Mending a Broken Heart

Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. K.A. Jackson et al., "Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells," J Clin Invest,107:1395-1402, June 2001. (Cited in 122 papers) A.A. Kocher et al., "Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function," Nat Med, 7: 430-6, April 2001. (Cited in 119 papers) In February, a three-inch nail punctured the heart of 16-year-old Dmitri Bonnville. The resulting swelling caused a heart attack, and though the boy lived, doctors feared that surviving tissue in the organ would not last. Cardiologists at William Beaumont Hospital in Royal Oak, Michigan, injected the boy with hematopoietic stem cells, hoping to repair his heart; it was the first trial of its kind in the United States.1 Two years ago, the general perception was that adult stem cells could not differentiate into other tissues, says Margaret Goodell, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston. Even though researchers have used bone marrow-derived stem cells for more than 30 years in transplants to treat leukemia, other cancer types, and genetic immune deficiencies, it was not known whether adult-derived stem cells would prove to be powerful and general therapeutic tools. Goodell and her team at Baylor helped serve notice to the scientific community that adult stem cells hold more promise than originally given credit.2 Along with second Hot Paper author Silviu Itescu and other researchers, Goodell pioneered the application of adult bone marrow-derived stem cells in tissue regeneration. Itescu and his team from Columbia University's Presbyterian Medical Center demon-strated that such derived angioblasts might reduce the morbidity and mortality associated with left ventricular remodeling.3 These Hot Papers "confirm that hematopoietic stem cells could be used to repair the heart after a heart attack," says David T. Harris, stem cell bank director, University of Arizona, and scientific director of the San Bruno, Calif.-based Cord Blood Registry, which collects and stores cord blood samples. SHOT IN THE DARK Goodell's original intent was to investigate whether bone marrow-derived stem cells could differentiate into skeletal muscle cells, and whether stem cells in the muscle might differentiate into blood. "We had a collaborator who knew how to make specific injuries in cardiac muscle, so we thought we would try there," Goodell recalls. "At the time, it was just a shot in the dark." In an early study, the researchers ground muscle from mice and included it in a bone marrow transplant.4 They followed the marked cells and found that they generated a surprising amount of blood. "We saw all this activity," says Goodell. "So we thought that these stem cells in muscle were normally going to give rise to muscle, and we were putting them in a different environment to form blood." THE BEST LAID PLANS: Schematic outline of experimental strategy used by the Goodell team. Bone marrow hematopoietic stem cells were taken from a Rosa26 mouse (blue), which expresses the lacZ transgene, and transplanted into a lethally irradiated congenic mouse. Ligation of a coronary artery was later performed, creating an infarction in the mouse heart. Two to four weeks later, the hearts were analyzed for lacZ-cell incorporation. (Reprinted with permission, J Clin Invest, 107:1395-1402, 2001) But their interpretation was wrong. The ground-up muscle cells did indeed produce blood, but the responsible party was blood-forming stem cells naturally present in the muscle. Nevertheless, the red herring proved fruitful. "That's what led us to the idea that [bone marrow-derived] stem cells could make cardiac muscle, too," Goodell adds. Goodell's team took mouse-derived, enriched hematopoietic stem cells from bone marrow and transplanted them into terminally irradiated mice. After damaging the animals' hearts, the researchers injected cells expressing lacZ, allowing the team to track them. Differentiated cardiomyocytes descended from the donor cells made up 0.02% of the damaged region, while donor-derived endothelial cells made up 3.3% of the cells in small surrounding blood vessels.2 The additions led to little functional improvement, but Goodell believes that further studies to enhance uptake could bolster its therapeutic promise. Others have joined the work, with varying results. "There's no good consensus in the field about why there is variability in [uptake]," says Goodell. GIVE 'EM MORE BLOOD Itescu's group had taken a less circuitous route. They wanted to prove that stem cells introduced into a heart could improve the secondary damage that follows a heart attack. After an infarction, surviving cells must work hard to compensate for lost tissue; they exhaust themselves and die, leading to progressive damage. "You lose more and more of the heart, and you go into heart failure," says Itescu. This is what the Michigan doctors feared. Bone marrow-derived cells can induce angiogenesis, thus rescuing oxygen-starved tissues. Itescu's team worked to identify the specific cells responsible. "We had the specific markers. Our hypothesis was that the reason people go into heart failure after a heart attack is that there isn't enough blood vessels to supply the heart cells not killed during an initial heart attack, because they have to work harder to compensate for the tissue that is lost. You want to protect that piece of heart from secondary [damage], and you can do that by [adding] blood vessels." In embryos, hemangioblasts give rise to blood vessels and blood cells. The cells express vascular endothelial growth factor receptor-2 (VEGFR-2) and form colonies in response to VEGF. Itescu reasoned that cells from adult bone marrow with similar properties would share similar markers. The team injected human volunteers with granulocyte-colony stimulating factor (G-CSF) to trigger the release of bone marrow into the blood stream. Using antibodies attached to magnetic beads to isolate cells expressing CD34, a transmembrane protein, the researchers further refined the cells until they identified five surface molecules that characterize bone marrow-derived hemangioblasts. STAINED HEART: Sections of mouse hearts after injury showing small lacZ-positive capillaries. In images e through l the sections were costained with markers for endothelial cells: flt-1 (green) and I-cam1 (red). (Reprinted with permission, J Clin Invest, 107:1395-1402, 2001) Researchers expanded the cells in vitro and injected them into rats that had an induced heart attack. Within 48 hours the cells had infiltrated the damaged site, and at two weeks showed a significant increase in the microvascularity and in the numbers of angioblasts and capillaries in the damaged area, with the human-derived cells accounting for 20% to 25% of the capillary vasculature. Moreover, the rats' heart function improved. After 15 weeks, the treated rats showed reduced heart function of 18% to 34%, compared with 48% to 59% in control groups. "[The results] change the dogma that there is a natural progression of cells working harder and subsequently dying, that you can't interrupt that phase. We've shown you can interrupt that phase, if you give them more blood," says Itescu. In a related, highly cited paper, researchers from New York Medical College and two NIH institutes found that bone marrow transplants, injected directly into the heart, resulted in 68% of new myocardium in an infarcted area.5 WORKING IN TANDEM Bone marrow-derived stem cells have been shown to differentiate into a wide variety of tissue types, including bone, cartilage, fat, tendon, and skeletal and cardiac muscle. Itescu's work suggests similar possibilities for regenerating other organs. "We're certainly looking at this as a generic model for various organisms," he says. Itescu and others are pressing on with clinical trials, despite an absence of long-term data elsewhere. Itescu is characteristically confident: They'll use the same G-CSF-administered methodology. The team's success regarding the rat studies bodes well for clinical trials, Itescu says. "I anticipate it should work even better after we adjust the dosage." Some argue that researchers should understand better the underlying biology before clinical trials begin. Others disagree. "We need to do this in parallel," states Stephen J. Forman, director of hematology and bone marrow transplantation at City of Hope National Medical Center, Duarte, Calif. "I think that any data we get from the clinical trial feeds back directly into the laboratory." Itescu's team, following the same strategy, has identified the signals from the heart that attract the stem cells, mapping the receptors that guide the cells from systemic circulation into the heart. The results are in press. At Baylor, Goodell's team is investigating the ability of skeletal-muscle stem cells to differentiate into endothelial cells and smooth muscle cells. They have teased apart one population that is especially adept at generating endothelial cells from a population with a predilection towards smooth muscle cells.6 As research continues to play out in the lab, Bonnville's condition may tell more about the promise this research holds. After the transplant, doctors noted a slight increase in the left-ventricle's ejection fraction, a measure of the heart's ability to pump blood. But, doctors were cautious about attributing improvement to the procedure and intend to evaluate Bonnville again in about 60 days. Jim Kling (jkling@nasw.org) is a freelance writer in Washington, DC. References 1. N. Wade, "Doctors use bone marrow stem cells to repair a heart," The New York Times, A:20, March 7, 2003. 2. K.A. Jackson et al., "Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells," J Clin Invest, 107:1395-1402, June 2001. (Cited in 122 papers) 3. A.A. Kocher et al., "Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function," Nat Med, 7:430-6, April 2001. (Cited in 119 papers) 4. K.A. Jackson et al. "Hematopoietic potential of stem cells isolated from murine skeletal muscle," Proc Natl Acad Sci, 96:14482-6, 1999. 5. D. Orlic et al., "Bone marrow cells regenerate infarcted myocardium," Nature, 410:701-5, April 5, 2001. (Cited in 270 papers) 6. S.M. Majka et al, "Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration," J Clin Invest, 111:71-9, January 2003.

Mending a Broken Heart

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