The DanCell clinical trial was conducted about seven years ago at the Odense University Hospital, Odense, Denmark by a clinical research team led by Axel Diederichsen. The DanCell study examined 32 patients with severe ischemic heart failure who had received two rounds of bone marrow stem cell treatments.
The DanCell study was small and uncontrolled. However, because the vast majority of stem cell-based clinical trials have examined the efficacy of stem cell treatments in patients who have recently experienced a heart attack, this study was one of the few that examined patients with chronic heart failure.
In this study, patients had an average ejection fraction of 33 ± 9%, which is in the cellar – normal ejection fractions in healthy patients are in the 50s-60s. Therefore, these are patients with distinctly “bad tickers.” All 32 patients received two repeated infusions (4 months apart) of their own bone marrow stem cells, but these stem cell infusions were quantitated to determine the number of “CD34+” cells and the number of “CD133+” cells. CD34 is a cell surface protein found on bone marrow hematopoietic stem cells, but it by no means exclusive to HSCs. CD133 is also a cell surface protein found, although not exclusively, on the surfaces of cells that form blood vessels and blood vessels cells as well.
Initially, patients showed no improvements in heart function after 12 months. However, when patients were classified according to those who received the most or the least number of CD34+ cells, a curious thing emerged: those who received more CD34+ cells had a better chance of surviving than those who received fewer CD34+ cells.
Is this a fluke? To determine if it was, Diederichsen and his colleagues followed these patients for 7 years after the bone marrow infusion. When Diederichsen and his colleague recorded the number of deaths and compared them with the number of CD34+ cells infused, the pattern once again held true. The CD34+ cell count and CD133+ cell count did not significantly correlate with survival, but the CD34+ cell count alone was significantly associated with survival. In the authors own words: “decreasing the injected CD34 cell count by 10 increases the mortality risk by 10%.”
The conclusions of this small and admittedly uncontrolled study: “patients might benefit from intracoronary stem cell injections in terms of long-term clinical outcome.”
Three things to consider: Patients with heart conditions have poorer quality bone marrow stem cell numbers. Therefore, allogeneic stem cells might be a better way to go with this patient group. Secondly, the Danish group used Lymphoprep to prepare their bone marrow stem cells, which has been used in other failed studies, and the stem cell quality was almost certainly an issue in these cases (see the heart chapter in my book The Stem Cell Epistles for more information). Therefore, independent tests of the bone marrow quality are probably necessary as well or a different isolation technique in general. Also, a controlled trial must be run in order to confirm the efficacy of bone marrow stem cell infusions for patients with chronic ischemic heart disease. Until them, all we can conclude is that intracoronary injections of a high number of CD34+ cells may have a beneficial effect on chronic ischemic heart failure in terms of long-term survival.
Researchers from the famed Mayo Clinic, in collaboration with scientists at a biopharmaceutical biotechnology company in Belgium have invented a specialized catheter for transplanting stem cells into a beating heart.
This new device contains a curved needle with graded openings along the shaft of the needle. The cells are released into the needle and out through the openings in the side of the needle shaft. This results in maximum retention of implanted stem cells to repair the heart.
“Although biotherapies are increasingly more sophisticated, the tools for delivering regenerative therapies demonstrate a limited capacity in achieving high cell retention in the heart,” said Atta Behfar, the lead author of this study and a cardiologist. “Retention of cells is, of course, crucial to an effective, practical therapy.”
Researchers from the Mayo Clinic Center for Regenerative Medicine in Rochester, MN and Cardio3 Biosciences in Mont-Saint-Guibert, Belgium, collaborated to develop the device. Development of this technology began by modeling the dynamic motions of the heart in a computer model. Once the Belgium group had refined this computer model, the model was tested in North America for safety and retention efficiency.
These experiments showed that the new, curved design of the catheter eliminates backflow and minimizes cell loss. The graded holes that go from small to large diameters decrease the pressures in the heart and this helps properly target the cells. This new design works well in healthy and damaged hearts.
Clinical trials are already testing this new catheter. In Europe, the CHART-1 clinical trial is presently underway, and this is the first phase 3 trial to examine the regeneration of heart muscle in heart attack patients.
These particular studies are the culmination of years of basic science research at Mayo Clinic and earlier clinical studies with Cardio3 BioSciences and Cardiovascular Centre in Aalst, Belgium, which were conducted between 2009 and 2010. This study, the C-CURE or Cardiopoietic stem Cell therapy in heart failURE study examined 47 patients, (15 control and 32 experimental) who received injections of bone marrow-derived mesenchymal stem cells from their own bone marrow into their heart muscle. Control patients only received standard care. After six months, those patients who received the stem cell treatment showed an increase in heart function and the distance they could walk in six minutes. No adverse effects were observed in the stem cell recipients.
This study established the efficacy of mesenchymal stem cell treatments in heart attack patients. However, other animal and computer studies established the efficacy of this new catheter for injecting heart muscle with stem cells. Hopefully, the results of the CHART-1 study will be available soon.
Postscript: The CHART-2 clinical trial is also starting. See this video about it.
Using induced pluripotent stem cells to have heart muscle cells is one of the goals of regenerative medicine. Successful cultivation of heart muscle cells from a patient’s own cells would provide material to replace dead heart muscle, and could potentially extend the life of a heart-sick patient.
Unfortunately, induced pluripotent stem cells, which are made by applying genetic engineering techniques to a patient’s own adult cells, like embryonic stem cells, will cause tumors when implanted into a living organism. To beat the problem of tumor formation, scientists must be able to efficiently isolate the cells that have properly differentiated from those cells that have not differentiated.
A new paper from a laboratory the Emory University School of Medicine in Atlanta, Georgia, have used “molecular beacons” to purify heart muscle cells from induced pluripotent stem cells, thus bringing us one step closer to a protocol that isolates pure heart muscle cells from induced pluripotent stem cells made from a patient’s own cells.
Molecular beacons are nanoscale probes that fluoresce when they bind to a cell-specific messenger RNA molecule. Because heart muscle cells express several genes that are only found in heart muscle cells, Kiwon Ban in the laboratory of Young-Sup Yoon designed heart muscle-specific molecular beacons and used them to purify heart muscle cells from cultured induced pluripotent stem cells from both mice and humans.
The molecular beacons made by this team successfully isolated heart muscle cells from an established heart muscle cell line called HL-1. Then Ban and co-workers applied these heart-specific molecular beacons to successfully isolate heart muscle cells that were made from human embryonic stem cells and human induced pluripotent stem cells. The purity of their isolated heart muscle cells topped 99% purity.
Finally, Ban and others implanted these heart muscle cells into the hearts of laboratory mice that had suffered heart attacks. When heart muscle cells that had not been purified were used, tumors resulted. However, when heart muscle cells that had been purified with their molecular beacons were transplanted, no tumors were observed and the heart function of the mice that received them steadily increased.
Because the molecular beacons are not toxic to the cells, they are an ideal way to isolate cells that have fully differentiated to the desired cell fate away from potentially tumor-causing undifferentiated cells. in the words of Ban and his colleagues, “This purification technique in combination with cardiomyocytes (heart muscle cells) generated from patient-specific hiPSCs will be of great value for drug screening and disease modeling, as well as cell therapy.”
Mesenchymal stem cells have the ability to improve the heart after a heart attack. However can repeated administrations of mesenchymal stem cells cause an increased benefit to the heart after a heart attack?
A collaborative research project between the Royal Adelaide Hospital, the University of Adelaide in South Australia, and the Mayo Clinic in Rochester, Minnesota has administered mesenchymal stem cells multiple times to rodents after a heart attack to determine if administering these stem cells multiple times after a heart attack increases the performance of the heart.
The experimental procedure was relatively straight-forward. Three groups of mice were evaluated by means of cardiac magnetic resonance imaging (MRI). Then all three were given heart attacks by tying off the left anterior descending artery. Immediately after the heart attack, two groups were injected with one million mesenchymal stem cells into the heart. The third group was injected with ProFreeze (a cryopreservation solution). One week later, a second set of heart MRIs were taken, and the first and third group of mice received injections of ProFreeze and the third group received another one million mesenchymal stem cells. All animals were given two more heart MRIs one week later and two weeks after that. One month after the initial heart attacks, the mice were euthanized and their hearts were sectioned and examined.
Those mice that did not receive injections of mesenchymal stem cells showed a precipitous drop in their heart performance. The ejection fraction (average percent of blood pumped from the heart) dropped from around 60% to about 20% and then stayed there. Those mice treated with one round of mesenchymal stem cells (MSCs) after their ejection fractions drop from 60% to about 35% after one week, and then stayed there. Those animals that received two shots of MSCs have their ejection fractions drop from around 60% to about 41%. Thus the administration of a second round of MSCs did significantly increase the performance of the heart.
The heart also shows tremendous structural improvements as a result of MSC transplantation. These improvements are even more dramatic in those mice that received two doses of MSCs. The mass of the heart and the thickness of the walls of the heart are greater in those animals that received two MSC doses, than those that received only one dose. Secondly, the size of the heart scar is smallest in those animals that received two doses of MSCs. Third, the density of blood vessels was MUCH higher in the animals that received two MSC doses. Also, the tissue far from the infarction in those animals that had received two doses of MSCs showed twice the density of blood vessels per cubic millimeter of heart tissue than those animals that had only received one injection of MSCs. Therefore, additional transplantations of MSCs increase blood vessel density, decrease the size of the heart scar and increase the thickness of the walls of the heart.
MSCs have the capacity to heal the heart after a heart attack. The degree to which they heal the heart differs from patient to patient, but additional treatments have the capacity to augment the healing capacities of these cells. Also, in this experiment, the mice received someone else’s MSCs. This is known as “allogeneic” transplantation, and it is an important concept, since older patients, diabetic patients, or those who have had a heart attack typically have MSCs that do not perform well. Therefore to receive MSCs from a donor is a way around this problem.
The problem with this experiment is that it was done in mice, and they were injected directly into the heart tissue. Such a procedure is almost certainly impractical for human patients. Instead, intracoronary delivery is probably more practical, but here again, repeated releasing cells into the coronary arteries increases the risk of clogging them. Therefore, it is probably necessary to administer the second dose of MSCs some time after the first dose. To calibrate when to administer the second dose, large animal experiments will be required.
Thus, while this experiment looks interesting and hopeful, more work is required to make this usable in humans. It does, however, establish the efficacy of repeated allogeneic MSC transplantations, which is an important feature of these experiments.
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.
Embryonic stem cells have the capacity to differentiate into every cell in the adult body. One cell type into which embryonic stem cells (ESCs) can be differentiated rather efficiently is cardiomyocytes, which is a fancy term for heart muscle cells. The protocol for making heart muscle cells from ESCs is well worked out, and the conversion is rather efficient and the purification schemes that have been developed are also rather effective (for example, see Cao N, et al., Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions. Cell Res. 2013 Sep;23(9):1119-32. doi: 10.1038/cr.2013.102 and Mummery CL et al., Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res. 2012 Jul 20;111(3):344-58).
Using these cells in a clinical setting has two large challenges. The first is that embryonic stem cell derivatives are rejected by the immune system of the recipient, thus setting up the patient for a graft versus host response to the implanted tissue, thus making the patient even sicker than when they started. The second problem is that heart muscle cells made from ESCs are immature and cause the heart to beat abnormally fast thus causing “tachyarrythmias” and died within the first two weeks after the transplant (see Liao SY, et al., Heart Rhythm 2010 7:1852-1859).
Both of these problems are large problems, but the laboratory of Ronald Li at the University of Hong Kong at used a genetic engineering trick to make heart muscle cells from mouse embryonic stem cells to seemingly fix this problem.
Li and his colleagues engineered mouse ESCs with a gene for a potassium rectifier channel that could be induced with drugs. Then they differentiated these genetically ESCs into heart muscle cells. This potassium rectifier channel (Kir2.1) is not present in immature heart muscle cells and putting it into these cells might cause them to beat at a slower rate.
These engineered ESC-derived heart muscle cells were tested for their electrophysiological properties first. Without the drug that induces KIR2.1, the heart muscle cells showed very abnormal electrical properties. However, once the drug was added, their electrical properties looked much more normal.
Then they induced heart attacks in laboratory animals and implanted their engineered ESC-derived heart muscle cells 1 hour after the heart attacks were induced. Animals not given the drug to induce the expression of Kir2.1 faired very poorly and had episodes of tachyarrythmia (really fast heart beat) and over half of them died by 5 weeks after the implantation. Essentially the implanted animals did worse than those animals that had had a heart attack that were not treated. However, those animals that were given the drug that induces the expression of Kir2.1 in heart muscle cells did much better. The survival rate of these animals was higher than the untreated animals after about 7 weeks after the procedure. Survival rates increased by only a little, but the increase was significant. Also, the animals that died did not die of tachyarrythmias. In fact the rate of tachyarrythmias in the animals given the inducing drug (which was doxycycline by the way) had significantly lower levels of tachyarrythmia than the other two groups.
Other heart functions were also significantly affected. The ejection fraction in the animals that ha received the Kir2.1-expression heart muscle cells was 10-20% higher than the control animals. Also the density of blood vessels was substantially higher in both sets of animals treated with ESC-derived heart muscle cells. The echocardiogram of the hearts implanted with the Kir2.1-expressing heart muscle cells was altogether more normal than that of the others.
This paper is a significant contribution to the use of ESC-derived cells to treat heart patients. The induction of heart arrhythmias by ESC-derived heart muscle cells is a documented risk of their use. Li and his colleagues have effectively eliminated that risk in this paper by forcing the expression of a potassium rectifier channel in the ESC-derived heart muscle cells. Also, because these cells were completely differentiated and did not have any interloping pluripotent cells in their culture, tumor formation was not observed.
There are a few caveats I would like to point out. First of all, the increase in survival rate above the control is not that impressive. The improvement in heart function parameters is certainly encouraging, but because the survival rates are not that higher than the control mice that received no treatment, it appears that these benefits were only conferred to those mice who survived in the first place.
Secondly, even though the heart attacks were induced in the ventricles of the heart, Li and his colleagues injected a mixture of heart muscle cells that included atrial, ventricular, nodal and heart fibroblasts. This provides an opportunity for beat mismatches and a “substrate for ventricular tachycardia” as Li puts it. In the future, the transplantation of just ventricular heart muscle cells would be cleaner experiment. Since these mice were not observed long enough to observe potential arrythmias that might have arisen from the presence of a mixed population in the ventricle.
Finally, in adapting this to humans might be difficult, since the hearts of mice beat so much faster than those of humans. It is possible that even if human cardiomyocytes were engineered with Kir2.1-type channels, that arrythmias might still be a potential problem.
Despite all that, Li’s publication is a large step forward.
Stem cell treatments for heart attacks can improve heart function after a heart attack. This has been repeated shown in laboratory animals and human trials have also established the efficacy of bone marrow-based heart treatments. Despite these successes, there are some indications that the improvements wrought by bone marrow stem cell transplants into the heart are not stable, and the functional increases caused by it are transient.
To determine is functional improvements in the heart are transient or stable, Jaroslaw Kaspzak and his colleagues at the Medical University of Lodz, Poland have published a study in which they treated 60 heart attack patients and then tracked them for two years. Their study was published in the journal Kardiologia Polska, which, fortunately, is in English, since I do not read Polish.
In this study, 60 heart attack patients were treated with primary angioplasty and randomly assigned to two groups. The first group consisted of 40 patients who were treated with standard care, and bone marrow stem cell transplants. The bone marrow cells were harvested 3-11 days after the heart attack. The bone marrow cells were administered to the heart by means of intracoronary catheters (over the wire balloon catheter) very near to the area of the infarct (the area in the heart damaged by the heart attack). The second group of 20 patients were treated with standard care. All patients were subjected to echocardiography before treatment and 1, 3, 6, 12, and 24 months after treatment. Additionally, the percentages of patients who experienced subsequent heart attacks, admission to the hospital for heart failure, or revascularization, was tabulated two years after the treatment.
Just after the heart attack, the heart function of both groups was essentially the same. The fraction of blood pumped from the heart (ejection fraction) in the treated group was 35% ± 6% and 33% ± 7% in the control group (normal is 55% – 70%). The volume of blood left in the heart after it contracts (end systolic volume) was 95 milliliters ± 39 milliliters in the treated group and 99 milliliters ± 49 milliliters in the control group (normal is 50-60 milliliters). The amount of blood in the heart after it fills (end diastolic volume) was 149 ± 48 milliliters in the treated group and 151 ± 65 milliliters in the control group (normal is 120-130 milliliters). The end diastolic volume (EDV) is a measure of the firmness of the heart walls. A damaged heart is not as firm and its flaccid walls expand greatly and take up more volume, which puts further strain upon the heart. Thus a DECREASE in the EDV is an indication of improvement in the heart. Nevertheless, it is clear that before the treatment regimes were instigated, the average medical conditions of the two groups was essentially the same, at least when it comes to the heart.
The results of each treatment strategy are rather telling. The ejection fraction in the control group increased 3.7% one moth after the heart attack, 4.7% by 6 months, 4.8% at 12 months, and 4.7% at 24 months. This this group saw its greatest increase six months after the heart attack and although this increase was stable, it was modest at best. The bone marrow-treated group, however, saw an average ejection fraction increase of 7.1% after one month, 9.3% at 6 months, 11.0% after one year, and 10% after two years. Thus the bone marrow-treated group not only showed a much faster and more robust increase in injection fraction, but an increase that was sustained two years after the procedure. Also, the treated group saw half the percentage of deaths due to cardiac events (5%) than that observed in the control group (10%). The percentage of hospitalizations for heart failure in the treated group (3%) was 20% of that seen in the control group (15%). The rates of revasculaizations and new heart attacks was essentially the same in both groups.
This study joins other long-term studies that have demonstrated long-term improvements in heart attack patients treated with heart infusions with bone marrow-derived stem cells. The REPAIR-AMI clinical trial, which examined 204 heart attack patients, showed stable, long-term benefits that lasted for at least two years for those patients who had been treated with infusions of bone marrow stem cells. Other studies have not found no significant differences between heart attack patients treated with standard care and those who also received bone marrow infusions. However, there are probable explanations for many of these failures. The ASTAMI study that failed to show significant differences between the two groups not only transplanted a lower number of cells than this present study and the successful REPAIR-AMI study. Secondly, the negative FINCELL study used patients whose average ejection fractions were 59% ± 11%. Clinical studies that have tested bone marrow heart infusions have established that those patients with lower ejection fractions are helped the most by them. This is the case of the negative HEBE study; the patients with the lowest ejection fraction showed the greatest improvements relative to the control group, but these improvements were swamped out by those with higher ejection fractions that were not helped nearly as much. Third, the meta-analysis of Martin-Rendon showed that the best time period to treat heart attack patients was 4-7 days after the heart attack. In the HEBE study, patients received bone marrow infusions 7 days after angioplasty. How soon after the heart attack was the angioplasty performed? This is not reported, probably because it varied from patient to patient. Nevertheless, this places the treatment outside the optimum established by other experiments.
Thus, once again, we see that bone marrow treatments for hearts are safe, and effective, and they convey long-term benefits to patients who receive them. Much work remains, since only some people consistently benefit from these treatments. Why is this the case? Only more work will tell.