Erik M. Shapiro

In vivo detection of single cells by MRI

The use of high‐relaxivity, intracellular contrast agents has enabled MRI monitoring of cell migration through and homing to various tissues, such as brain, spinal cord, heart, and muscle. Here it is shown that MRI can detect single cells in vivo, homing to tissue, following cell labeling and transplantation. Primary mouse hepatocytes were double‐labeled with green fluorescent 1.63‐μm iron oxide particles and red fluorescent endosomal labeling dye, and injected into the spleens of recipient mice. This is a common hepatocyte transplantation paradigm in rodents whereby hepatocytes migrate from the spleen to the liver as single cells. One month later the animals underwent in vivo MRI and punctuated, dark contrast regions were detected scattered through the livers. MRI of perfused, fixed samples and labeled hepatocyte phantoms in combination with histological evaluation confirmed the presence of dispersed single hepatocytes grafted into the livers. Appropriate controls were used to determine whether the observed contrast could have been due to dead cells or free particles, and the results confirmed that the contrast was due to disperse, single cells. Detecting single cells in vivo opens the door to a number of experiments, such as monitoring rare cellular events, assessing the kinetics of stem cell homing, and achieving early detection of metastases. Magn Reson Med, 2006. Published 2006 Wiley‐Liss, Inc.

Sizing it up: Cellular MRI using micron‐sized iron oxide particles

There is rapidly increasing interest in the use of MRI to track cell migration in intact animals. Currently, cell labeling is usually accomplished by endocytosis of nanometer‐sized, dextran‐coated iron oxide particles. The limitations of using nanometer‐sized particles, however, are that millions of particles are required to achieve sufficient contrast, the label can be diluted beyond observability by cell division, and the label is biodegradable. These problems make it difficult to label cells other than macrophages in vivo, and to conduct long‐term engraftment studies. It was recently demonstrated that micron‐sized iron oxide particles (MPIOs) can be taken up by a number of cell types. In this study we examined the MRI properties of single MPIOs with sizes of 0.96, 1.63, 2.79, 4.50, and 5.80 μm. Furthermore, the capacity of cells to endocytose these MPIOs was investigated, and the MRI properties of the labeled cells at 7.0 and 11.7 Tesla were measured as a function of image resolution and echo time (TE). Cells labeled with MPIOs generally contained iron levels of ∼100 pg, which is approximately threefold higher than those obtained with the best strategies to label cells using nanometer‐sized particles. On occasion, some cells had levels as high as ∼400 pg. We demonstrate that these large particles and the cells labeled with them can be detected by spin echo (SE)‐based imaging methods. These measurements indicate that MPIOs should be useful for improving cell tracking by MRI. Magn Reson Med 53:329–338, 2005. Published 2005 Wiley‐Liss, Inc.

23Na MRI accurately measures fixed charge density in articular cartilage.

One of the initiating steps of osteoarthritis is the loss of proteoglycan (PG) molecules from the cartilage matrix. One method for assessing cartilage integrity, therefore, is to measure the PG content or fixed charge density (FCD) of cartilage. This report shows the feasibility of calculating FCD by 23Na MRI and introduces MRI protocols for human studies, in vivo. 23Na MRI was used to measure the sodium concentration inside bovine patellar cartilage. The sodium concentration was then converted to FCD (mM) by considering ideal Donnan equilibrium. These FCD measurements were compared to FCD measurements obtained through standard dimethylmethylene blue PG assays. There was a high correlation (slope = 0.89, r2 = 0.81) between the FCD measurements obtained by 23Na MRI and those obtained by the PG assays. These methods were then employed in quantifying the FCD of articular cartilage of human volunteers in vivo. Two imaging protocols were compared: one using a birdcage coil, the other using a transmit/receive surface coil. Both methodologies gave similar results, with the average sodium concentration of normal human patellar cartilage ranging from ∼240 to 260 mM. This corresponds to FCDs of –158 mM to –182 mM. Magn Reson Med 47:284–291, 2002.

Magnetic resonance imaging of the migration of neuronal precursors generated in the adult rodent brain

Neural progenitor cells (NPCs) reside within the subventricular zone (SVZ) in rodents. These NPCs give rise to neural precursors in adults that migrate to the olfactory bulb (OB) along a well-defined pathway, the rostral migratory stream (RMS). Here we demonstrate that these NPCs can be labeled, in vivo, in adult rats with fluorescent, micron-sized iron oxide particles (MPIOs), and that magnetic resonance imaging (MRI) can detect migrating neural precursors carrying MPIOs along the RMS to the OB. Immunohistochemistry and electron microscopy indicated that particles were inside GFAP(+) neural progenitor cells in the SVZ, migrating PSA-NCAM(+) and Doublecortin(+) neural precursors within the RMS and OB, and Neu-N(+) mature neurons in the OB. This work demonstrates that in vivo cell labeling of progenitor cells for MRI is possible and enables the serial, non-invasive visualization of endogenous progenitor/precursor cell migration.

In vivo labeling of adult neural progenitors for MRI with micron sized particles of iron oxide: quantification of labeled cell phenotype.

The subventricular zone (SVZ) is a continual source of neural progenitors throughout adulthood. Many of the animal models designed to study the migration of these cells from the ventricle to places of interest like the olfactory bulb or an injury site require histology to localize precursor cells. Here, it is demonstrated that up to 30% of the neural progenitors that migrate along the rostral migratory stream (RMS) in an adult rodent can be labeled for MRI via intraventricular injection of micron sized particles of iron oxide (MPIOs). The precursors migrating from the SVZ along the RMS were found to populate the olfactory bulb with all three types of neural cells; neurons, oligodendrocytes, and astrocytes. In all cases 10-30% of these cells were labeled in the RMS en route to the olfactory bulb. Ara-C, an anti-mitotic agent, eliminated precursor cells at the SVZ, RMS, and olfactory bulb and also eliminated the MRI detection of the precursors. This indicates that the MRI signal detected is due to progenitor cells that leave the SVZ and is not due to non-specific diffusion of MPIOs. Using MRI to visualize neural progenitor cell behavior in individual animals during plasticity or disease models should be a useful tool, especially in combination with other information that MRI can supply.

Magnetic poly(lactide-co-glycolide) and cellulose particles for MRI-based cell tracking

Biodegradable, superparamagnetic microparticles and nanoparticles of poly(lactide‐co‐glycolide) (PLGA) and cellulose were designed, fabricated, and characterized for magnetic cell labeling. Monodisperse nanocrystals of magnetite were incorporated into microparticles and nanoparticles of PLGA and cellulose with high efficiency using an oil‐in‐water single emulsion technique. Superparamagnetic cores had high magnetization (72.1 emu/g). The resulting polymeric particles had smooth surface morphology and high magnetite content (43.3 wt % for PLGA and 69.6 wt % for cellulose). While PLGA and cellulose nanoparticles displayed highest r  2* values per millimole of iron (399 sec−1 mM−1 for cellulose and 505 sec−1 mM−1 for PLGA), micron‐sized PLGA particles had a much higher r  2* per particle than either. After incubation for a month in citrate buffer (pH 5.5), magnetic PLGA particles lost close to 50% of their initial r  2* molar relaxivity, while magnetic cellulose particles remained intact, preserving over 85% of their initial r  2* molar relaxivity. Lastly, mesenchymal stem cells and human breast adenocarcinoma cells were magnetically labeled using these particles with no detectable cytotoxicity. These particles are ideally suited for noninvasive cell tracking in vivo via MRI and due to their vastly different degradation properties, offer unique potential for dedicated use for either short (PLGA‐based particles) or long‐term (cellulose‐based particles) experiments. Magn Reson Med, 2011.

Cellular Magnetic Resonance Imaging: Nanometer and Micrometer Size Particles for Noninvasive Cell Localization

SummaryThe use of nanometer and micrometer-sized superparamagnetic iron oxide particles as cellular contrast agents allows for the noninvasive detection of labeled cells on high-resolution magnetic resonance images. The development and application of these techniques to neurologic disorders is likely to accelerate the development of cell transplantation therapies and allow for the detailed study of in vivo cellular biology. This review summarizes the early development of iron oxide—based cellular contrast agents and the more recent application of this technology to noninvasive imaging of cellular transplants. The ability of this technique to allow for the noninvasive detection of in vivo transplants on the single-cell level is highlighted.

Convertible manganese contrast for molecular and cellular MRI.

We describe here the use of inorganic manganese based particles as convertible MRI agents. As has been demonstrated with iron oxide particles, manganese oxide and manganese carbonate particles can be internalized within phagocytotic cells, being subsequently shuttled to endosomes and/or lysosomes. As intact particles, only susceptibility‐induced MRI contrast is exhibited, most often seen as dark contrast in susceptibility‐weighted images. Modulation of MRI contrast is accomplished by the selective degradation of these particles within the endosomal and lysosomal compartments of cells. Upon particle deconstruction in the endosomes and lysosomes, the dissolved Mn2+ acts as a T1 agent, eliciting bright contrast in T1‐weighted images. This modulation of MRI contrast is demonstrated both in vitro in cells in culture, and also in vivo, in rat brain. These particles are the potential building blocks for an entire class of new environmentally responsive MRI contrast agents. Magn Reson Med 60:265–269, 2008.

Controlled aggregation of ferritin to modulate MRI relaxivity.

Ferritin is an iron storage protein expressed in varying concentrations in mammalian cells. The deposition of ferric iron in the core of ferritin makes it a magnetic resonance imaging contrast agent, and ferritin has recently been proposed as a gene expression reporter protein for magnetic resonance imaging. To date, ferritin has been overexpressed in vivo and has been coexpressed with transferrin receptor to increase iron loading in cells. However, ferritin has a relatively low T(2) relaxivity (R(2) approximately 1 mM(-1)s(-1)) at typical magnetic field strengths and so requires high levels of expression to be detected. One way to modulate the transverse relaxivity of a superparamagnetic agent is to cause it to aggregate, thereby manipulating the magnetic field gradients through which water diffuses. In this work, it is demonstrated by computer simulation and in vitro that aggregation of ferritin can alter relaxivity. The effects of aggregate size and intraaggregate perturber spacing on R(2) are studied. Computer modeling indicates that the optimal spacing of the ferritin molecules in aggregate for increasing R(2) is 100-200 nm for a typical range of water diffusion rates. Chemical cross-linking of ferritin at 12 A spacing led to a 70% increase in R(2) compared to uncross-linked ferritin controls. To modulate ferritin aggregation in a potentially biologically relevant manner, ferritin was attached to actin and polymerized in vitro. The polymerization of ferritin-F-actin caused a 20% increase in R(2) compared to unpolymerized ferritin-G-actin. The R(2)-value was increased by another 10% by spacing the ferritin farther apart on the actin filaments. The modulation of ferritin aggregation by binding to cytoskeletal elements may be a useful strategy to make a functional reporter gene for magnetic resonance imaging.

Detection of the anoxic depolarization of focal ischemia using manganese‐enhanced MRI

Mismatch between diffusion‐ and perfusion‐weighted MRI was used to indicate a treatable area following focal ischemia, called the penumbra. Activity‐induced manganese contrast MRI has been reported as a new visualization method for neural activation using manganese ions as a depolarization‐dependent contrast agent. It is well known that energy failure induced by cerebral ischemia produces anoxic depolarization. The purpose of this study was to detect manganese accumulation caused by permanent middle cerebral artery occlusion (MCAO) of rat brain and to compare regional differences between manganese accumulation and decreased apparent diffusion coefficient (ADC). The ratios of signal intensity of manganese‐enhanced MRI in the ipsilateral cortex to that in the contralateral cortex were 171.0 ± 17.5% in MCAO group and 108.4 ± 13.2% in the sham group. In addition, the enhanced region was much smaller than the area which was detected as having a reduced ADC. Magn Reson Med 50:7–12, 2003.