Pre-clinical evaluation of Magnetically labeled Cells for Cellular MRI

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Abstract

Previously we combined commonly used Protamine Sulfate (Pro) with superparamagnetic dextran coated iron oxide nanoparticle (SPIO) Ferumoxides (FE), a complex is formed that can be used to magnetically label stem cells and other mammalian cells. Cells take up FEPro complexes by macropinocytosis. Early detection of labeled cells in vivo by cellular MRI requires the development of novel pulse sequences or image processing to improve the sensitivity to low numbers of iron oxide labeled cells in tissues. We previously revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. To validate this new approach human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) were labeled. Labeling for 4 hours using 100g/ml of Fe and 3g/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. However, ferumoxides were taken off the market place and ferumoxytol (F) an FDA approved iron oxide nanoparticle used to treat iron deficiency anemia for chronic kidney disease was introduced in 2009. Ferumoxytol (F) is a nanoparticle of 17-31 nm in diameter with the 6 nm iron oxide crystals core and has been also used in experimental and clinical trials as a macrophage imaging agent as well as blood pool agent with MRI. In late 2009, we characterized the physiochemical properties of F and P nanocomplexes in water. Fs zeta potential is -55mV while protamine (P) is 7.2mV in water. The two nanocomplexes form greater than 400 nm particles of FP via electrostatic interactions; however we could not label cells with any ratio of the two drugs. The addition of heparin sulfate (H) resulted in our ability to label cancer cell lines in culture. Heparin-based nanocomplexes have been shown to self-assemble with a variety of cationic molecules and have been used for drug delivery, tissue engineering or for prolonging circulating half-life of an agent. Although it was counterintuitive to add heparin to PF to facilitate endosomal incorporation in cells, the addition of heparin resulted in the formation of HPF nanocomplexes that were endocytosed by cells. Transmission electron microscopy of HPF revealed that these nanocomplexes were spheroid shaped with the HP in center surrounded by F. Incubating stem cells and T-cells in HFP nanocomplexes in serum free media for 2 hours followed by complete media resulted in cell labeling. HPF labeling did not impair the cells viability, proliferative capacity, apoptotic rate, activation, phenotypic surface marker expression, or capacity to differentiate. MRI at 3T of HPF labeled BMSC implanted in the rat brain demonstrated the ability to detect 1000 cells with a 50% decrease in T2* in the rat brain compared to the surrounding parenchyma. The HPF labeling technique has been scaledup, and the NIH Cell Processing Section cGMP facility was able to label BMSCs in biofactories with no changes in BMSC function or viability of the labeled cryopreserved cell product. The HPF labeling method should facilitate the monitoring by MRI of transplanted cells in clinical trials. In order to determine if it were possible to use a small molecule to determine if multipotential stem cells differentiated a fluorescently labeled sigma-2 (σ(2)) receptor probe, SW120 was used to evaluate σ(2) receptor expression in human stem cells (SC), including: bone marrow stromal, neural progenitor, amniotic fluid, hematopoietic, and embryonic stem cells. The sigma-2 (σ(2)) receptor is a potential biomarker of proliferative status of solid tumors. Specific synthetic probes using N-substituted-9-azabicyclo 3.3.1nonan-3α-yl carbamate analogs have been designed and implemented for experimental cancer diagnosis and therapy. Stem cells were incubated with at fluorescently tagged SW120 molecule for short periods of time and we were able to demonstrate rapid binding and incorporation into the SC. We evaluated the intensity of SW120 and a proliferation marker 5-ethynyl-2'-deoxyuridine (EdU) relative to cell passage number and multi-potency and showed that significantly higher σ(2) receptor density among proliferating stem cells relative to lineage-restricted cell types. Cellular internalization of the σ(2) receptor in stem cells was consistent with receptor-mediated endocytosis and confocal microscopy indicated SW120 specific co-localization with a fluorescent marker of lysosomes in all SC imaged. These results suggested that the sigma -2 receptor could be used to monitor stem cell differentiation in vivo and plans are underway to develop a molecular imaging probe based on the SW120 backbone for noninvasive tracking of stem cell differentiation by MRI or positron emission tomography.