Seattle, WA - Researchers in Washington say they have overcome two key hurdles that have barred the successful differentiation of human embryonic stem (HES) cells into cardiomyocytes and their subsequent survival after transplantation. The group, having successfully created a highly purified cardiomyocyte culture and ensured high survival of the cells, went on to demonstrate not only that the cells then developed into heart muscle in rats but that, compared with rats that did not receive this cell line, rats that received the purified cells experienced some degree of recovery in infarcted muscle and did not develop heart failure.

"We showed that these cells could survive and form new heart muscle in an injured heart, and that if you do that, it prevents the development of heart failure," senior author on the study, Dr Charles E Murry (University of Washington, Seattle), told heartwire.

Murry and colleagues, with first author Dr Michael A LaFlamme (University of Washington), published their findings online August 26, 2007 in Nature Biotechnology.

As Murry described to heartwire, the first step in the experiment was to develop a technique for improving the purity of the HES-derived cardiomyocytes, from less than 1% of the cells differentiating into cardiomyocytes to 30% to 50%. The second challenge was slashing the high rates of cardiomyocyte death after transplantation.

"We'd previously been transplanting human heart-muscle cells into uninjured hearts, and we got very robust grafts, so we got a false sense of confidence," Murry said. "When we turned then to try this in infarcted hearts, we found that this is a very inhospitable environment for the growth of these cells—only maybe 15% of the animals had any detectable human heart muscle in them, and those were far too small to affect heart function. So that was a bummer."

The three primary hypothetical culprits in the death of the implanted cells, he explained, were ischemia; inflammation; and loss of cell contact with the extracellular matrix: cardiomyocytes are usually anchorage-dependent but had to be detached from their substrate in order to be injected into the rat infarcts. Ultimately, investigators created a "prosurvival cocktail" that targeted multiple possible factors affecting cell survival and provided a scaffold for injected cells to cling to.

To test the ability of the cells to form new heart muscle and attenuate infarct-related left ventricular dysfunction, the investigators injected their purified cells and prosurvival cocktail into four-day-old rat infarcts, killed the rats at 28 days, and assessed the impact of the transplanted cells using echocardiography and other imaging tests. They found that rats that had received the purified cells had attenuation of left-ventricular end-diastolic and end-systolic diameters and improved ejection fractions as well as significant increases in systolic wall thickening in the infarct zones. "What we found is that this stopped progression of heart failure from four to 28 days but didn't bring it all the way back to normal. We stopped the progression, but we didn't completely reverse it."

Murry predicts, however, that if they were to follow the animals for longer, the area of new muscle formation might increase, potentially leading to greater functional improvement. Ongoing studies are looking at whether larger doses of cells might lead to greater improvements, as well as at mechanisms of graft development, including whether implanted cardiomyocytes actually beat in synch with native heart cells.

Ultimately, says Murry, "The notion is that this would be a way, if a patient came in and had suffered a heart attack, that there could be an off-the-shelf product ready that is a highly purified, well-characterized form of human heart-muscle cells that could be transplanted into their hearts to prevent heart failure from developing after a heart attack."

But there are many more hurdles ahead, he acknowledged. For one, investigators conducted this study in an immunosuppressed cell line, something not possible in humans, for obvious reasons. "That's the biggest drawback to this approach, right now," Murry observed. "The notion is that it would have to be restricted to patients who are sick enough that we'd be willing to immunosuppress them. At the moment, we're looking at long-term immunosuppression until we can figure out how to make the cells stealthy so they can sort of fly under the radar, immunologically, or how to teach the body to be tolerant of them in a variety of ways. People are working hard on this, but it's not something that's ready for practice."

Investigators also need to redo their experiments in a larger animal model to make sure their results can be "scaled up" to hearts more similar to humans. Larger models would also enable them to better investigate side effects, including arrhythmias. In the current study, investigators saw no evidence of arrhythmias, but these are harder to spot in an animal with a heart rate of 450 beats per minute. Survival was no different in treated rats compared with control animals; the authors also saw no increase in tumors or any suggestion that cardiomyocytes were distributed or growing elsewhere in the bodies of the test animals.