Approximately 700,000 Americans suffer a heart attack every year and stem cells have the potential to heal the damage wrought by a heart attack. Stem cells therapy has tried to take stem cells cultured in the laboratory and apply them to damaged tissues.
In the case of the heart, transplanted stem cells do not always integrate into the heart tissue. In the words of Jeffrey Spees, Associate Professor of Medicine at the University of Vermont, “many grafts simply didn’t take. The cells would stick or would die.”
To solve this problem, Spees and his colleagues examined ways to increase the efficiency of stem cell engraftment. In his experiments, Spees and others used mesenchymal stem cells from bone marrow. Mesenchymal stem cells are also called stromal cells because they help compose the spider web-like filigree within the bone marrow known as “stroma.” Even though the stroma does not make blood cells, it supports the hematopoietic stem cells that do make all blood cells. Here is a picture of bone marrow stroma to give you an idea of what it looks like:
Stromal cells are known to secrete a host of molecules that protect injured tissue, promote tissue repair, and support the growth and proliferation of stem cells.
Spees suspected that some of the molecules made by bone marrow stromal cells could enhance the engraftment of stem cells patches in the heart. To test this idea, Spees and others isolated proteins from the culture medium of bone marrow stem cells grown in the laboratory and tested their ability to improve the survival and tissue integration of stem cell patches in the heart.
Spees tenacity paid off when he and his team discovered that a protein called “Connective tissue growth factor” or CTGF plus the hormone insulin were in the culture medium of these stem cells. Furthermore, when this culture medium was injected into the heart prior to treating them with stem cells, the stem cell patches engrafted at a higher rate.
“We broke the record for engraftment,” said Spees. Spees and his co-workers called their culture medium from the bone marrow stem cells “Cell-Kro.” Cell-Kro significantly increases cell adhesion, proliferation, survival, and migration.
Spees is convinced that the presence of CTGF and insulin in Cell-Kro have something to do with its ability to enhance stem cell engraftment. “Both CTGF and insulin are protective,” said Spees. “Together they have a synergistic effect.”
Spees is continuing to examine Cell-Kro in rats, but he wants to take his work into human trials next. His goal is to use cardiac stem cells (CSCs) from humans, which already have a documented ability to heal the heart after a heart attack. See here, here, and here.
“There are about 650,000 bypass surgeries annually,” said Spees. “These patients could have cells harvested at their first surgery and banked for future application. If they return for another procedure, they could then receive a graft of their own cardiac progenitor cells, primed in Cell-Kro, and potentially re-build part of their injured heart.”
Globally, thousands of heart patients have been treated with stem cells from bone marrow and other sources. While many of these patients have been helped by these treatments, the results have been inconsistent, and most patients only show a modest improvement in heart function.
The reason for these sometimes underwhelming results seems to result from the fact that implanted stem cells either die soon after they are delivered to the heart or washed out. Since the heart is a pump, it is constantly contracting and having fluid (blood) wash through it. Therefore, it is one of the last places in the body we should expect implanted stem cells to stay put.
To that end, cardiology researchers a Emory University in Atlanta, Georgia have packaged stem cells into small capsules made of alginate (a molecule from seaweed) to keep them in the heart once they are implanted there.
W. Robert Taylor, professor of medicine and director of the cardiology division at Emory University School of Medicine, and his group encapsulated mesenchymal stem cells in alginate and used them to male a “patch” that was applied to the hearts of rats after a heart attack. Taylor’s group compared the recovery of these animals to those rats that had suffered heart attacks, but were treated with non-encapsulated cells, or no cells at all. The rats treated with encapsulated cells not only showed a more robust recovery, but they had larger numbers of stem cells in their hearts and showed better survival.
Of this work, Taylor said, “This approach appears to be an effective way to increase cell retention and survival in the context of cardiac cell therapy. It may be a strategy applicable to many cell types for regenerative therapy in cardiovascular medicine.
Readers of this blog might remember that I have detailed before the inhospitable environment inside the heart after a heart attack. Oxygen levels are low because blood vessels have died, and roving white blood cells are gobbling up cell debris and releasing toxic molecules while they do it. Also the dying cells have released a toxic cocktail of molecules that make the infarcted area very inhospitable. Injecting stem cells into this region is an invitation for more cells to die. Previous experiments have shown that preconditioning stem cells either by genetically engineering them to withstand high stress levels of by growing them in high-stress conditions prior to implantation can increase their survival in the heart.
Taylor also pointed out that the mechanical forces of the contracting heart can squeeze them and displace them from the heart, much like pinching a watermelon seed between your fingers causes it to slip out. “These cells are social creatures and like to be together,” said Taylor. “From some studies of cell therapy after myocardial infarction, one can estimate that more than 90 percent of the cells are lost in the first hour. With numbers like that, it’s easy to make the case that retention is the first place to look to boost effectiveness.”
Encapsulation keeps the mesenchymal stem cells together in the heart and “keeps them happy.” Encapsulation, however, does not completely cut off the cells from their environment. They can still sense the cardiac milieu and release growth factors and cytokines while they are protected from marauding white blood cells and antibodies that might damage, destroy, or displace them.
Alginate already has an impressive medical pedigree as a biomaterial. It is completely non-toxic, and chefs use it to make edible molds to encase other types of tasty morsels. Dentists use alginate to take impressions of a patient’s teeth and it is also used a component of wound dressings. One of Taylor’s co-authors, an Emory University colleague named Collin Weber has used alginate to encapsulate insulin-producing islet-cells that are being tested in clinical trials with diabetics.
Encasing cells in alginate prevents them from replacing dead cells, but mesenchymal stem cells tend to do the majority of their healing by means of “paracrine” mechanisms; that is to say, mesenchymal stem cells tend to secrete growth factors, cytokines and other healing molecules rather than differentiating into heart cells. Mesenchymal stem cells can be isolated from bone marrow or fat.
One month after suffering from a heart attack, those rats that had suffered a heart attack saw their ejection fractions (a measure of how much volume the heart pumps out with every beat) fell from an average of 72% to 34%. However, rats treated with encapsulated mesenchymal stem cells saw an increase in their ejection fractions from 34% to 56%. Those treated with unencapsulated mesenchymal stem cells saw their ejection fractions rise to 39%.
One of the main effects of implanted stem cells is the promotion of the growth of new blood vessels. In capsule-treated rats, the damaged area of the heart had a blood vessel density that was several times that of the hearts of control animals. Also, the area of cell death was much lower in the hearts treated with encapsulated MSCs.
The encapsulated stem cells seem to stay in the heart for just over ten days, which is the time is takes for the alginate hydrogels to break down. Taylor said that he and his lab would like to test several different materials to determine how long these capsules remain bound to the patch.
The goal is to use a patient’ own stem cells as a source for stem cell therapy. Whatever the source of stem cells, a patient’s own stem cells must be grown outside the body for several days in a stem cell laboratory, much like Emory Personalized Immunotherapy Center in order to have enough material for a therapeutic effect.
Cardiac Troponin I-interacting Kinase or TNNI3K is an enzyme that was initially identified in fetal and adult heart tissue, but was undetectable in other tissues. The function of this enzyme remains unknown, but Chinese scientists showed that overexpression of TNNI3K in cultured heart muscle cells causes them to blow up and get large (hypertrophy). Earlier this year, a research team from Peking Union Medical College showed that overexpression of TNNI3K in mice caused enlargement of the heart (Tang H., et al., J Mol Cell Cardiol 54 (2013): 101-111). These results suggested that TNNI3K is a potential therapeutic target for heart attack patients.
To that end, Ronald Vagnozzi and his colleagues in the laboratory of Thomas Force at Temple University School of Medicine and their collaborators designed small molecules that can inhibit TNNI3K activity, and these small molecules decrease cardiac remodeling after a heart attack in rodents. Large animal trials are planned next.
In the first experiments of this paper, Vagnozzi and others showed that the levels of TNNI3K in the heart increase after a heart attack. Measurements of TNNI3K protein levels failed to detect it in all tissue other than the heart. Furthermore, it was present throughout the heart, and mainly in heart muscle and not in blood vessels, fibroblasts, and other types of non-muscle heart tissues.
Next, Vagnozzi and others measured TNNI3K protein levels in heart transplant patients. The heart tissues of these patients, who had badly dysfunctional hearts showed higher than usual levels of TNNI3K protein. Thus, TNNI3K is associated with heart tissue and is up-regulated in response to heart dysfunction.
The next experiment examined the effects of overexpressing the human TNNI3K gene in mice. While the overexpression of TNNI3K did not affect heart function of structure under normal circumstances, under pathological conditions, however, this is not he case. If mice that overexpressed TNNI3K where given heart attacks and then “reperfused,” means that the blood vessel that was tied off to cause the heart attack was opened and blood flowed back into the infarcted area. In these cases, mice that overexpressed TNNI3K had a larger area of cell death in their hearts than their counterparts that did not overexpress TNNI3K. The reason for this increased cell death had to do with the compartment in the cell that generated most of the energy – the mitochondrion. TNNI3K causes the mitochondria in heart muscle cells to go haywire and kick out all kinds of reactive oxygen-containing molecules that damage cells.
Cell damage as a result of reactive oxygen-containing molecules (known as reactive oxygen species or ROS) activates a pathway in heart cells called the “p38” pathway, which leads to programmed cell death.
Once Vagnozzi and his colleagues nailed down the function of TNNI3K in heart muscle cells after a heart attack, they deleted the gene that encodes TNNI3K and gave those TNNI3K-deficient mice heart attacks. Interestingly enough, after a heart attack, TNNI3K-deficient mice showed much small dead areas than normal mice. Also, the levels of the other mediators of TNNI3K-induced cell death (e.g., oxygen-containing molecules, p38, ect.) were quite low. This confirms the earlier observations that TNNI3K mediates the death of heart muscle cells after a heart attack, and inhibiting TNNI3K activity decreases the deleterious effects of a heart attack.
And now for the pièce de résistance – Vagnozzi and his crew synthesized small molecules that inhibited TNNI3K in the test tube. Then they gave mice heart attacks and injected these molecules into the bellies of the mice. Not only were the infarcts, or areas of dead heart muscle cells small in the mice injected with these TNNI3K inhibitors, but the heart of these same mice did not undergo remodeling and did not enlarge, showed reduced scarring, and better ventricular function. This is a proof-of-principle that inhibiting TNNI3K can reduce the pathological effects of a heart attack.
This strategy must be tested in large animals before it can move to human trials, but the strategy seems sound at this point, and it may revolutionize the treatment of heart attack patients.
Scientists at Tulane University in New Orleans, La. (US) have completed a study that suggests that stem cells derived from cortical, or compact bone do a better job of regenerating heart tissue than do the heart’s own stem cells.
The study, led by Steven R. Houser, Ph.D., FAHA, director of Tulane’s School of Medicine’s Cardiovascular Research Center (CVRC), could potentially lead to an “off the rack” source of stem cells for regenerating cardiac tissue following a heart attack.
Cortical bone stem cells (CBSCs) are considered some of the most pluripotent cells in the adult body. These cells are naïve and ready to differentiate into just about any cell type. However, even though CBSCs and similar pluripotent stem cells retain the ability to develop into any cell type required by the body, they have the potential to wander off course and land in unintended tissues. Cardiac stem cells, on the other hand, are more likely to stay in their resident tissue.
To determine how CBSCs might behave in the heart, Houser’s team, led by Temple graduate student Jason Duran, collected the cells from mouse tibias (shin bones), expanded them in the lab and then injected them into back the mice after they had undergone a heart attack.
The cells triggered the growth of new blood vessels in the injured tissue and six weeks after injection had differentiated into heart muscle cells. While generally smaller than native heart cells, the new cells had the same functional capabilities and overall improved survival and heart function.
Similar improvements were not observed in mice treated with cardiac stem cells, nor did those cells show evidence of differentiation.
“What we did generates as many questions as it does answers,” Dr. Houser said. “Cell therapy attempts to repopulate the heart with new heart cells. But which cells should be used, and when they should be put into the heart are among many unanswered questions.”
The next step will be to test the cells in larger animal models. The current study was published in the Aug. 16 issue of Circulation Research.
The first patient has been treated in a groundbreaking medical trial in Ottawa, Canada, that uses a combination of stem cells and genes to repair tissue damaged by a heart attack. The first test subject is a woman who suffered a severe heart attack in July and was treated by the research team at the Ottawa Hospital Research Institute (OHRI). Her heart had stopped beating before she was resuscitated, which caused major damage to her cardiac muscle.
The therapy involves injecting a patient’s own stem cells into their heart to help fix damaged areas. However, the OHRI team, led by cardiologist Duncan Stewart, M.D., took the technique one step further by combining the stem cell treatment with gene therapy.
“Stem cells are stimulating the repair. That’s what they’re there to do,” Dr. Stewart said in an interview. “But what we’ve learned is that the regenerative activity of the stem cells in these patients with heart disease is very low, compared to younger, healthy patients.”
Stewart and his colleagues will supply the stem cells with extra copies of a particular gene in an attempt to restore some of that regenerative capacity. The gene in question encodes an enzyme called endothelial nitric oxide synthase (eNOS). Nitric oxide is a small, gaseous molecule that is made from the amino acid arginine by the enzyme nitric oxide synthase. Nitric oxide or NO signals to smooth muscle cells that surround blood vessels to relax, which causes blood vessels to dilate and this increases blood flow. In the damaged heart, NO also helps build up new blood vessels, which increase healing of the cardiac muscle. Steward added, “That, we think, is the key element. We really think it’s the genetically enhanced cells that will provide the advantage.”
The study will eventually involve 100 patients who have suffered severe heart attacks in Ottawa, Toronto and Montreal.
After a heart attack, inflammation in the heart kills off heart muscle cells and fibroblasts in the heart make a protein called collagen, which forms a heart scar. The heart scar does not contract and does not conduct electrochemical signals. The scar will contract over time, but its presence can lead to abnormal heart rhythms, also known as arrhythmias. Arrythmias can be fatal, since they can cause a heart attack. To prevent a heart attack, physicians will treat heart attack patients with a group of drugs called beta-blockers that slow down the heart rate and protect the heart from the deleterious effects of norepinephrine (secreted by the sympathetic nerve inputs to the heart). An alternative treatment is digoxin or digitalis, which is a chemical found in foxglove. Digitalis inhibits ion pumps in heart muscle cells and slows the heart and the force of its contractions. Digitalis, however, interacts with a whole shoe box fill of drugs, has a very long half-life, and is hard to dose. Therefore it is not the first choice.
Given all this, helping the heart to make a smaller heart scar is a better strategy for treating a heart after a heart attack. To accomplish this, you need to inhibit the heart fibroblasts that make the heart scar in the first place. Secondly, you must move something into the place of the dead cells. Otherwise, the heart could burst or scar tissue will move into the area anyway.
To that end, Yigang Wang and his colleagues at the University of Cincinnati Medical Center in Ohio have published an ingenious paper in which they tried two different strategies to reduce the size of the heart scar, which concomitantly increased the colonization of the heart by induced pluripotent stem cells engineered to express a sodium-calcium exchange pump.
Previously, Wang and his colleagues used a patch to heal the heart after a heart attack. The patch consisted of endothelial cells, which make blood vessels, induced pluripotent stem cells engineered to make a sodium-calcium exchange pump called NCX1, and embryonic fibroblasts. This so-called tri-cell patch makes new blood vessels, establishes new heart muscle, and the foundational matrix molecules to form a platform for beating heart muscle.
In order to get these cells to spread throughout the injured heart, Wang and others used a reagent that specifically inhibits heart fibroblasts. They used a small non-coding RNA molecule. A group of microRNAs called miR-29 family are downregulated after a heart attack. As it turns out, these microRNAs inhibit a group of genes that involved in collagen deposition. Therefore, by overexpressing miR-29 microRNAs, they could prevent collagen deposition and reduce scar formation.
The experimental design in this paper is rather complex. Therefore, I will go through it slowly. First, they tried to overexpress miR-29 microRNAs in cultured heart fibroblasts and sure enough, they inhibited collagen synthesis. Cells overexpressing miR-29 made less than a third of the collagen of their normal counterparts. When they placed these fibroblasts into the heart and induced heart attacks, again, they made significantly less collagen when they were expressing miR-29.
Then they used their miR-29 RNAs by injecting them directly into the heart before inducing a heart attack, and then after the heart attack, they applied the tri-patch. Their results were significant. The scar size was smaller (almost one-third the size of the controls), and the density of blood vessels was much higher in the tri-patched hearts treated with miR-29. The induced pluripotent stem cells differentiated into heart muscle cells and spread throughout the heart. Heart function measures also consistently went up too. The echiocardiograph before more normal, the ejection fraction went up, the % shortening of the heart muscle fibers was increased, and the relaxation phase of the heart (diastole) also was not so puffy (see graphs and figures below).
There is a cautionary note to this study. Inhibiting collagen formation after a heart attack could create soft fragile regions of the heart that are subject to rupture should the vascular systolic pressure increase. While that threat was not observed in this study, human hearts, which are much larger, would be much more susceptible to such a mishap. Therefore, while this study is interesting and suggest a strategy in humans, it requires more testing and refinement before anyone can even think about applying it to humans.
Scientists from the United Kingdom have, for the first time, highlighted the natural regenerative abilities of a group of stem cells that live in our hearts. This particular study shows that these cells are responsible for repairing and regenerating muscle tissue that has been damaged by a heart attack. Such damage to the heart can lead to heart failure.
There is a robust debate as to the regenerative capacity of cardiac stem cells (CSCs) in the hearts a adult human beings. While many scientists are convinced that CSCs in the hearts of newborns have good regenerative ability, many remain unconvinced that adult CSCs can do similar things (see Zaruba, M.M., et al., Circulation 121, 1992–2000 and Jesty, S.A., et al., Proc. Natl. Acad. Sci. USA 109, 13380–13385). Nevertheless, an earlier paper showed that when introduced into heart muscle after a heart attack, CSCs will regenerate the lost heart muscle and blood vessels lost in the infarct (see Beltrami, A.P., et al., Cell 114, 763–776). Resolving this disagreement requires a different type of experiment.
In this paper, Bernardo Nadal-Ginard and colleagues from the and his collaborators at the Stem Cell and Regenerative Biology Unit at the Liverpool John Moores University in Liverpool and his collaborators from Italy used a different way to affect the heart. When heart attacks are experimentally induced in the heart of rodents, the infarcts are large and they kill off large numbers of CSCs. Therefore, Nadal-Ginard and others induced severe diffuse damage of the heart muscle that also spared the CSCs. They gave the mice a large dose of a drug called isoproterenol, which acts as a “sympathomimetic.” This is confusing science talk that simply means that the drug speeds the heart rate to the point where the heart muscle exhausts itself and then starts to die off. This treatment, however, spares the CSCs (see Ellison, G.M., et al., J. Biol. Chem. 282, 11397–11409).
When the heart muscle was damaged, the CSCs differentiated into heart muscle cells and other heart-specific cells and repaired the damage in the heart. Also, the repairing cells were in the heart and were not the result of bone marrow stem cells that migrated to the bone marrow, thus putting to rest a controversy that has lasted for some years that CSCs are the result of bone marrow stem cells that migrate to the heart.
Elimination of CSCs prevents heart repair after heart damage. If, however, these heart-based stem cells are replaced after damage, the heart repairs itself and the heart recovers its function, anatomical integrity, and cellular structure.
In other experiments, removal of cardiac stem cells (CSCs) and re-injection after a heart attack shows that the CSCs can home in and repair the damaged heart.
Since Nadal-Ginard showed that CSCs have a capacity to home to the damaged heart, less invasive treatments might be possible and that these treatments might even prevent heart failure after a heart attack in the future.
In a healthy heart, the quantity of CSCs is sufficient to repair heart muscle tissue. However, once the heart is damaged many of the CSCs are also damaged and cannot multiply or produce new muscle tissue. In these cases it could be possible to replace damaged CSCs with new ones that have been grown in the laboratory and administered intravenously.,
These new approaches involved maintaining or increasing the activity of CSCs in order to renew heart muscle and replace old, damaged cells. This new strategy will only require intravenous administration of CSCs and not require open heart procedures that require such a long time to recover.
These findings are very promising. The nest step is a clinical trial, which is due to start early 2014 and is aimed at assessing the safety and effectiveness of CSCs for preventing and treating heart failure in humans.