How best to isolate and define tissue-specific stem cells is often contentious, but the field of cardiac regeneration tends to be especially discordant. At a recent meeting on cardiovascular regenerative medicine sponsored by the U.S. National Institutes of Health (NIH), academic researchers disagreed on questions of the most fundamental nature, such as whether stem cells persist in the adult heart.

Credit: original painting by Jon Marquette

Perhaps the most heated debate is the merit of clinical trials treating persons who have suffered a heart attack with autologous, or patient-derived, bone marrow cells, given that the fate of such engrafted cells is still unknown. In the preclinical arena the survival success and ability to repair heart function of engrafted cardiomyocytes are far from clear. Still, the NIH is poised to devote tens of millions of dollars to start clinical trials aimed at this question in the United States, and clinical trials are continuing in Germany.

Myocardial infarction (MI) is one of the leading causes of death and disability worldwide. Scar tissue caused by infarctions constrains the heart and reduces its pumping capacity. Scientists hope to create healing cardiomyocytes, either by engrafting stem cell-derived cardiomyocytes or preparations of adult stem cells (mesenchymal or hematopoietic) into damaged areas or by stimulating resident stem cells (assuming they exist) to form new cardiomyocytes.

Follow the cells

The most fundamental question is whether stem cells exist in the adult heart. Ken Chien has long been an advocate of genetic marking experiments to define heart progenitors, and his approach has yielded by far the best-characterized population of heart progenitors. These cells express a marker called Islet 1 (Isl1). The most rigorous test of whether these cells are in fact multipotent stem cells came from a study in which his laboratory made transgenic mice that expressed a green fluorescent protein or other traceable marker in Isl1+ cells. He found evidence for a hierarchy of multipotent and bipotent Isl1+ heart progenitor cells, which disappear shortly after birth1,2. At this meeting he reported that, late during development, their descendants are primarily located in the atrial and outflow-tract region of the heart, the regions least likely to be damaged during a heart attack, which primarily affects the left ventricular chamber.

Scientists hope to create healing cardiomyocytes, either by engrafting stem cell-derived cardiomyocytes into damaged areas or by stimulating resident stem cells (assuming they exist) to form new cardiomyocytes.

As Ronglih Liao elegantly explained, things get a bit woollier when looking for other heart progenitors in adults, a famously nonregenerative organ. She described work from other groups showing five different ways to isolate presumptive cardiac stem cells, either by growing cardiospheres or by isolating cells that express various markers, namely c-kit, Isl1, or Sca1. Another class of progenitors express a proton-pumping protein that extrudes certain dyes, providing another type of marker, which is also observed in hematopoietic stem cells (HSCs; such cells are termed side population or SP cells).

Richard Harvey proposed that a population of epicardial cells expressing the PDGF receptor are cardiac progenitors. In contrast, the Isl+ cells, which are only present in the embryo and neonates, appear to be distinct from these other populations with more restricted locations. There are large overlaps between the Sca1 and SP populations, and 5–10% of SP cells are c-kit+. Further complicating matters, the cell populations giving rise to cardiospheres are heterogeneous.

Not only is it difficult to determine what cell types a single cell might become, it is also difficult to ascertain individual cell types, let alone the relationships between various progenitors. Are they related? Are they distinct populations of stem cells, kicking in at different stages of development, or populating different regions of the heart? Do their phenotypes drift during culture? Although in vitro studies showing that cardiosphere cells can form cardiomyocytes are intriguing, genetic lineage mapping, of the kind that Chien performed for Isl1+ cells, is the most rigorous way to confirm that these cells are multipotent, to determine where and when they reside in the heart, and to determine the hierarchy that links them. Comparing microarray and proteomic data on different progenitors might help resolve which ones are related or identical populations.

Preserving grafts

For several years scientists have been able to culture cardiomyocytes from embryonic stem cells and other tissue, and now they are starting to engraft them into animal models to repair damaged hearts in MI models in mice. Chuck Murry reported grafts expanded 7-fold in size over 4 weeks when cells were injected into healthy hearts in rats. However, grafts would not form in rat hearts with infarcted hearts. Instead, cells died and had no effect on function.

His lab spent a year and a half assessing possible techniques to improve survival of the grafts. They devised a “cocktail” of prosurvival factors and cell death inhibitors to protect the engrafted cells. This seemed to do the trick, Murry said. He reported in Nature Biotechnology that engrafted cardiomyocytes survived and improved heart function after infarctions3. However, his team has not yet had time to assess the long-term survival of the cells. At the this year's meeting of the International Society for Stem Cell Research, Christine Mummery reported that whereas grafts improved function for a few weeks; benefits disappear by 13 weeks (reported in this June Nature Reports Stem Cells meeting report). She also found that the grafts do not functionally couple with the existing tissue, critical to creating a graft that can coordinate with the host's beating heart. It will be important to confirm that Murry's approach overcomes these problems.

Trials for clinicians

By far the most divisive question amongst the conference attendees was whether it is premature to test stem cell injection in human heart patients in clinical trials. Andreas Zeiher is spearheading a large clinical trial with 1,000 patients, injecting their own bone marrow into their hearts. So far modest improvements in heart function have been seen in smaller studies. Whereas some scientists at the conference believed that the benefits of the treatment, though modest at best, would add up to significant long-term benefits for patients, thus justifying going ahead with clinical trials. Others like Jonathan Epstein and Ken Chien believe such trials should wait for better understanding of what happens to the injected cells in the heart and why they are beneficial. They would like to see more robust improvements in animal models before using cell therapies in human patients.

To this end, Loren Field tried to monitor what happens to cardiomyocytes and bone marrow-derived HSCs when they are injected into the heart. Engrafted cells must be in synchrony with the host's heart tissue for the heart to beat properly, and Field's team used sophisticated imaging techniques that allowed him to monitor calcium fluxes in individual cells, which synchronize cardiomyocytes in the beating heart, aiming to see how well the donor and engrafted cells were coupled. He found that calcium fluxes differ between the mouse and human cells. When human embryonic stem cell-derived cardiomyocytes engrafted into SCID mice the cells survived but beat independently of the host tissue. When he injected bone marrow-derived HSCs into mouse hearts with MI, however, he found that the marrow-derived cells lack calcium transients and thus are unlikely to have formed cardiomyocytes. If this is the case in the human clinical trials in which HSCs were injected into hearts, then any improvement occurring is not due to formation of new muscle cells by the injected blood cells, as has been proposed by Piero Anversa and others.

Richard Lee talked about his collaboration with Claude Lechene for a multi-isotope imaging technique that could allow very precise mass spectrometric measurements of cell turnover and fate in human patients. Such imaging techniques will be essential to determine the fate of engrafted cells, as well as the properties and lineage hierarchies of natural progenitors.

Eduardo Marbán presented promising preclinical results from human myocardial biopsy samples, which contain a mixture of cells, including cells that proliferate in culture, forming “cardiospheres”. He uses a catheter to remove a 20-mg plug of tissue from human surgical patients. In culture, cardiospheres formed at the rate of about a million cells in 30 days. His group has induced MIs in pigs, taken biopsy samples from them, cultured the cardiospheres, introduced the reporter gene LacZ, and then infused cells into the animals. The labeled cells grew not only into blood vessels but also into cardiomyocytes and smooth muscle. The treatment reduced infarct size in the damaged hearts. Although the effects were small, Marbán believes it could be therapeutic. He is working with an NIH-funded consortium to prepare clinical-grade cells for an anticipated clinical trial.

Caution ahead

The divisions in the cardiovascular regeneration field on the question of when clinical trials are appropriate portend the future of stem cell research, at least as it is done responsibly (all bets are off when it comes to ongoing rather speculative clinical trials for spinal cord injury). A basic lesson is that finding evidence for the presence of a cell surface marker does not prove the existence or function of a cell, and it may not even prove the existence of a marker. Thus, it is hard to define starting populations of cells and what they ultimately become.

Understanding the fate of engrafted cells and ways to promote their survival will be crucial for any kind of cell replacement therapy to work, although the damaged heart may be an especially hostile environment. But with heart disease both common and deadly, basic scientists are finding that many clinicians are eager to pull their experiments into patients, and most of the more clinically oriented scientist-clinicians, particularly those using autologous cells to engraft, view the key output as improvement in patient survival. To them the more academic issues, such as mechanism, are secondary. This dichotomy will be fascinating and informative to follow.