Bobbi K. Lewis

Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells.

Magnetic resonance (MR) tracking of magnetically labeled stem and progenitor cells is an emerging technology, leading to an urgent need for magnetic probes that can make cells highly magnetic during their normal expansion in culture. We have developed magnetodendrimers as a versatile class of magnetic tags that can efficiently label mammalian cells, including human neural stem cells (NSCs) and mesenchymal stem cells (MSCs), through a nonspecific membrane adsorption process with subsequent intracellular (non-nuclear) localization in endosomes. The superparamagnetic iron oxide nanocomposites have been optimized to exhibit superior magnetic properties and to induce sufficient MR cell contrast at incubated doses as low as 1 μg iron/ml culture medium. When containing between 9 and 14 pg iron/cell, labeled cells exhibit an ex vivo nuclear magnetic resonance (NMR) relaxation rate (1/T2) as high as 24–39 s−1/mM iron. Labeled cells are unaffected in their viability and proliferating capacity, and labeled human NSCs differentiate normally into neurons. Furthermore, we show here that NSC-derived (and LacZ-transfected), magnetically labeled oligodendroglial progenitors can be readily detected in vivo at least as long as six weeks after transplantation, with an excellent correlation between the obtained MR contrast and staining for β-galactosidase expression. The availability of magnetodendrimers opens up the possibility of MR tracking of a wide variety of (stem) cell transplants.

Investigation of low frequency drift in fMRI signal

Low frequency drift (0.0-0.015 Hz) has often been reported in time series fMRI data. This drift has often been attributed to physiological noise or subject motion, but no studies have been done to test this assumption. Time series T*2-weighted volumes were acquired on two clinical 1.5 T MRI systems using spiral and EPI readout gradients from cadavers, a normal volunteer, and nonhomogeneous and homogeneous phantoms. The data were tested for significant differences (P = 0.001) from Gaussian noise in the frequency range 0.0-0.015 Hz. The percentage of voxels that were significant in data from the cadaver, normal volunteer, nonhomogeneous and homogeneous phantoms were 13.7-49.0%, 22.1-61.9%, 46.4-68.0%, and 1.10%, respectively. Low frequency drift was more pronounced in regions with high spatial intensity gradients. Significant drifting was present in data acquired from cadavers and nonhomogeneous phantoms and all pulse sequences tested, implying that scanner instabilities and not motion or physiological noise may be the major cause of the drift.

Magnetic intracellular labeling of mammalian cells by combining (FDA-approved) superparamagnetic iron oxide MR contrast agents and commonly used transfection agents.

Mammalian stem cells or other cells are being considered for use for infusion or transplantation into tissue for purposes of repair or for revascularization or therapeutic approaches (i.e., genetically altered cells) (1–5). Dextrancoated superparamagnetic iron oxide (SPIO) nanoparticles, a distinct class of MR contrast agents, cannot be used to efficiently label stem cells or other mammalian cells in vitro in their native unmodified form (6–8). Previously, we demonstrated that by conjugating antigenspecific internalizing monoclonal antibodies to the surface, dextran coating cells could be magnetically labeled during their normal expansion in culture (6). This magnetic labeling approach is limited because it requires the availability of an internalizing monoclonal antibody that recognizes a specific cell surface antigen. It is suitable only for labeling of cells that express the targeted receptor and is commonly species specific. Other approaches have involved the synthesis and modification of ultrasmall SPIO (USPIO or MION) particles with tat-proteins facilitating the incorporation into the cells, although this involves also a (synthetic) protein derivative (7,8). An alternative approach is the complex synthesis of a superparamagnetic iron oxide (SPIO) coated with dendrimers or “magnetodendrimers”, which will non-specifically label most mammalian cells (9). The dendrimer coating of the iron oxide nanoparticles provides the needed high affinity for cellular membranes for the construction of suitable cellular contrast agents, as dendrimers are commonly used as non-viral transfection agents (10,11). Magnetodendrimers can be used to efficiently and non-specifically label mammalian stem cells and cancer cells (12,13), however, they are not widely available or approved by the Food and Drug Administration (FDA). Over the past 10 years there has been significant research in developing new transfection agents (TA) including cationic peptides, dendrimers, poly-amines and lipids for nonviral transfection of DNA into the nucleus (10,14,15). These transfection agents are being developed to overcome the problem of the endosomal capture of the TA-DNA complex and inefficient release of the targeted material into the nucleus (14,15). TA’s are macromolecules with molecular weights from 1 to 10 kilo Dalton possessing an electrostatic charge. Based upon efficient labeling of mammalian cells by magnetodendrimers (12,13), we hypothesize that commercially available macromolecular transfection agents would coat via electrostatic interaction with dextran-coated iron oxide MR contrast agents and chaperon these nanoparticles into cells. We present here the magnetic labeling results of combining dendrimers and other commercially available TA’s with (FDA-approved) dextran-coated iron oxide MR contrast agents (Feridex and MION-46L). Acad Radiol 2002; 9(suppl 2):S484–S487

In Vivo Trafficking and Targeted Delivery of Magnetically Labeled Stem Cells

Targeted delivery of intravenously administered genetically altered cells or stem cells is still in an early stage of investigation. We developed a method of delivering iron oxide (ferumoxide)-labeled mesenchymal stem cells (MSCs) to a targeted area in an animal model by applying an external magnet. Rats with or without an external magnet placed over the liver were injected intravenously with ferumoxide-labeled MSCs and magnetic resonance imaging (MRI) signal intensity (SI) changes, iron concentration, and concentration of MSCs in the liver were monitored at different time points. SI decreased in the liver after injection of MSCs and returned gradually to that of control rat livers at approximately day 29. SI decreases were greater in rats with external magnets. Higher iron concentration and increased labeled cell numbers were detected in rat livers with external magnets. The external magnets influenced the movement of labeled MSCs such that the cells were retained in the region of interest. These results potentially open a new area of investigation for delivering stem cells or genetically altered cells.

Methods for magnetically labeling stem and other cells for detection by in vivo magnetic resonance imaging

Superparamagnetic iron oxide (SPIO) nanoparticles are being used for intracellular magnetic labeling of stem cells and other cells in order to monitor cell trafficking by magnetic resonance imaging (MRI) as part of cellular-based repair, replacement and treatment strategies. This review focuses on the various methods for magnetic labeling of stem cells and other mammalian cells and on how to translate experimental results from bench to bedside.

COMBINATION OF TRANSFECTION AGENTS AND MAGNETIC RESONANCE CONTRAST AGENTS FOR CELLULAR IMAGING: RELATIONSHIP BETWEEN RELAXIVITIES, ELECTROSTATIC FORCES, AND CHEMICAL COMPOSITION

The purpose of this study was to investigate the changes in electrostatic and magnetic resonance (MR) properties observed when MR contrast agents (CAs) (Feridex®, MION‐46L, or G5‐dendrimer‐DOTA‐Gd) are combined with transfection agents (TAs) under various conditions for use as a CA‐TA complex basis for cellular labeling and MRI. CAs were incubated with various classes of TAs for 0–48 hr in solutions of varying concentrations and pH values. NMR relaxation rates (1/T1, 1/T2), MRI and zeta potential (ZP) of CA‐TA solutions were measured. TAs decreased the 1/T1 and 1/T2 of G5‐DOTA‐Gd, Feridex®, and MION‐46L by 0–95%. Altering the pH of G5‐DOTA‐Gd‐TA decreased the T1‐weighted signal intensity (SI) on MRI from 0 to 78%. Measured ZP values for G5‐DOTA‐Gd, Feridex®, and MION‐46L were −51, −41, and −2.0 mV, respectively. The TA LV had a negative ZP, while the other TAs had ZPs ranging from +20 to +65 mV. The alteration of the ZP and NMR relaxivities of the MR CAs, Feridex®, MION‐46L, and G5‐DOTA‐Gd by TAs has been demonstrated. These results enhance our understanding of the relationship between electrostatic and MR properties. Magn Reson Med 50:275–282, 2003. Published 2003 Wiley‐Liss, Inc.

Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging

We report on a new straightforward magnetic cell-labeling approach that combines three US Food and Drug Administration (FDA)-approved drugs—ferumoxytol, heparin and protamine—in serum-free medium to form self-assembling nanocomplexes that effectively label cells for in vivo magnetic resonance imaging (MRI). We observed that the ferumoxytol-heparin-protamine (HPF) nanocomplexes were stable in serum-free cell culture medium. HPF nanocomplexes show a threefold increase in T2 relaxivity compared to ferumoxytol. Electron microscopy showed internalized HPF in endosomes, which we confirmed by Prussian blue staining of labeled cells. There was no long-term effect or toxicity on cellular physiology or function of HPF-labeled hematopoietic stem cells, bone marrow stromal cells, neural stem cells or T cells when compared to controls. In vivo MRI detected 1,000 HPF-labeled cells implanted in rat brains. This HPF labeling method should facilitate the monitoring by MRI of infused or implanted cells in clinical trials.

In Vivo Transfer of Intracellular Labels from Locally Implanted Bone Marrow Stromal Cells to Resident Tissue Macrophages

Intracellular labels such as dextran coated superparamagnetic iron oxide nanoparticles (SPION), bromodeoxyuridine (BrdU) or green fluorescent protein (GFP) are frequently used to study the fate of transplanted cells by in vivo magnetic resonance imaging or fluorescent microscopy. Bystander uptake of labeled cells by resident tissue macrophages (TM) can confound the interpretation of the presence of intracellular labels especially during direct implantation of cells, which can result in more than 70% cell death. In this study we determined the percentages of TM that took up SPION, BrdU or GFP from labeled bone marrow stromal cells (BMSCs) that were placed into areas of angiogenesis and inflammation in a mouse model known as Matrigel™ plaque perfusion assay. Cells recovered from digested plaques at various time points were analyzed by fluorescence microscopy and flow cytometry. The analysis of harvested plaques revealed 5% of BrdU+, 5–10% of GFP+ and 5–15% of dextran+ macrophages. The transfer of the label was not dependent on cell dose or viability. Collectively, this study suggests that care should be taken to validate donor origin of cells using an independent marker by histology and to assess transplanted cells for TM markers prior to drawing conclusions about the in vivo behavior of transplanted cells.

Proton Magnetic Resonance Spectroscopic Imaging in Children With Recurrent Primary Brain Tumors

PURPOSE Proton magnetic resonance spectroscopic imaging ((1)H-MRSI) is a noninvasive technique for spatial characterization of biochemical markers in tissues. We measured the relative tumor concentrations of these biochemical markers in children with recurrent brain tumors and evaluated their potential prognostic significance. PATIENTS AND METHODS (1)H-MRSI was performed on 27 children with recurrent primary brain tumors referred to our institution for investigational drug trials. Diagnoses included high-grade glioma (n = 10), brainstem glioma (n = 7), medulloblastoma/peripheral neuroectodermal tumor (n = 6), ependymoma (n = 3), and pineal germinoma (n = 1). (1)H-MRSI was performed on 1. 5-T magnetic resonance imagers before treatment. The concentrations of choline (Cho) and N-acetyl-aspartate (NAA) in the tumor and normal brain were quantified using a multislice multivoxel method, and the maximum Cho:NAA ratio was determined for each patients tumor. RESULTS The maximum Cho:NAA ratio ranged from 1.1 to 13.2 (median, 4.5); the Cho:NAA ratio in areas of normal-appearing brain tissue was less than 1.0. The maximum Cho:NAA ratio for each histologic subtype varied considerably; approximately equal numbers of patients within each tumor type had maximum Cho:NAA ratios above and below the median. Patients with a maximum Cho:NAA ratio greater than 4.5 had a median survival of 22 weeks, and all 13 patients died by 63 weeks. Patients with a Cho:NAA ratio less than or equal to 4.5 had a projected survival of more than 50% at 63 weeks. The difference was statistically significant (P =.0067, log-rank test). CONCLUSION The maximum tumor Cho:NAA ratio seems to be predictive of outcome in children with recurrent primary brain tumors and should be evaluated as a prognostic indicator in newly diagnosed childhood brain tumors.

A pilot study of recombinant insulin-like growth factor-I in seven multiple sclerosis patients

The purpose of this open-label, crossover study was to determine the safety and efficacy of recombinant insulin-like growth factor-1 (rhIGF-1) using magnetic resonance imaging (MRI) and clinical measures of disease activity in seven multiple sclerosis (MS) patients. Monthly clinical and MRI examinations were performed during a 24-week baseline and a 24-week treatment period with rhIGF-1. The primary outcome measure was contrast enhancing lesion (CEL) frequency on treatment compared to baseline. Secondary outcome measures included clinical and MRI measures of disease activity including: white matter lesion load (WMLL), magnetization transfer ratio (MTR), T1-Hypointensity volume, cervical spine cross-sectional area and proton magnetic resonance spectroscopic (MRS) imaging for determining regional metabolite ratios. rhIGF-1 (Cephalon) was administered at a dose of 50 mg subcutaneously twice a day for 6 months. rhIGF-1 was safe and well tolerated with no severe adverse reactions. There was no significant difference between baseline and treatment periods for any MRI or clinical measures of disease activity. Although rhIGF-1 did not alter the course of disease in this small cohort of MS patients, the drug was well tolerated. Further studies using rhIGF-1 alone or in combination with other therapies may be of value because of the proposed mechanism of action of this growth factor on the oligodendrocyte and remyelination.