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.
The heart receives nerve input from several nerves. Some of these inputs come from the branches of the autonomic nervous system. If that sounds cryptic, just think of the word “automatic.” In other words, the things your body does without you consciously thinking about it are largely directed by the autonomic nervous system: digestion, breathing, the beating of your heart, and so on are all things that our body does without us consciously thinking about it.
The autonomic nervous system consists of two branches, the sympathetic and the parasympathetic branches of the autonomic nervous system. With respect to the heart, the sympathetic nerve inputs to the heart accelerate the heart beat and the force of the heart’s contractions. The parasympathetic inputs to the heart slow the heartbeat, but do not have any direct effect on the force of the heart’s contractions.
The sympathetic nerves that connect to the heart release the neurotransmitters epinephrine and norepinephrine. These neurotransmitters bind to receptors on the surface of heart muscle cells in order to elicit their stimulatory responses. The receptors that bind epinephrine and norepinephrine are called “adrenergic” receptors because they bind epinephrine, which used to be called “adrenaline.” When pharmacists talk about “adrenergic” stimulation, they mean receptors that bind to epinephrine and norepinephrine (for the sake of brevity, I am going to abbreviate these two molecules as Epi/NE).
Now if all this seems confusing, I am sorry, but it is going to get worse. You see there are different flavors of adrenergic receptors. There are alpha and beta adrenergic receptors. Both alpha and beta adrenergic receptors bind Ep/NE, but the specific responses they elicit can differ, depending on the cell and the machinery it has to respond to the bound receptor. A quick example might help make this clear. If you get an asthma attack, you can breathe in a product called Primatene Mist, which is simply aerosolized epinephrine. Epi, in your lungs, causes the smooth muscles that surround your breathing passages to relax and your breathing passages dilate. This allows you to breath much more easily. However, that same molecule, Epi, will cause your heart to beat faster and harder. The same molecule – Epi – elicits two completely distinct responses from two tissues. This is due to the fact that the heart has one type of adrenergic receptor on the surfaces of its cells (so-called beta1 adrenergic receptors), and the bronchial smooth muscle has a distinct beta adrenergic receptor the on the surfaces of its cells (so-called beta2 adrenergic receptors).
I realize that this is a very long introduction, but it is necessary in order to talk about the paper that I found. In this paper, scientists in Mark Sussman’s laboratory at the San Diego Heart Research Institute have examined cardiac progenitor cells (CPCs) from male mice and their response to beta adrenergic stimulation. You see, once we are born, adrenergic stimulation causes the heart to grow and mature. However, once the heart muscle cells mature, this stimulation no longer causes the heart to enlarge in the same way that heart normally does shortly after birth, although the heart is still capable of remodeling in response to constant aerobic exercise. However, after a heart attack, the secretion of Epi/Ne tends to drive deterioration of the heart. Therefore, a common drug strategy to treat heart attack patients is to prescribe a class of drugs called “beta blockers,” which protect the heart from the deleterious effects of adrenergic stimulation after a heart attack. However, the effects of adrenergic stimulation on CPCs is unknown, and Sussman’s laboratory used cultured CPCs to determine the effects of adrenergic stimulation on CPCs.
CPCs are a stem cell population that resides in the heart. A respectable corpus of literature has shown that CPCs can differentiate into various heart-specific cell types and replace dying heart muscle. Our hearts do not recover properly after a heart attack because the CPCs healing capacities are overwhelmed after a heart attack (See Leri A, Kajstura J, and Anversa P, Circulation Research 109 (2011) 941-61 for an excellent summary of the physiological tasks performed by CPCs).
In the Sussman paper, cultured CPCs from mice and humans were cultured in the laboratory. It was quickly discovered that CPCs do NOT express beta1 adrenergic receptors on their surfaces, but beta2 adrenergic receptors. You might smirk and this and say “so what?” However this is significant for the following reason: Early in their lives, heart muscle cells expression beta2 adrenergic receptors, but they later switch to exclusive expression of beta1 adrenergic receptors. They express beta2 adrenergic receptors during that time when they can rapidly divide and respond to the needs of the heart. CPCs express beta 2 adrenergic receptors only when they are in their undifferentiated state. Once they differentiate, they switch to beta1 adrenergic receptors.
Secondly, Sussman and his crew discovered that stimulation of the beta2 adrenergic receptors on the surfaces of CPCs caused them to divide. Sussman and others used a molecule called fenoterol, which binds very tightly to beta2 adrenergic receptors and activates them.
Third, once the CPCs were differentiated into heart muscle cells, they no longer expressed beta2 adrenergic receptors, but expressed beta 1 adrenergic receptors. Did this change the response of the cells to adrenergic stimulation? YES. Instead of dividing in response to adrenergic stimulation, the cells were much more sensitive to dying. To make sure that this result was not a fluke, Sussman and others engineered CPCs to express beta1 adrenergic receptors, and, sure enough, those cells were also sensitized to cell death upon expression of beta1 adrenergic receptor.
This is all fine and dandy for a culture dish, but can this make a difference in a living animal? Sussman used a specific mouse strain called TOT. These mice have a special pathology in that their hearts enlarge and start to not work very well once they are exposed to large quantities of Epi/NE. Can beta blockers prevent this enlargement of the heart in TOT mice? It definitely can. However, Sussman wanted to know what happened to the CPCs. Therefore, they broke the mice into three groups. Two groups received metoprolol and the third did not. Then four weeks later, one TOT mouse group that had received metoprolol and another that had not received transplantations of marked CPCs into their hearts (the CPCs glowed). Then they examined the CPCs two weeks after the implantation. The CPCs in non-metoprolol-treated TOT mice took a beating. However, in the metoprolol-treated mice, the CPCs were three times more prevalent and showed overall lower levels of programmed cell death. There was less DNA synthesis in the hearts of metoprolol-treated animals, indicating that there was less of a need for replacement of dead cells.
These results indicate that beta blockers do more than protect the heart from excessive Epi/NE after a heart attack. They also protect the CPCs in the heart, and that could be an even more significant contribution to the life of the heart after a heart attack. It is might be possible to direct or even augment the activity of CPCs in the heart after a heart attack to accelerate cardiac healing. That would be a tremendous step in cardiac healing.
Bone Morphogen Protein 2 (BMP2) is a powerful signaling molecule that is made during development, healing, and other significant physiological events. During the development of the heart, BMP2 modulates the activation of cardiac genes. In culture, BMP2 can protect heart muscle cells from dying during serum starvation. Can BMP2 affect hearts that have just experienced a heart attack?
Scientists from the laboratories of Karl Werdan and Thomas Braun at the Max Planck Institute or Heart and Lung Research in Bad Nauheim, Germany have addressed this question in a publication in the journal Shock.
In this paper, Henning Ebelt and his colleagues Gave intravenous BMP2 to mice after a heart attack. CD-1 mice were subjected to LAD-ligation to induce a heart attack (LAD stands for left anterior descending coronary artery, which is tied shut to deprive the heart muscle of oxygen). 1 hour after the heart attack, mice were given 80 microgram / gram of body weight of intravenous recombinant BMP2. The hearts of some animals were removed 5-7 days after the heart attack, but others were examined 21 days after the heart attack to determine the physiological performance of the hearts. Control animals were given intravenous phosphate buffered saline.
The extirpated hearts were analyzed for cell death, and the size of their heart scars. Also, protein expression analyses showed the different proteins expressed in the heart muscle cells as a result of BMP2 treatment. Also, the effects of BMP2 on cultured heart muscle cells was ascertained.
The results showed that BMP2 could protect cultured heart muscle cells from dying in culture if they when they were exposed to hydrogen peroxide. Hydrogen peroxide mimics stressful conditions and under normal circumstances, cultured heart muscle cells pack up and die in the presence of hydrogen peroxide (200 micromolar for those who are interested). However, if cultured with 80 ng / mL BMP2, the survival of cultured heart muscle cells greatly increased.
When it came to the hearts of mice that were administered iv BMP2, the BMP2-administered mice survived better and had a smaller infarct size (almost 50% of the heart in the controls and less than 40% in the BMP2-administered hearts). When the degree of cell death was measured in the mouse hearts, those hearts from mice that were administered BMP2 showed less cell death (as determined by the TUNEL assay). BMP2 also increased the beat frequency and contractile performance of isolated heart muscle cells.
FInally, the physiological parameters of the BMP2-treated animals were slightly better than in the control animals. The improvements were consistent, but not overwhelming.
Interestingly, when the proteins made by the hearts of BMP2- and PBS-administered animals were analyzed, there were some definite surprises. BMP2 normally signals to cells by binding a two-part receptor that sticks phosphates on itself, and in doing so, recruits “SMAD” proteins to it that end up getting attached to them. The SMAD proteins with phosphates on them stick together and go to the nucleus where they activate gene expression.
However, the heart muscle cells of the BMP2-administered mice did not contain heavily phosphorylated SMAD2, even though they did show phosphorylated SMAD1, 5, & 8. I realize that this may sound like Greek to you, but it means this: Different members of the BMP superfamily signal to cells by utilizing different combinations of phosphorylated SMADs. The related signaling molecule, TGF-beta (transforming growth factor-beta), increases scar formation in the heart after a heart attack. TGF-beta signals through SMAD2. BMP2 does not signal through SMAD2, and therefore, elicits a distinct biological response than TGF-beta.
These results show that BMP2 administration after a heart attack decreases cell death and decreases the size of the heart scar. There might be a clinical use for BMP2 administration after a heart attack.
See Henning Ebelt, et al., Shock 2013 Apr;39(4):353-60.
Atsushi Asakura and his colleagues at the University of Minnesota Stem Cell Institute have extended some of their earlier findings in a paper that appeared in PLoS One last year. This paper is almost a year old by now, but its results are fascinating and are definitely worth examining.
In 2007, Asakura published a paper with the Canadian researcher Michael A. Rudnicki in the Proceedings of the National Academy of Sciences. In this paper, Asakura and his colleagues examined the ability of muscle satellite cells from MyoD- mice to integrate into injured muscle. I realize that last sentence just sounded like gobbledygook, to some of you, but I will try to put the cookies on a lower shelf.
Satellite cells constitute a stem cell population within skeletal muscle. They are a small population of muscle-making stem cells found in skeletal muscle and they express a whole host of muscle-specific genes (e.g., desmin, Pax7, MyoD, Myf5, and M-cadherin). Satellite cells are responsible for muscle repair, but previous work has shown that there are at least two populations of satellite cells in skeletal muscle. One population rapidly contributes to muscle repair, whereas the other population is more stem cell-like and remains longer in an undifferentiated state in the recipient muscle (see Beauchamp JR , et al (1999) J Cell Biol 144:1113–1122; Kuang S , et al (2007) Cell 129:999–1010). Presently, it is not clear which population is more efficient in repairing continuously degenerating muscle.
MyoD is a gene that encodes a protein that binds to DNA and activates the expression of particular genes. It plays a vital role in regulating muscle differentiation, and belongs to a family of proteins known as myogenic regulatory factors or MRFs. All MRFs are bHLH or basic helix loop helix transcription factors, and they act sequentially in muscle differentiation. MRF family members include MyoD, Myf5, myogenin, and MRF4 (Myf6). MyoD is one of the earliest genes that indicates a cell has committed to become a muscle cell. MyoD is expressed in activated satellite cells, but not in quiescent (sleeping) satellite cells. Strangely, even though MyoD marks myoblast commitment, muscle development is not dramatically prevented in mouse mutants that lack the MyoD gene. However, this is likely to result from functional redundancy from Myf5. Nevertheless, the combination of MyoD and Myf5 is vital to the success of muscle production.
Therefore, Asakura and his crew decided to isolated muscle satellite cells from mice that lacked functional copies of the MyoD gene. Making such mice is labor intensive, but doable with mouse embryonic stem cell technology. When such MyoD- mice were made, Asakura and others isolated the satellite cells from these mice and characterized them. They discovered in their 2007 paper, that the satellite cells from the MyoD- mice were much more stem cell-like than satellite cells from MyoD+ mice. The MyoD- satellite cells grew better in culture, integrated into injured muscles better and survived better than their MyoD+ counterparts.
Why is this important? Because when it comes to treating degenerative muscle diseases like muscular dystrophy, finding the best cell is crucial. MyoD+ satellite cells have been used, but they are limited in the amount of muscle repair they provide. MyoD- cells might be a better option for treating a disease like muscular dystrophy.
Or for that matter, what about the heart? Finding the right cell to treat the heart after a heart attack has proven difficult. There are some things bone marrow cells do well, and other things they do not do well when it comes to regenerating the heart. Likewise, there are some things mesenchymal do well and other things they do not do well when placed in a damaged heart. Can MyoD- satellite cells do a better job than either of these types of stem cells?
That was the question addressed in the 2012 Nakamura paper that was published in PLoS One. Clinical trials that have treated heart attack patients with injections of MyoD+ satellite cells into the heart have shown that such treatments can improve heart function, but usually only transiently. They also prevent remodeling of the heart after a heart attack. However, two larger studies failed to produce significant improvements in heart function compared to the placebo, and patients who received the satellite cell transplants were also susceptible to very fast heart beats (tachycardia). Because of these downsides, the excitement for transplanting muscle satellite cells into the heart has waned.
So how did MyoD- satellite cells do? All the laboratory animals used in this experiment (BALB/c mice) were given heart attacks, and injected with either MyoD+ or MyoD- satellite cells. The hearts of animals injected with MyoD- satellite cells were compared with animals whose hearts were injected with MyoD+ satellite cells.
In culture, the MyoD- satellite cells grew better than the MyoD+ cells. When injected into the heart, the MyoD- cells integrated into the heart muscle and spread throughout the heart muscle much more robustly than the MyoD+ cells. The MyoD- cells were also much less susceptible to cell death and survived better than their MyoD+ counterparts.
Functionally speaking for the heart, animals that had received transplantations of MyoD- satellite cells had higher ejection fractions, small areas of dead heart tissue, lower end systolic and end diastolic volumes, and more normal echocardiograms. Even though MyoD- cells differentiated into skeletal muscle and not heart muscle (no surprise there), the MyoD- cells induced a very substantial quantity of new blood vessels to sprout in the scar area.
From these experiments, it seems that the MyoD- satellite cells are superior to the MyoD+ satellite cells for treating heart after a heart attack. These cells secrete a whole host of factors that aid the heart in healing and also structurally support the heart and prevent remodeling.
Might it be possible to use such cells in human trials? Asakura notes that engineering MyoD- satellite cells would be impractical for human clinical purposes, but it might be possible to downregulate MyoD expression with drugs (bromodeoxyuridine) or other reagents (RNAi or Id protein transformation).
This work shows that there is a better way to use muscle satellite cells for heart treatments. It simply requires you to remove MyoD function, and the cells will grow and spread throughout the heart better, and more robustly augment heart function and healing.
Stephen Worthley from the Cardiovascular Investigation Unit at the Royal Adelaide Hospital in Adelaide, Australia and his colleagues have conducted a timely experiment with rodents that examines the effects of dosage and timing on stem cell treatments in the heart after a heart attack.
Mesenchymal stem cells from bone marrow and other sources have been used to treat the heart of laboratory animals and humans after a heart attack. The optimal timing for such a treatment remains uncertain despite a respectable amount of work on this topic. Early intervention (one week) seems offer the best hope for preserving cardiac function, but the heart at this stage is highly inflamed and cell survival is poor. If treatment is delayed (2-3 weeks after the heart attack), the prospects for cell survival are better, but the heart at this time is undergoing remodeling and scar formation. Therefore, stem cell therapy at this time seems unlikely to work. Human clinical trials seem to suggest that mesenchymal stem cell treatment 2-3 weeks after a heart attack does no good (see Traverse JH, et al JAMA 2011;306:2110-9). The efficacy of the delivering mesenchymal stem cells to the heart at these different times has also not been compared.
If that degree of uncertainty is not enough, dosage is also a mystery. Rodent studies have used doses of one million cells, but studies have not established a linear relationship between efficacy and dose, and higher dosages seem to plateau in effectiveness (see Dixon JA, et al Circulation 2009;120(11 Suppl):S220-9). High doses might even be deleterious.
So what is the best time to administer after a heart attack, and how much should be administered? These are not trivial questions. Therefore a systematic study is required and laboratory animals such as rodents are required.
In this study, five groups of rats were given heart attacks by ligation of the left anterior descending artery, and two groups of rats received bone marrow-derived mesenchymal stem cells immediately after the heart attack. The first group received a low dose (one million cells) and the second group received twice as many cells. The three other groups received their treatments one week after the heart attack. The third group received the low dose of stem cells received the low dose of cells (one million cells), and the fourth group received the higher dose (two million cells). The fifth group received no such cell treatment.
All mesenchymal stem cells were conditioned before injection by growing them under low oxygen conditions. Such pretreatments increase the viability of the stem cells in the heart.
The results were interesting to say the least. when assayed four weeks after the heart attacks, the hearts of the control animals showed a left ventricular function that tanked. The ejection fraction fell to 1/3rd the original ejection fraction (~60% to ~20%) and stayed there. The early high dose animals showed the lowest decrease in ejection fraction (-8%). The early low dose group showed a greater decrease in ejection fraction. Clearly dose made a difference in the early-treated animals with a higher dose working better than a lower dose.
In the later-treated animals, dose made little difference and the recovery was better than the early low dose animals. when ejection fraction alone was considered. However, when other measures were considered, the picture becomes much more complex. End diastolic and end systolic volumes were all least increased in the early high dose animals, but all four groups show significantly lower increases than the controls. The mass of the heart, however, was highest in the late high-dose animals as was ventricular wall thickness.
When the movement of the heart walls were considered, the early-treated animals showed the best repair of those territories of the heart near the site of injection, but the later-treated animals showed better repair at a distance from the site of injection. The same held for blood vessel density: higher density in the injected area in the early-treated animals, and higher blood vessel density in those areas further from the site of injection in the later-treated animals.
The size of the heart scar clearly favored the early injected animals, which the lower amount of scarring in the early high dose animals. Finally when migration of the mesenchymal stem cells throughout the heart was determined by using green fluorescent protein-labeled mesenchymal stem cells, the later injected mesenchymal stem cells were much more numerous at remote locations from the site of injection, and the early treated animals only had mesenchymal stem cells at the site of injection and close to it.
These results show that the later doses of mesenchymal stem cells improve the myocardium further from the site of the infarction and the early treatment improve the myocardium at the site of the infraction. Cell dosage is important in the early treatments favoring a higher dose, but not nearly as important in the later treatments, where, if anything, the data favors a lower dose of cells.
Mesenchymal stem cells affect the heart muscle by secreting growth factors and other molecules that aids and abets healing and decreases inflammation. However, research on these cells pretty clearly shows that they modulate their secretions under different environmental conditions (see for example, Thangarajah H et al Stem Cells 2009;27:266-74). Therefore, the cells almost certainly secrete different molecules under these conditions.
In order to confirm these results, similar experiments in larger animals are warranted, since the rodent heart is a relatively poor model for the human heart as it beats much faster than human hearts.
See James Richardson, et al Journal of Cardiac Failure 2013;19(5):342-53.
Julie Chao is from the Department of Biochemistry and Molecular Biology, at the Medical University of South Carolina. Dr. Chao and her colleagues have published a paper in Circulation Journal about genetically modified mesenchymal stem cells and their ability to help heal a heart that has just experienced a heart attack.
Several laboratories have used mesenchymal stem cells (MSCs), particularly from bone marrow, to treat the hearts of laboratory animals that have recently experienced a heart attack. However, heart muscle after a heart attack is a very hostile place, and implanted MSCs tend to pack up and die soon after injection. Therefore, such injected cells do little good.
To fix this problem, researchers have tried preconditioning cells by growing them in a harsh environment or by genetically engineering them with genes that can increase their tolerance of harsh environments. Both procedures have worked rather well. In this paper, Chao and her group engineered bone marrow-derived MSCs to express the genes that encode “tissue kallikrein” (TK). TK circulates throughout our bloodstream but several different types of cells also secrete it. It is an enzyme that degrades the protein “kininogen” into small bits that have several benefits. Earlier studies from Chao’s own laboratory showed that genetically engineering TK into the heart improved heart function after a heart attack and increased the ability of MSCs to withstand harsh conditions (see Agata J, Chao L, Chao J. Hypertension 2002; 40: 653 – 659; Yin H, Chao L, Chao J. Journal of Biol Chem 2005; 280: 8022 – 8030). Therefore, Chao reasoned that using MSCs engineered to express TK might also increase the ability of MSCs to survive in the post-heart attack heart and heal the damaged heart.
In this paper, Chao and others made adenoviruses that expressed the TK gene. Adenoviruses place genes inside cells, but they do not integrate those genes into the genome of the host cell. Therefore, they are safer to use than retroviruses. Chao and others used these TK-expressing adenoviruses to infect tissue and MSCs.
When TK-expressing MSCs were exposed to low-oxygen conditions, like what cells might experience in a post-heart attack heart, the TK-expressing cells were much heartier than their non-TK-expressing counterparts. When injected into rat hearts 20 minutes after a heart attack had been induced, the TK-expressing MSCs showed good survival and robust TK expression. Control hearts that had been injected with non-TK-expression MSCs or had not been given a heart attack showed no such elevation of TK expression.
There were also added bonuses to TK-expressing MSC injections. The amount of inflammation in the hearts was significantly less in the hearts injected with TK-expressing MSC injections compared to the controls. There were fewer immune cells in the heart 1 day after the heart attack and the genes normally expressed in a heart that is experiencing massive inflammation were expressed at lower levels relative to controls, if they were expressed at all.
Another major bonus to the injection of TK-expressing MSCs into the hearts of rats was that these cells protected the heart muscle cells from programmed cell death. To make sure that this was not some kind of weird artifact, Chao and her team placed the TK-expressing MSCs in culture with heart muscle cells and then exposed them to low-oxygen tension conditions. Sure enough, the heart muscle cells co-cultured with the TK-expressing MSCs survived better than those co-cultured with non-TK-expressing MSCs.
Finally, when the hearts of the rats were examined 2 weeks after the heart attack, it was clear that the enlargement of the heart muscle (so-called “remodeling”) occurred in animals that had received non-TK-expressing MSCs or had received no MSCs at all, but did not occur in the hearts of rats that had received injections of TK-expressing MSCs. The heart scar was also significantly smaller in the hearts of rats that had received injections of TK-expressing MSCs, and had a greater concentration of new blood vessels. Apparently, the TK-expressing MSCs induced the growth of new blood vessels by recruiting EPCs to the heart to form new blood vessels.
In conclusion, the authors write that “MSCs genetically-modified with human TK are a potential therapeutic for ischemic heart diseases.”
Getting FDA approval for genetically engineered stem cells will not be easy, but TK engineering seems much safer than some of the other modifications that have been used. Also the vascular and cardiac benefits of this gene seem clear in this rodent model. Pre-clinical trials with larger animals whose cardiac physiology is more similar to humans is definitely warranted and should be done before any talk of human clinical trials ensues.
By aggressively treating heart attack patients soon after their episodes, clinicians have been able to reduce early mortality from heart attacks. However, the survival of these patients tends to create a whole new set of issues for them and their hearts. Chronic heart failure is a common aftermath of a heart attack for heart attack survivors. (see Kovacic JC and Fuster V., Clin Pharmacol Ther 2011;90:509-18).
Since the heart muscle (myocardium) has only a limited capacity to regenerate after a heart attack, multifaceted treatments have emerged that are designed to relieve symptoms and improve the patient’s clinical status. In particular, therapies target impaired contractility of the heart and the ability of the heart to handle the workload without enlarging. However, these treatments do not address the loss of heart muscle that underlies all heart attacks (see McMurray JJ. Systolic heart failure. N Engl J Med 2010;362:228-38). To address the loss of contracting heart tissue, stem cells, traditionally isolated from bone marrow, have been used in several clinical trials. However, the results of these studies have been highly variable, since most bone marrow stem cells placed in a heart after a heart attack, die soon after implantation.
To improve the ability of bone marrow stem cells to repair the heart, Andre Terzic from the Mayo Clinic Center for Regenerative Medicine has designed a special cocktail to induce mesenchymal stem cells from bone marrow to become more heart-friendly. This cocktail consisted of the following growth factors: TGFβ1, BMP-4, Activin-A, retinoic acid, IGF-1, FGF-2, α-thrombin and IL-6. Mesenchymal stem cells were cultured for 10 days in this cocktail and then tested for heart-specific genes.
Terzic calls this procedure “cardiopoiesis,” and when he subjected bone marrow mesenchymal stem cells (BM-MSCs) to this procedure, they expressed a cadre of genes that is normally found in developing heart cells (Nkx2-5, MEF2C, GATA4, TBX5, etc.). In an earlier publication, Terzic and his colleagues transplanted BM-MSCs from heart patients into the hearts of mice that had suffered a heart attack and compared the effects of these cells on the heart, with BM-MSCs that had undergone this guided cardiopoiesis protocol. The results were astounding. Not only did the function of the hearts that had received the guided cardiopoiesis M-MSCs much more normal than those had had received the untreated BM-MSCs, but post-mortem examination of the hearts showed that the hearts that had received guided cardiopoiesis BM-MSCs contained human heart muscle cells integrated into the heart muscle tissue (Atta Behfar, et al., J Am CollCardiol. 2010 August 24; 56(9): 721–734). Therefore, this procedure, cried out for a clinical trial, and data from such a trial has already been reported.
In a paper from February 2013 (Bartunek J, et al., Journal of the American College of Cardiology (2013), doi: 10.1016/j.jacc.2013.02.071), Terzic and his team has reported on the administration of BM-MSCs into the hearts of 34 heart patients. Of these patients, 21 were implanted with their own BM-MSCs that had undergone guided cardiopoiesis and the other 12 received standard therapy for heart patients with no transplanted cells.
The results from this study were striking to say the least. According to Terzic, “The benefit to patients who received cardiopoietic stem cell delivery was significant.” Cardiologist Charles Murry wrote in an editorial, “Six months after treatment, the cell therapy group had a seven percent absolute improvement in EF (ejection fraction) over baseline, versus a non-significant change in the control group. The improvement in EF is dramatic, particularly given the duration between the ischemic injury and cell therapy. It compared favorably with our most potent therapies in heart failure.”
This clinical trial, known as the C-CURE trial, which stands for Cardiopoietic Stem Cell Therapy in Heart Failure. was an international, multi-center trial that treated enrolled patients from hospitals in Belgium, Serbia, and Switzerland. This trial represents the culmination of almost a decade of work by Terzic and others. “Discovery of rare stem cells that could inherently promote heart regeneration provided a critical clue. In following this natural blueprint, we further developed the know-how needed to convert patient-derived stem cells into cells that can reliably repair a failing heart.”
For this trial, Mayo Clinic partnered with Cadio3 Biosciences, which is a bio-science company in Mort-Saint-Guilbert, Belgium. This company provided advance product development, manufacturing scale-up, and clinical trial execution. Adaptation of this exciting new technology to the clinic could mean a new exciting fix for heart patients.
In a clinical trial that is probably one of the first of its kind, researchers from the laboratory of Marc Penn at the Summa Cardiovascular Institute in Akron, Ohio, activated the stem cells of heart failure patients by means of gene therapy.
Penn and his colleagues delivered a gene that encodes stromal-cell derived factor-1 or SDF-1. SDF-1 is a member of the chemokine family of signaling proteins, and chemokines are proteins that direct cells to get up and move somewhere. Thus, for stem cells, SDF-1 acts as a kind of “homing” signal.
In this unique study, Penn and his collaborators introduced SDF-1 into the heart in order to summon stem cells to the site of injury and enhance the body’s stem cell-based repair process. In a typical stem cell-based study, researchers extract and expand the number of cells, then deliver them back to the subject, but in this study, no stem cells were extracted. Instead they were summoned to the site of injury by SDF-1.
Marc Penn, professor of medicine at Northeast Ohio Medical University in Rootstown, Ohio and the director of research at Summa Cardiovascular Institute said of his clinical trial: “We believe stem cells are always trying to repair tissue, but they don’t do it well — not because we lack stem cells but, rather, the signals that regulate our stem cells are impaired.”
Previous research by Penn and colleagues has shown SDF-1 activates and recruits the body’s stem cells to sites of injury and this increases healing. Under normal conditions, SDF-1 is made after an injury but its effects are short-lived. For example, SDF-1 is naturally expressed after a heart attack but this augmented expression of SDF-1 only lasts only a week.
In the study, researchers attempted to re-establish and extend the time that SDF-1 could stimulate patients’ stem cells. The trial enrolled 17 NYHA Class III heart failure patients, with left ventricular ejection fractions less than 40% and an average time from heart attack of 7.3 years. Three escalating JVS-100 doses were evaluated: 5 mg (cohort 1), 15 mg (cohort 2) and 30 mg (cohort 3). The average age of the participants was 66 years old.
Researchers injected one of three doses of the SDF-1 gene (5mg, 15mg or 30mg) into the hearts of these patients, and monitored them for up to a year. Four months after treatment, they found:
1. Patients improved their average distance by 40 meters during a six-minute walking test.
2. Patients reported improved quality of life.
3. The heart’s pumping ability improved, particularly for those receiving the two highest doses of SDF-1 compared to the lowest dose.
4. No apparent side effects occurred with treatment.
According to Penn, “We found 50 percent of patients receiving the two highest doses still had positive effects one year after treatment with their heart failure classification improving by at least one level,” Penn said. “They still had evidence of damage, but they functioned better and were feeling better.”
Penn’s study suggests that our stem cells have the potential to induce healing without having to be taken out of the body. Penn said, “Our study also shows gene therapy has the potential to help people heal their own hearts.”
At the start of the study, participants didn’t have significant reversible heart damage, but lacked blood flow in the areas bordering their damaged heart tissue. The study’s results — consistent with other animal and laboratory studies of SDF-1 — suggest that SDF-1 gene injections can increase blood flow around an area of damaged tissue, which has been deemed irreversible by other testing.
In further research, Penn and his team are comparing results from heart failure patients receiving SDF-1 with patients who are not receiving SDF-1. If the trial goes well, the therapy could be widely available to heart failure patients within four to five years, Penn said.
Human amniotic fluid stem cells (hAFSCs) have been isolated from the “water” that surrounds the baby when it is born. Amniotic fluid is the material is lost when a pregnant woman’s “water breaks.” If the amniotic fluid is retrieved before it ruptures, a specific stem cell population can be isolated from it, and these stem cells grow very well in culture, and can differentiate into a multiple of adult cell types.
When it comes to the heart, hAFSCs have a bit of a mixed record. One publication from Anthony Atala’s laboratory showed that implantation of hAFSCs into the heart of a laboratory animal after a heart attacked was followed by the formation of bony nodules in the heart tissue (see Chiavegato et al., J Mol Cell Cardiol. 42 (2007) 746-759). However, a follow-up publication, showed that the conditions used in the previous experiments caused the formation of bony nodules in the heart regardless of whether or not hAFSCs were implanted into the heart (Delo DM et al., Cardiovasc Pathol 2011 20(2):e69-78). Other papers showed that implanted cAFSCs could protect the heart from further deterioration (Bollini S et al., Stem Cells Dev. 2011 20(11):1985-94). However, a perennial problem is the poor retention of the cells in the heart after injection. Therefore, one group tried implanting hAFSCs into cellular goo (extracellular matrix). This caused the hAFSCs to stay put in the heart and differentiate into heart muscle cells and blood vessels (Lee WY et al., Biomaterials. 2011 32(24):5558-6).
On the heals of this success comes a paper from Taiwanese researchers who have embedded hAFSCs into polylactic-co-glycolic acid (PLGA) beads and implanted these into the heart of a laboratory animal after a heart attack. These beads are made of material that is completely biogradable, but the hAFSCs survive and grow well in them. Also, once they are implanted into the heart, the beads are large enough to prevent them from being displaced. Once the beads disintegrate inside the heart tissue, the cells are already so deeply implanted into the heart tissue, that they do not become washed out by circulating blood and other fluids.
The implanted hAFSCs differentiated into heart muscle cells and blood vessels. The blood vessels density in these hearts of the hAFSC implanted animals twice that of the control animals in the area of the infarct and almost three times that of the control outside the area of the infarct. The scar shrunk in the hAFSC-implanted hearts by ~30%, and the structure of the hAFSC-implanted hearts was much more robust and thick relative to the controls. Finally, the contraction of the heart muscle was (4 weeks after treatment) twice as strong in the hAFSC-treated hearts compared to the control. Ejection faction was not measured, and that is a deficiency in this paper, but all the cardiac parameters that were measured were vastly improved in the hAFSC-treated hearts relative to the untreated controls.
This paper shows that the porous PLGA beads are not toxic, deliver cells to the chosen target, and quickly disintegrate without affecting the target tissue, in this case the heart. Clearly hAFSCs have a part to play in the future of regenerative medicine.
Stem cell scientists from the University of Maryland, Baltimore have used bone marrow mesenchymal stem cells (MSCs) to treat sheep that had suffered a heart attack. They found that the injected stem cells prevented the heart from deteriorating.
This work was a collaboration between the laboratories of Mark Pittenger, ZhonGjun Wu and Bartley Griffith from the Department of Surgery and the Artificial Organ Laboratory.
After a heart attack, the region of the heart that was deprived of oxygen undergoes cell death and is replaced by a heart scar. However, the region next to the dead cells also undergo problematic changes. The cells in these regions adjacent to dead region must contract more forcibly in order to compensate for the noncontracting dead region. These cells enlarge, but some undergo cell death due to inadequate blood supply. There are other changes that can occur, such as abnormalities in Calcium ion handling and poor contractability.
Thus, the problems that result from a heart attack can spread throughout the heart and cause heart failure. In this experiment, the U of Maryland scientists injected MSCs into the sheep hearts four hours after a heart attack to determine if the stem cells could prevent the region adjacent to the dead heart cells from deteriorating.
In this experiment, bone marrow MSCs were isolated from sheep bone marrow and put through a battery of tests to ensure that they could differentiate into bone, cartilage, and fat. Once the researchers were satisfied that the MSCs were proper MSCs, they induced heart attacks in the sheep, and then injected ~200 million MSCs into the area right next to the region of the heart that died.
After 12 weeks, tissue biopsies from these sheep hearts were taken and examined. Also, the sheep hearts were measured for their heart function and structure.
The sheep that did not receive any MSC injections continued to deteriorate and showed signs of stress. The cells adjacent to the dead region expressed a cadre of genes associated with increased cell stress. Furthermore, there was increased cell death and evidence of scarring in the region adjacent to the death region. There was also evidence of Calcium ion-handling problems in the adjacent tissue and increased cell death.
On the other hand, the hearts of the sheep that had received injections of MSCs into the area adjacent to the dead region showed a reduced expression of those genes associated with increased cell stress. Also, these hearts contracted better than those that had not received stem cell injections. There was also less cell death, less scarring, and no evidence of Calcium ion-handling problems.
Changes that occur in the heart after a heart attack are collectively referred to as “remodeling.” Remodeling begins regionally, in those areas near the dead heart cells, but these deleterious changes spread to the rest of the heart, resulting in heart failure. The injections of MSCs into the area next to the dead region clearly prevented remodeling from occurring.
This pre-clinical study is a remarkable study for another reason: the MSCs used in this study were allogeneic. Allogeneic is a fancy way of saying that they did not come from the same animal that suffered the heart attack, but from some other healthy animal. Therefore, the delivery of a donor’s MSCs into the heart of a heart attack patient could potentially prevent heart remodeling.
The main problem with this experiment is that the MSCs were injected directly into the heart muscle. In humans, such a procedure requires special equipment and carries potential risks that include perforation of the heart wall, rupture of the heart wall, or further damaging the heart muscle. Therefore, if such a technology could be adapted to a more practical delivery system in humans, then certainly human clinical trials should be forthcoming.
See Yunshan Zhao, et al., “Mesenchymal stem cell transplantation improves regional cardiac remodeling following ovine infarction.” Stem Cells Translational Medicine 2012;1:685-95.
A study from the laboratory of Armand Keating at the University of Toronto and Princess Margaret Hospital has compared the ability of umbilical cord stem cells and bone marrow stem cells to repair the hearts of laboratory animals after a heart attack. The umbilical cord stem cells showed a clear superiority to bone marrow stem cells when it came to repairing heart muscle.
Keating used human umbilical cord perivascular cells (HUCPVCs) for his experiment, and these cells are widely regarded as a form of umbilical cord mesenchymal stem cell that surround the umbilical cord blood vessels.
Transplantation of cells from either bone marrow or umbilical cord into the heart soon after a heart attack improved the function and structure of the heart. However, functional measurements showed that the HUCPVCs were twice as effective as bone marrow stem cells at repairing the heart muscle.
Keating added: “We are hoping that this translates into fewer people developing complications of heart failure because their muscle function after a heart attack is better.”
In addition to further pre-clinical tests, Keating and his research team hope to initiate clinical trials with human patients within 12-18 months. Keating is also interested in testing the ability of umbilical cord stem cells to heal the hearts of those cancer patients who have experienced heart damage as a result of chemotherapy. In such patients, chemotherapy rids their bodies of cancer, but the cure is worse than the cancer, since the drugs also leave the patients with a severely damaged heart. Such stem cell transplantations could potentially strengthen the hearts of these patients, and give them a new lease on life. My own mother died from congestive heart failure as a result of an experimental arsenic treatment that killed her heart muscle. My mother suffered from chronic myelogenous disease and the arsenic was meant to kill off all the rogue cells in her bone marrow, but instead it killed her heart. If such a stem treatment were available then, my mother might still be with me.
A large and very well designed and carefully controlled clinical trial known as TIME has failed to demonstrate any benefit for infusions of bone marrow stem cells into the heart 3-7 days after a heart attack. This study comes on the heals of a similar clinical study known as LateTIME, which stands for Late Timing In Myocardial infarction Evaluation, and tested the effects of bone marrow stem cells infusions into the heart of heat attack patients 2-3 weeks after a heart attack.
LateTIME enrolled 87 heart attack patients, and harvested their bone marrow stem cells. The stem cells were delivered into the hearts through the coronary arteries, but some received a placebo. All patients had their ejection fractions measured, their heart wall motions in the damaged areas of the heart and outside the damaged areas and the size of their infarcts. There were no significant changes in any of these characteristics after six months. Because another large clinical study known as the REPAIR-AMI study showed significant differences between heart attack patients that had received the placebo and those that had received bone marrow stem cells 3-7 days after a heart attack, this research group, known as the Cardiovascular Cell Therapy Research Network (CCTRN), sponsored by the National Institutes of Health, decided the test their bone marrow infusions at this same time frame.
TIME was similar in design to LateTIME. This study enrolled 120 patients that had suffered a heart attack and all patients received either an infusion of 150 million bone marrow stem cells or a placebo within 12 hours of bone marrow aspiration and cell processing either 3 days after the heart attack to 7 days. The researchers examined the changes in ejection fraction, movement of the heart wall, and the number of major adverse cardiovascular events plus the changes in the infarct size.
The results were resoundingly negative. At 6 months after stem cell infusion, there was no significant increase in ejection fractions versus the placebo and no significant treatment effect on the function of the left ventricle in either the infarct or the border zones. These findings were the same for those patients that received bone marrow stem cell infusions 3 days after their heart attack or 7 days after their heart attacks. Fortunately, the incidence of major adverse events were rare among all treatment groups.
Despite the negative results for these clinical trials, there are a few silver linings. First of all, the highly controlled nature of this trial sets a standard for all clinical trials to come. A constant number of stem cells were delivered in every patient, and because the stem cells were delivered soon after they were harvested, there were no potential issues about bone marrow storage.
Jay Traverse, the lead author of this study, made this point about this trial: “With this baseline now set, we can start to adjust some of the components of the protocol to grow and administer stem cell [sic] to find cases where the procedure may improve function. For example, this therapy may work better in different population groups, or we might need to use new cell types or new methods of delivery.”
When one examines the data for this study, it is clear that some patients definitely improved dramatically, whereas others did not. Below is a figure from the Traverse et al paper that shows individual patient’s heart function data 6 months after the stem cell infusions.
From examining these data even cursorily, it is clear that some patients improved dramatically while others tanked. Traverse is convinced that bone marrow stem cell infusions help some people, but not others (just like any other treatment). He is convinced that by mining these data, he can begin to understand who these patients are who are helped by bone marrow stem cell transplants and who are not. Also, the stem cells of these patients have been stored. Hopefully, further work with them will help Traverse and his colleagues clarify what, if anything, about the bone marrow of these patients makes them more likely to help their patients and so on.
There are some possible explanations for these negative results. Whereas the positive REPAIR-AMI used the rather labor-intensive Ficoll gradient protocols for isolating mononculear cells from bone marrow aspirates, the TIME trials used and automated system for collecting the bone marrow mononuclear cells. Cells isolated by the automated system have neither been tested in an animal model of heart attacks, nor established as efficacious in a human study of heart disease. Therefore, it is possible that the bone marrow used in this study was largely dead. Secondly, the cell products were kept in a solution that had a heparin concentration that is known to inhibit the migratory properties of mononuclear cells (See Seeger et al., Circ Res 2012 111(7): 1385-94). Therefore, there is a possibility that the bone marrow used in this study was no good. Until the bone marrow stem cells collected by this method are confirmed to be efficacious, judgment must be suspended.