to allow real-time tracking with gradient echo magnetic resonance imaging, but SPIO are approved for (diagnostic) imaging and not for (therapeutic) cell labeling in vivo. In this study, we investigated the possibility of labeling stem cells with an SPIO approved for patient use, albeit in a novel manner by enhancing uptake with the use of a transfection agent, also approved for patient use. Although there are many reports of hematopoietic stem cells being labeled with SPIO, there is some controversy regarding the efficiency of this and whether undifferentiated CD34þ progenitor (stem) cells are able to take up iron in the absence of a transfection agent to enhance the process. Methods. Human CD34þ cells were treated in vitro as follows: incubation with (i) medium only (control), (ii) ferumoxide (Endorem) and (iii) ferumoxide (Endorem) plus exposure to a transfection agent (protamine sulfate). Cells were incu- bated for 2, 4 and 24 hours and assessed for viability, differentiation capacity and visualized in vitro with 3-T magnetic resonance imaging. The cells were also analyzed by means of flow cytometry and morphology examined by electron microscopy. Results. CD34þ hematopoietic progenitor cells can internalize ferumoxide (Endorem) independently of a transfection agent. However, uptake of ferumoxide is enhanced after exposure to protamine sulfate. Iron labeling of CD34þ cells in this manner does not affect cell viability and does not appear to affect the potential of the cells to grow in culture. Iron-labeled CD34þ cells can be visualized in vitro on 3-T magnetic resonance image scanning. Conclusions. Endorem and protamine sulfate can be combined to promote iron oxide nanoparticle uptake by CD34þ cells, and this methodology can potentially be used to track the fate of cells in a clinical trial setting because both compounds are (separately) approved for clinical use. Key Words: CD34þ cells, Endorem, ferumoxide, MRI, protamine sulfate Introduction Stem and progenitor cells provide increasing potential for treatment of a variety of conditions including Parkinson disease (1), multiple sclerosis (2), spinal cord injury (3) and vascular diseases such as myocardial infarction and stroke (4,5). Despite experiences gained from (human) clinical trials, stem cell fate in the context of tissue repair or regeneration in vivo is poorly understood. Particular interest has been generated in the field of autologous hematopoietic stem cells because these can be obtained through bone marrow aspiration (CD34þ cells) or through mobilization into the circulation with the use of granulocyte-colony stimulating factor (6). Labeling cells with iron and tracking them in vivo with magnetic resonance imaging (MRI) creates the potential for “real-time” migration patterns to be established. Superparamagnetic iron oxide particles (SPIO) can be taken up into cells to allow tracking with gradient echo MR imaging, and, although it has been reported that “hematopoietic stem cells” have been labeled with SPIO (7e10), it is not clear whether undifferentiated CD34þ cells are able to take up iron particles without a transfection agent (substances that efficiently shuttle particles into the cell). SPIOs are negatively charged and do not adhere to cell surface membranes; therefore a transfection agent could be used to electrostatically combine with the dextran coating of the SPIO, modifying the surface charge and Correspondence: Dr D. Rhodri E. Jones, University of Nottingham, Division of Gastrointestinal Surgery, E Floor, West Block, Queen’s Medical Center, Nottingham NG7 2UH, UK. E-mail: d.r.e.jones@nottingham.ac.uk Cytotherapy, 2013; 15: 384e390 (Received 8 June 2012; accepted 23 October 2012) ISSN 1465-3249 Copyright Ó 2013, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2012.10.016