In a series of experiments in animals, researchers at Johns Hopkins have successfully used a technique that tracks mesenchymal stem cells via magnetic resonance imaging (MRI) to monitor the progress of the cells in repairing tissue scarred by heart attack.

The Johns Hopkins findings, presented in November at the American Heart Association's Scientific Sessions and published in a supplement to the journal Circulation, are believed to be the first demonstration of how the technique, which labels the cells with minuscule iron oxide particles, can be used to assess the clinical benefit -- if any -- of cell-based therapies.

According to senior investigator and veterinary radiologist Dara Kraitchman, V.M.D., Ph.D., "The technique has potentially broader applications and benefits for patient care because MRI technology is widely available and avoids the discomfort and risk of infection from biopsies, the standard method used in therapy checkups."

In a related study, also presented at the meeting, the Johns Hopkins team showed that a more advanced technique used with MRI, called inversion recovery with on-resonant water suppression, or IRON for short, could be used to monitor iron-labeled stem cells and to guide deployment of a stent, a device that widens arteries at risk of clogging and prompting a heart attack.

Previous Johns Hopkins research on animals whose hearts had been injected with adult stem cells showed that heart function was restored to its original condition within two months, and more than 75 percent of dead scar tissue disappeared, having been replaced with healthy-looking heart tissue. Clinical studies are now under way at Johns Hopkins and elsewhere to find out if similar benefits result in humans.

"It is still a scientific puzzle as to whether adult stem cells develop into new and healthy heart tissue, or exactly how long their healing effects last, but MRI offers the best chance for determining just how well the therapy works at repairing damaged hearts," Kraitchman says.

The researchers made stem cells visibly distinct from all others by labeling them with a metallic compound made up of iron oxide nanoparticles, one thousandth of a millimeter in diameter, which can be permanently taken up within cells and, unlike most other metals, seen by MRI.

In the latest study, 13 dogs underwent surgery to create heart muscle damage similar to what happens in a naturally occurring heart attack. Six were treated with iron-oxide-labeled stem cells and seven served as study controls, receiving no stem-cell injections.

Mesenchymal bone marrrow stem cells, known to give rise to a variety of cell types, were injected across three regions of the hearts to find out if injecting one region or another made a difference in how well the heart recovered. The sites included heart-attack-damaged muscle consisting of mostly dead scar tissue, and normal, undamaged heart tissue, as well as tissue at the border area between the scar and normal tissue, called the peri-infarction zone.

Kraitchman, an associate professor at The Johns Hopkins University School of Medicine, says that in the peri-infarction zone, some life remains in the tissue, including a working vascular supply with some capillaries.

For two months after injection, the animals were monitored by cardiac MRI at weekly intervals.

The damaged area decreased significantly, by 20 percent in both groups, showing that healing had occurred. However, the size, or mass, of left ventricular tissue decreased by 2.5 percent in the stem-cell group, while the control group lost more than 20 percent of its mass, indicating that the stem-cell-treated hearts were maintaining their muscle strength during the healing process while control hearts were showing steady signs of failure and reduced function.

MRI also showed that stem cells were incorporated into the heart tissue itself, mostly in the peri-infarction zone. Measurements of the heart's pumping function also improved in the same region.

"Our results show that MRI tracking of mesenchymal stem cells can be used
- as a replacement to surgical biopsy - to verify that such cell-based therapies reached damaged areas of the heart and were able to effect repair and improve heart function," says study senior co-investigator Jeff Bulte, Ph.D., an associate professor in radiology at Hopkins who developed the labeling method.

In other experiments in rabbits and dogs, the researchers successfully used the IRON method, which was developed at Hopkins, to track relatively small numbers of stem cells in the body and to deploy a metallic stent, a mesh-like device that opens blood vessels narrowed by fat and calcium buildup.

"Physicians must confirm that potential therapies, whether they are cell based or involve devices, are delivered as planned to the targeted organ or other part of the body," says lead investigator Wesley Gilson, Ph.D., a postdoctoral research fellow at Johns Hopkins. Gilson's work was recognized at the heart meeting, where he was a finalist for the prestigious Melvin Judkins Young Clinical Investigator Award.

With IRON, conventional MRI technology is adapted to reveal images of ever smaller numbers of cells, avoiding image artifacts that mimic the appearance of iron-labeled cells.

Scientists were able to visualize metal objects, which previously appeared as dark spots on the MRI screen, by suppressing the vast majority of the conventional image produced from water molecules (or so-called on-resonant signal), the most common substance in the body.

By eliminating the water-based signals, the scientists were left only with the signal produced from metal objects (or so-called off-resonant signal), such as prosthetic screws, metal clips or stents.

In effect, Gilson says, the machine was made sensitive to iron molecules in the formerly unseen region. "We effectively shut out what we could easily see, to focus on what remained, and it worked. What formerly appeared on MRI as signal voids, or hyperintense signals, became clearly visible."

To validate the new technique, the Johns Hopkins team successfully differentiated between different concentrations of iron-labeled stem cells, extracted from a dog's bone marrow. In lab testing, the various concentrations, ranging from hundreds of cells to 2 million mesenchymal stem cells per 100 microliters of fluid, were injected into infarcted heart tissue from a dog, and then made visible by MRI using IRON. In another related experiment, injections of iron-labeled stem cells were made into a leg of a rabbit to see if they could be tracked in the body with the IRON-MRI technique.

Results from both experiments showed that the new method could distinguish each concentration from the other and also track the labeled stem cells in the beating heart and limb.

To test other applications of MRI using IRON, the Johns Hopkins team tracked delivery of an implantable cardiac device, a stent, in a dog. The researchers successfully deployed a conventional, stainless-steel stent, with a catheter, to one of the animal's main arteries. In conventional MRI, the stainless-steel stent would produce a large signal void that blurs the image of surrounding tissue. With MRI using IRON, however, the stent appeared as a bright object, similar to how it would appear if tracked by X-ray imaging, the traditional method used to deploy the devices.

"The ready availability of MRI machines means that IRON could be widely introduced into clinical practice within a relatively short time," says Gilson.

Funding for this study was provided by the National Institutes of Health.
Additional support came from Osiris Therapeutics, of Baltimore, Md., which developed the process for preparing the adult mesenchymal stem-cell product extracted from bone marrow that was used in the study.

Besides Kraitchman, Bulte and Gilson, other researchers involved in these studies were Matthias Stuber, Ph.D.; Lawrence Hofmann, M.D.; Dorota Kedziorek, M.D.; and Parag Karmarkar, M.S.