T H E   N I H    C A T A L Y S T      J A N U A R Y   -   F E B R U A R Y   2000


by Celia Hooper
Original magnetic tagging system developed by the LDRR team and their colleagues shuttles nanoparticles of iron oxide (MION-46L) into cells via a monoclonal antibody (OX-26) to the cell’s transferrin receptor. Cells internalize the superparamagnetic nanoparticles, ultimately leading to the visible marking of the cells during magnetic resonance imaging.
Three-dimensional reconstruction magnetic resonance image of rat spinal cord shows distribution of magnetically tagged cells 10 days after the tagged oligodendrocyte progenitor cells were injected into a myelin-deficient animal. This image was captured by Peter van Gelderen of the NIH In Vivo NMR Center using a 4.7 Tesla MR imager.

Staff scientist Jeff Bulte showed this picture of one of his daughter’s Pokémon game cards at a recent professional meeting, but strenuously denies that the Pokémon character was the inspiration for LDRR’s maghemite cell-tagging system.

The Clinical Center’s Laboratory of Diagnostic Radiology Research (LDRR) is on to something big, and the excitement surfaces quickly when you talk to lab chief Joe Frank or staff scientist Jeff Bulte. Along with colleagues at NIH and at the University of Wisconsin, Bulte and Frank have developed an elegant New Age version of what old-school microscopists called a vital dye—that is, a substance that marks cells without damaging them.

The New Age twist is that the "dye" in this case is a magnetic label that allows researchers to image tagged cells in living animals, noninvasively, without radioiosotopes, at a microscopic level, using magnetic resonance imaging (MRI).

If LDRR’s work pans out, it could yield the label of choice for tracking cells in vivo. Beyond the tracking of oligodendrocyte progenitor cells injected into the rat—LDRR’s starting point—Frank envisions the technique being used to trace a variety of stem cells and transgenic cells that have been harvested, cultured, or manipulated in some way outside the body and then injected back in. This could include "any stem cell, tumor cell, or other transplantable cells–for instance, islet cells or specific subpopulations of T cells to evaluate temporal-spatialmigration in autoimmune disease," Frank suggests.

Many types of cells, he says, will take up the nanoparticles of oxidized iron in LDRR’s magnetic tagging system. If the appropriate number of cells are then injected back into a living body and then home in on any soft tissue, Bulte and Frank predict they will generate detectable signal changes on MRI."People have wanted to do this for a long time," says Bulte, and he himself could barely believe it when the research team first injected and imaged magnetically tagged cells in an animal.

"I thought I was looking at charcoal particles" that had been used to mark the injection site "or some other artifact," he recalls. The key to LDRR’s success, according to Frank, was getting a high degree of magnetic labeling of cells.

The LDRR group constructed their first-generation magtag by attaching a nanoparticle of maghemite to a monoclonal antibody to the rat’s transferrin receptor–a receptor found on almost all cells in all species of animals. The cell surface receptor binds the antibody and then rapidly internalizes the iron, stashing it away in endosomes. The iron oxide in the tag is superparamagnetic, the LDRR scientists explain. This means that cells loaded with the tag are magnetized only when they are in the strong magnetic field of an MR scanner. Tagged cells retain no magnetic memory after being scanned and will not clump with one another or with metallic materials.

The researchers believe the nano-particles of iron eventually get used by the cell and then recycle through the animal’s iron pool. The team has been able to follow magtagged oligodendrocyte progenitor cells for 42 days after transplantation into the rat. They have watched the cells slowly migrate from the injection site.

Bulte says there is almost no chance of overloading an experimental subject with iron. Humans, for example, typically have 4 grams of iron in their bodies, and experimental dosing would be in the microgram range. Bulte found that the iron tag did not impair the migration or other functions of labeled cells. In fact, he says, "you can take these cells back and put them in culture" and they will thrive. He points out that birds, butterflies, bacteria, and other organisms with innate directional sense all use nanoparticles of iron in their internal compasses.

In their initial experiments, Frank, Bulte, and Peter van Gelderen of the NIH In Vivo NMR Center viewed the mag-tagged cells with a 4.7 Tesla MR instrument. But they have since shown that they can follow injected cells with a 1.5 Tesla instrument—a field strength that can be used clinically in humans. This field strength showed signal changes from a few millimeters of rat spinal cord bearing 50,000 or so tagged cells injected during the group’s experiments.

These days, Frank says, clinical MRI has a resolution of 1 mm in the human brain, and the team expects to be able to reach this level of resolution, or better, when magtagged cells make their way into clinical applications.

For animal imaging, the potential resolution could be much greater, however, with the higher field strength MR scanners now being installed in the In Vivo NMR Center. With the current 4.7 Tesla instrument, the team can resolve patches as small as 78 micrometers. At least in animal models, they expect to be able to resolve as few as five to 100 tagged cells in living animals, depending on the location of the cells within tissue. Bulte says one factor boosting the system’s sensitivity is a "blooming" or magnetic susceptibility effect–an amplification of signal from surrounding water molecules that occurs when tagged cells are excited in the MR field.

Fittingly, the first research employing the new technique involved a central ervous system study that appeared in the final issue of the Proceedings of the National Academy of Science in the Decade of the Brain [PNAS 96:15256–15261, 1999]. The work sprang from LDRR’s research on repair of dysmyelination and demyelination–damage or loss of the myelin sheath around axons seen in multiple sclerosis and other neurological diseases.

Su-Chun Zhang and Ian Duncan of the University of Wisconsin’s School of Veterinary Medicine injected oligodendrocyte precursor (CG-4) cells into the spinal cord of myelin-deficient rats. Because the injected cells had been magtagged, the researchers were, for the first time, able to follow the migration and integration of the cells into the nervous system noninvasively.

The distribution of magtagged cells on their three-dimensional MR images correlated closely with myelination in the rats, shown subsequently by sacrificing the rats, dissecting the spinal cord, and looking at the distribution of the cells with traditional histopathological techniques 14 days post-transplantation. Bulte says the high resolution, MRI-computer-generated sections of tissue were actually more helpful in interpreting the traditional anatomic sections, rather than vice versa.

Since establishing the efficacy of their technique, the LDRR scientists have gone on to document the migration of oligodendrocyte precursors in young normal rats. Along with their collaborators, they are planning studies to track migration and remyelination of stem-cell derived oligospheres injected into a myelin-deficient dog developed by the Wisconsin group.

Frank says he foresees no major technical barriers that would block use of magnetic tags in humans. "It’s just a matter of time." Tracking neural stem cells inserted in the brain or spinal cord to repair neurodegeneration or trauma-related injury would be natural extensions of LDRR’s magtag work.

In tandem with their substantive research, LDRR scientists have also been improving upon and developing the original magtag technique, for which they filed a patent application. They now have a second-generation magtag that is not dependent on species-specific monoclonal antibodies or even on the transferrin receptor. Like the first-generation tag, the new marker–a coated polymeric iron compound–shows high affinity for cell membranes and will work on any cell type.

Although the magtags have been developed to be used on a broad range ofcells and to applied ex vivo, Frank says it should be possible to adapt the tagging system to target individual cell types via specific receptors and to label these cell populations in vivo. But targeted delivery of magtags would be another frontier with additional hurdles and probably couldn’t be explored for a few years–at least until the researchers have mapped the research continent they just discovered.

Bulte observes that the group is not the first to use magnetism in navigating uncharted territory: "Human explorers made their greatest discoveries only after the compass was invented."


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