Jan. 3, 2005 HOUSTON - Regeneration of damaged hearts using blood stem cells now appears to be clinically promising, say Texas researchers who show that in mice, human stem cells use different methods to morph into two kinds of cells needed to restore heart function - cardiac muscle cells that contract the heart as well as the endothelial cells that line blood vessels found throughout the organ.
Using a sophisticated way of examining the "humanness" of mouse heart cells, researchers report in the December 21 issue of the journal Circulation (which was published online December 13) that two months after mice with ailing hearts were treated with human stem cells, about two percent of cells in their heart showed evidence of a human genetic marker.
Furthermore, researchers described, for the first time, how these human master cells use different ways to become two distinct kinds of cells needed in the heart. Human stem cells primarily "fuse" onto mouse cardiac cells to produce new muscle (myocyte) cells that have both human and mouse DNA. But to form new blood vessel cells, they "differentiate" or mature by themselves, presumably to patch damaged mouse blood vessels with human cells.
These findings should help resolve debate within the field as to whether stem cell transfer actually creates new types of cells that last within a heart, says the study's lead author Edward T. H. Yeh, M.D., professor and chair of The University of Texas M. D. Anderson's Department of Cardiology.
"We have shown that these stem cells create both types of tissue needed to repair areas of damage, that they use two different ways to develop them, and that these cells can persist for up to a year, which is a long time in the life of a mouse," he says.
"Most of all, this study is important because it begins to explain why stem cells can help a heart heal," he adds. "Clinical trials that use bone marrow stem cells in people with heart damage have shown promise, but no one knows how it works. This starts to provide an explanation."
Yeh and his research team, which includes investigators from the Texas Heart Institute, have been looking for a relatively simple way to help restore the functioning of hearts damaged by chemotherapy, which can occur in up to 10 percent of cancer patients treated with such drugs, he says. "Deriving stem cells from bone marrow is a complicated matter. It would be much easier for patients if the stem cells were taken from blood. It would be as simple as a blood donation."
Last year, Yeh published a study in Circulation showing human stem cells that expressed a protein (CD34+) known to be associated with stem cells could help treat mice that had been given an artificially induced heart attack, compared to a control group of mice that did not receive the stem cells. These mice do not have an immune system, so that they would not reject the human stem cells (taken from cells left over from banking human blood.) They found that in the treated mice, new cardiac myocytes had developed at the edge of damaged tissue, and several layers of new blood vessel tissue had also grown.
In this study, Yeh wanted to find out how these new cells were formed, and how many were created. So they again induced a heart attack in immune-deficient mice and treated them with human CD34+ stem cells, and then sacrificed some of the animals after two months to look at their hearts. This time, however, instead of using the traditional method of examining heart tissue by slicing the organ, the researchers devised a way to sort all of the heart cells based on their expression of marker proteins. Among the markers they looked for were HLA (human leukocyte antigens) proteins, found only on human cells; troponin T, which identifies a myocyte, whether human or mice; and VE-cadherin to pinpoint endothelial cells in both species.
They found that one percent of the cells expressed both HLA and troponin T, which meant that they were human-derived cardiac myocytes. "That is a high frequency of new cells after only two months," Yeh says. The total proportion of cells that expressed HLA was two percent, which meant that the other one percent were human endothelial cells, which they further identified with the VE-cadherin marker. The researchers used PCR analysis to confirm the HLA findings, and then they stained the cells so that the human X chromosome would show up in one color, and the mouse X chromosome would be highlighted in a different hue.
The majority (73 percent) of HLA-positive myocytes contained both human and mouse DNA, and about 24 percent contained only human DNA. This suggests, says Yeh, that the CD34+ stem cells "fused" with existing mouse myocytes, and that the few cells without mouse DNA "either differentiated, or lost the mouse X chromosome when dividing." Such fusion has been seen in the liver when that organ repairs itself, Yeh notes. "Cell fusion has been important for muscle growth. Myocytes fuse to form a muscle and the muscle regrows fused."
He also says that cells with both mouse and human DNA are not diseased or cancerous, and appear to perform as needed, although more confirmatory work is needed before "we can declare them absolutely normal.
"This paper shows that fusion is a predominant event in muscle cell generation, and it may work by allowing a cell to enter the cell cycle and divide and produce new progeny, but all of this is new and needs to be studied further," Yeh says.
Cells that line the many vessels of the heart, however, developed differently in this experiment, Yeh says. More than 97 percent of endothelial cells with a human X chromosome showed no evidence of mouse DNA, which means that, once in the heart, they transdifferentiate, or morph directly into endothelial cells. "This is a fairly straightforward process for a stem cell, which is destined to become certain types of cell," he says.
Yeh adds that one experimental mouse has lived for more than a year, ever since the experiment was conducted "which suggests the beneficial nature of the stem cell treatment."
The work is continuing, he says. Researchers are now using diagnostic tests to study cardiac function in treated experimental mice and they are planning to test the power of other human blood stem cells to repair the heart.
The study was funded by M. D. Anderson Cancer Center. Co-authors include, from M. D. Anderson, Sui Zhang, M.D., Ph.D., Zeev Estrov, M.D., Dachum Wang, M.D., and Sean Raj; and James T. Willerson, M.D. from the The University of Texas Health Science Center at Houston, and Texas Heart Institute/St. Luke's Episcopal Hospital. Yeh shares a faculty appointment with UT Health Science Center at Houston and the Texas Heart Institute.
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