September 2004, Issue 3

Treating Heart Disease with a Patient's Own Stem Cells

Douglas W. Losordo, MD, associate professor of medicine at Tufts University School of Medicine and chief of cardiovascular research at Caritas St. Elizabeth's Medical Center, is currently directing the first US adult stem cell clinical trial for the treatment of heart disease. The phase I trial, a randomized double-blind placebo-controlled study, will involve 24 patients with severe myocardial ischemia for whom traditional therapies, such as bypass, angioplasty or stenting, have not been successful.

Myocardial ischemia is a debilitating and often painful condition characterized by poor blood flow to regions of the heart muscle. The trial is based on the hypothesis that circulating bone marrow-derived stem cells, the well-known source of new blood cells, are also capable of forming new blood vessels. The trial will involve stimulating a patient's bone marrow to release stem cells into the circulation, from which they will be collected, purified, and then injected directly into ischemic areas of the patient's heart. The researchers believe these cells will differentiate into blood vessel cells and develop a collateral blood supply for the oxygen-starved tissue. "Even though it's only a 24-patient study, by carefully defining the patient group through several parameters, I think we'll be able to detect what we're looking for, which is of course safety and some evidence that this hypothesis is correct, that these cells can actually improve tissue perfusion and make people feel better," says Losordo.

Bone marrow-derived stem cells capable of differentiating into blood vessel endothelial cells were first detected in adult human peripheral blood by Takayuki Asahara, MD, PhD, then a researcher in the laboratory of the late Jeffrey Isner at St. Elizabeth's Medical Center. Prior to this discovery in 1997, it was thought that endothelial stem cells existed only in embryos and that adults formed new blood vessels through the proliferation and migration of existing endothelial cells. Realizing the scientific and therapeutic possibilities of bone marrow-derived stem cells, Losordo's group immediately began doing laboratory studies and perfecting techniques in preparation for human trials. They have shown that injecting autologous stem cells into ischemic heart muscle results in new blood-vessel formation at the site of injection in several experimental models.

Endothelial stem cells are also known as endothelial progenitor cells (EPCs) and CD34-positive cells (for the CD34 marker on their surface). It is believed that stem cells have the potential to be any cell in the body. "The analogy I use is that it's like trains in a station," Losordo explains. "All the trains in the station have the potential to go anywhere, but as they go out on a certain track, the possibilities of places they can go become more limited, until they're on a definite journey to one location. The stem cells of the bone marrow are about the same. The true stem cells can be anything. Then they start to differentiate along a certain subdivision until they have a certain destiny."

Although they are not totipotent stem cells, with the capacity to form any tissue, the CD34 cells are pluripotent, with the capacity to form several tissues. So what makes the CD34 cells become blood vessel cells, rather than blood cells, when injected into an ischemic region of the heart? "Fortunately, environmental cues tell them what to become. When they get put into an ischemic tissue, the signals that are sent to these pluripotent cells say, 'We don't need another red blood cell here. We need a blood vessel cell.' So that's what triggers them to differentiate to a certain pathway, we think," says Losordo.

"As we looked closer and closer at these cells, it appeared that they were a natural response to an ischemic insult," says Losordo. "So when the blood supply to a tissue is interrupted, nature has a variety of mechanisms by which it tries to overcome that deficit of oxygen. One of the major efforts is the formation of a collateral blood supply. Gene therapy for angiogenesis uses the concept that when a blood vessel becomes blocked, certain genes are turned on and expressed at higher levels, as a way to encourage new blood vessel growth. It turns out that these stem cells are also part of that response. "

Besides being part of the body's response to ischemic insults, endothelial progenitor cells appear to be intimately involved in the everyday care and maintenance of the vasculature. In an elegant experiment that Losordo says is one of his favorite pieces of data, Haruchika Masuda, MD, PhD, a postdoctoral fellow in the laboratory, showed that the monthly growth of blood vessels in the uterus that occurs before menopause is contributed to significantly by these progenitor cells from bone marrow.

The current clinical trial is taking advantage of technology originally designed for missile tracking. The metal tip of the catheter used to inject the stem cells into the heart muscle is the "missile" that is tracked in three-dimensional space at a submillimeter level of precision. Moving the catheter along the inner surface of the ventricle allows the tracking software to create a map of the surface. "The catheter tip, being metal, is also able to continuously record the electrocardiogram of each [square millimeter] of muscle, and the magnitude of depolarization of that impulse tells us whether the muscle is dead or alive," says Losordo. "So we get a three-dimensional map of the inner surface of the ventricle showing us what's alive and what's scar tissue."

At the same time, the tracking device can measure and map the square millimeters of tissue during diastole and again during systole (when the heart contracts). Pieces of muscle that don't decrease in surface area during systole aren't contracting. "So now you have a map of viability and a map of contractility," says Losordo. "What we're interested in are the areas that are alive, so they're still conducting electricity, but they're not contracting. That tells us that it's an area of the heart muscle that's lacking a blood supply — it's hibernating. And that hibernating, severely ischemic muscle is what we target with the cell therapy."

The current clinical trial involves three dose levels of endothelial progenitor cells. For five consecutive days prior to stem cell collection, granulocyte colony stimulating factor (GCSF) is given to the patients to stimulate mobilization of stem cells from the bone marrow. On the fifth and sixth days, mononucleocytes are collected and separated to purify the endothelial progenitor cells. The procedure yields 70% to 95% pure CD34-positive stem cells. The primary endpoint is three months after stem cell injection, at which time the patient's symptoms will be evaluated, primarily by treadmill walking time. The results of this pilot study will help the researchers decide on the parameters of a larger, phase II study.

Losordo believes that a combination of gene and cell therapy might prove the most effective treatment for many patients. His group is currently studying some of these combination therapies. They are combining vascular endothelial growth factor (VEGF) gene therapy, which accelerates blood vessel recovery in ischemic tissue, with stimulation of bone marrow-derived progenitor cells. "The studies show that if you give a [stem cell] mobilizing agent along with gene therapy, then the impact of the gene therapy is increased by about 5-fold over what it is without the mobilizing agent. So it's a clue about a way to augment the therapy, but it's also a nice demonstration of the concept that these progenitor cells really are mediating the gene therapy effect of improving perfusion."

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