Ali S. Arbab

Intracytoplasmic tagging of cells with ferumoxides and transfection agent for cellular magnetic resonance imaging after cell transplantation: Methods and techniques

Background. Superparamagnetic iron oxides (SPIO) are being used to label cells for in vivo monitoring by magnetic resonance imaging (MRI). The purpose of this study is to present protocols using SPIO and a polycationic transfection agent for magnetic labeling of cells as a basis for cellular MRI. Methods. Various concentrations of ferumoxides (FE)–poly-l-lysine (PLL) complexes were used to magnetically label cells. Iron incorporation into cells along with cell viability and short- and long-term toxicity were evaluated. Results. Rapidly growing cell suspension and adherent cells were effectively labeled by means of endocytosis into endosomes at low concentrations of FE (25 &mgr;g/mL media) and PLL (0.75 &mgr;g/mL media). Hematopoietic stem cells and lymphocytes required higher concentrations of PLL (1.5 &mgr;g/mL) in serum-free media during initial FE-PLL complex formation before labeling the cells in culture. Total iron concentration in cells depended on the cell type, concentration of FE-PLL complexes in media, cellular density, and incubation time. Iron concentrations after overnight incubation with given FE at 25 &mgr;g/mL media resulted in, for example, T cells being labeled with 1 to 3 pg/cell of intracytoplasmic endosomal iron and 15 to 20 pg/cell of intracytoplasmic iron in mesenchymal stem cells compared with 0.01 to 0.1 pg/cell for unlabeled cells. Protocols developed for this study demonstrated no adverse effect on the cell viability, functional capacity, or toxicity. Conclusion. This technique can be used to label cells for in vivo MRI tracking of stem cells and lymphocytes. FE at a concentration of 25 to 50 &mgr;g/mL with a ratio of SPIO to PLL of 1:0.03 to 1:0.06 would be sufficient to label cells for cellular MRI.

Comparison of Transfection Agents in Forming Complexes with Ferumoxides, Cell Labeling Efficiency, and Cellular Viability

By complexing ferumoxides or superparamagnetic iron oxide (SPIO) to transfection agents (TAs), it is possible to magnetically label mammalian cells. There has been no systematic study comparing TAs complexed to SPIO as far as cell labeling efficiency and viability. This study investigates the toxicity and labeling efficiency at various doses of FEs complexed to different TAs in mammalian cells. Different classes of TAs were used, such as polycationic amines, dendrimers, and lipid-based agents. Cellular toxicity was measured using doses of TAs from 1 to 50 μg/mL in incubation media. Iron incorporation efficiency was measured by combining various amounts of FEs and different doses of TAs. Lipofectamine2000 showed toxicity at lowest dose (1 μg/mL), whereas FuGENE6 and low molecular weight poly-L-lysine (PLI.) showed the least toxicity. SPIO labeling efficiency was similar with high-molecular-weight PIX (388.1 kDa) and superfect, whereas FuGENE6 and low-molecular-weight PLL were inefficient in labeling cells. Concentrations of 25 to 50 μg/mL of FEs complexed to TAs in media resulted in sufficient endocytosis of the SPIO into endosomes to detect cells on cellular magnetic resonance imaging.

Bone marrow‐derived mesenchymal stromal cells accelerate wound healing in the rat

Bone marrow‐derived mesenchymal stromal cells (BMSCs) are multipotential stem cells capable of differentiation into numerous cell types, including fibroblasts, cartilage, bone, muscle, and brain cells. BMSCs also secrete a large number of growth factors and cytokines that are critical to the repair of injured tissues. Because of the extraordinary plasticity and the ability of syngeneic or allogeneic BMSCs to secrete tissue‐repair factors, we investigated the therapeutic efficacy of BMSCs for healing of fascial and cutaneous incisional wounds in Sprague–Dawley rats. Systemic administration of syngeneic BMSCs (2 × 106) once daily for 4 days or a single treatment with 5 × 106 BMSCs 24 hours after wounding significantly increased the wound bursting strength of fascial and cutaneous wounds on days 7 and 14 postwounding. Wound healing was also significantly improved following injection of BMSCs locally at the wound site. Furthermore, allogeneic BMSCs were as efficient as syngeneic BMSCs in promoting wound healing. Administration of BMSCs labeled with iron oxides/1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate fluorescent dye revealed that systemically administered BMSCs engraft to the wound. The increase in the tensile strength of wounds treated with BMSCs was associated with increased production of collagen in the wound. In addition, BMSC treatment caused more rapid histologic maturation of wounds compared with untreated wounds. These data suggest that cell therapy with BMSCs has the potential to augment healing of surgical and cutaneous wounds.

Preparation of Magnetically Labeled Cells for Cell Tracking by Magnetic Resonance Imaging

Publisher Summary This chapter describes the preparation of magnetically labeled cells for cell tracking by magnetic resonance imaging (MRI). Magnetic resonance (MR) tracking of magnetically labeled cells following transplantation or transfusion is a rapidly evolving new field. At one hand, MR cell tracking with its excellent spatial resolution can be used as a noninvasive tool to provide unique information on the dynamics of cell movement within and from tissues in animal disease models. As for MR contrast agents, gadolinium is the most effective paramagnetic contrast agent, owing to its seven unpaired electrons, but its relaxivity is far lower than the so-called superparamagnetic iron oxides. A significant improvement of labeling of nonphagocytic cells has been achieved by linking the particles to the human immunodeficiency virus (HIV) tat peptide. The distribution of magnetic microsphere-labeled porcine mesenchymal stem cells has been studied in a swine myocardial infarct model using MRI. Control cells are run side-by-side to determine the percent increased reactive oxygen species (ROS) production in the labeled cells. A transient increase in ROS production is also observed for the labeled cells.

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.

Magnetic Resonance Imaging and Confocal Microscopy Studies of Magnetically Labeled Endothelial Progenitor Cells Trafficking to Sites of Tumor Angiogenesis

AC133 cells, a subpopulation of CD34+ hematopoietic stem cells, can transform into endothelial cells that may integrate into the neovasculature of tumors or ischemic tissue. Most current imaging modalities do not allow monitoring of early migration and incorporation of endothelial progenitor cells (EPCs) into tumor neovasculature. The goals of this study were to use magnetic resonance imaging (MRI) to track the migration and incorporation of intravenously injected, magnetically labeled EPCs into the blood vessels in a rapidly growing flank tumor model and to determine whether the pattern of EPC incorporation is related to the time of injection or tumor size. Materials and Methods: EPCs labeled with ferumoxide–protamine sulfate (FePro) complexes were injected into mice bearing xenografted glioma, and MRI was obtained at different stages of tumor development and size. Results: Migration and incorporation of labeled EPCs into tumor neovasculature were detected as low signal intensity on MRI at the tumor periphery as early as 3 days after EPC administration in preformed tumors. However, low signal intensities were not observed in tumors implanted at the time of EPC administration until tumor size reached 1 cm at 12 to 14 days. Prussian blue staining showed iron‐positive cells at the sites corresponding to low signal intensity on MRI. Confocal microcopy showed incorporation into the neovasculature, and immunohistochemistry clearly demonstrated the transformation of the administered EPCs into endothelial cells. Conclusion: MRI demonstrated the incorporation of FePro‐labeled human CD34+/AC133+ EPCs into the neovasculature of implanted flank tumors.

Magnetic resonance imaging of labeled T-cells in a mouse model of multiple sclerosis†

Multiple sclerosis (MS) is a T cell–mediated autoimmune disease with early lesions characterized by mononuclear cellular infiltrate, edema, demyelination, and axonal loss that contribute to the clinical course of the disease. Experimental autoimmune encephalomyelitis (EAE) in the mouse is a valuable model with a similar disease course to relapsing‐remitting MS. The ability to detect the migration of encephalitogenic T cells into the central nervous system in EAE and MS would provide key information on these cells role in the development of lesions observed on magnetic resonance imaging (MRI). T cells were labeled for detection by magnetic resonance imaging using Food and Drug Administration–approved, superparamagnetic iron oxide nanoparticles (Ferumoxides) complexed to poly‐L‐Lysine (FE‐PLL). EAE was induced by adoptive transfer of either labeled or unlabeled T cells. After disease onset, FE‐PLL–labeled T cells were detected in the mouse spinal cord using in vivo and ex vivo cellular MRI. Excellent correlation was seen between MRI‐visible lesions in the spinal cord and histopathology. The results demonstrate that T cells labeled with FE‐PLL can induce EAE disease and can be detected in vivo in the mouse model. The magnetic labeling of cells opens the possibility of monitoring specific cellular phenotypes or pharmacologically or genetically engineered cells by MRI.

Investigation of neural progenitor cell induced angiogenesis after embolic stroke in rat using MRI

Using MRI, we investigated dynamic changes of brain angiogenesis after neural progenitor cell transplantation in the living adult rat subjected to embolic stroke. Neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat were labeled by superparamagnetic particles and intracisternally transplanted into the adult rat 48 h after stroke (n = 8). Before and after the transplantation, an array of MRI parameters were measured, including high resolution 3D MRI and quantitative T1, T1sat (T1 in the presence of an off-resonance irradiation of the macromolecules of brain), T2, the inverse of the apparent forward transfer rate for magnetization transfer (kinv), cerebral blood flow (CBF), cerebral blood volume (CBV), and blood-to-brain transfer constant (Ki) of Gd-DTPA. The von Willerbrand factor (vWF) immunoreactive images of coronal sections obtained at 6 weeks after cell transplantation were used to analyze vWF immunoreactive vessels. MRI measurements revealed that grafted neural progenitor cells selectively migrated towards the ischemic boundary regions. In the ischemic boundary regions, angiogenesis confirmed by an increase in vascular density and the appearance of large thin wall mother vessels was coincident with increases of CBF and CBV (CBF, P < 0.01; CBV, P < 0.01) at 6 weeks after treatment, and coincident with transient increases of K(i) with a peak at 2 to 3 weeks after cell therapy. Relative T1, T1sat, T2, and kinv decreased in the ischemic boundary regions with angiogenesis compared to that in the non-angiogenic ischemic region (T1, P < 0.01 at 6 weeks; T1sat, P < 0.05 at 2 to 6 weeks; T2, P < 0.05 at 3 to 6 weeks; kinvP < 0.05 at 6 weeks). Of these methods, Ki appear to be the most useful MR measurements which identify and predict the location and area of angiogenesis. CBF, CBV, T1sat, T1, T2, and kinv provide complementary information to characterize ischemic tissue with and without angiogenesis. Our data suggest that select MRI parameters can identify the cerebral tissue destined to undergo angiogenesis after treatment of embolic stroke with cell therapy.

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.

MRI detects white matter reorganization after neural progenitor cell treatment of stroke

We evaluated the effects of neural progenitor cell treatment of stroke on white matter reorganization using MRI. Male Wistar rats (n = 26) were subjected to 3 h of middle cerebral artery occlusion and were treated with neural progenitor cells (n = 17) or without treatment (n = 9) and were sacrificed at 5-7 weeks thereafter. MRI measurements revealed that grafted neural progenitor cells selectively migrated towards the ischemic boundary regions. White matter reorganization, confirmed histologically, was coincident with increases of fractional anisotropy (FA, P < 0.01) after stroke in the ischemic recovery regions compared to that in the ischemic core region in both treated and control groups. Immunoreactive staining showed axonal projections emanating from neurons and extruding from the corpus callosum into the ipsilateral striatum bounding the lesion areas after stroke. Fiber tracking (FT) maps derived from diffusion tensor imaging revealed similar orientation patterns to the immunohistological results. Complementary measurements in stroke patients indicated that FT maps exhibit an overall orientation parallel to the lesion boundary. Our data demonstrate that FA and FT identify and characterize cerebral tissue undergoing white matter reorganization after stroke and treatment with neural progenitor cells.