Stem Cell Therapy Replaces Dead Heart Muscle in Primates


The laboratory of Charles Murry at the University of Washington has used embryonic stem cells to make heart muscle cells that were then used to regenerate damaged hearts in non-human primates. This experiment demonstrates the possibility of using heart muscle cells derived from pluripotent stem cells, but it also underscores the many challenges that still must be overcome.

When the heart undergoes a heart attack or other types of damage, heart muscle cells begin to die off and these cells are not easy to replace. Heart muscle cells, also known as cardiomyocytes, do not readily replace themselves. Even though the heart has a resident stem cell population, (cardiac progenitor cells or CPCs) these heart-specific stem cells have a limited capacity to regenerate the heart. After a heart attack, as many as one billion cardiomyocytes or more die. The loss of so many beating heart muscle cells compromises heart function and can also lead to chronic heart failure and even death.

Physicians, cardiologists, and researchers have been on the lookout for new and improved procedures and technologies to replenish damaged heart tissue. Several different types of stem cells have shown promise in animal models and in human clinical trials. Stem cells from bone marrow have the ability to secrete a cocktail of molecules that stimulate heart regeneration. Whole bone marrow or isolated stem cell populations have shown variable, but statistically significant in patients who have had a recent heart attack. Unfortunately, stem cells from bone marrow do not have the ability to differentiate into heart muscle cells, and to maximize regeneration of the heart, damaged heart muscle must be replaced.

Human embryonic stem cells have proven promising in small animal models, but the long-term effects of embryonic stem cell-mediated improvements in some cases have proven to be transient. An additional problem with embryonic stem cell-derived heart muscle cells is their tendency to cause abnormal heart rates, otherwise known as arrhythmias.

Scientists in Murry’s laboratory tried to scale-up the production of cardiomyocytes from human embryonic stem cells in order to test the regenerative ability of these cells in a large animal model – non-human primates. These experiments were published online on April 30, 2014, in the journal Nature.

Murry’s team derived cardiomyocytes from genetically-engineered human embryonic stem cells that made a fluorescent calcium indicator that glowed in the presence of high calcium ion concentrations. With this fluorescent calcium indicator, Murray and his coworkers could track the calcium waves that mark the electrical activity of a beating heart. The animal subjects for this experiment were pigtail macaques (Macaca nemestrina) that had suffered heart damage and had been treated with drugs to suppress their immune systems. Five days later, the embryonic stem cell-derived cardiomyocytes were delivered in a surgical procedure to the damaged regions and surrounding border zones of the heart.

Over a 3-month period, the implanted cells infiltrated damaged heart muscle, matured, and organized themselves into muscle fibers in all the monkeys who received the treatment. An average of 40% of the damaged tissue was replaced by these grafts. Three-dimensional imaging showed that arteries and veins integrated into the grafts. Because sick hearts often contain clogged blood vessels, oxygen delivery to the damaged heart tissue was minimal. However, because these grafts contained integrated blood vessels, they would potentially be long-lasting.

Calcium activity studies showed that the heart muscle tissue within the grafts were electrically active and coupled to activity of the host heart. The grafts beat along with host muscle at rates of up to 240 beats per minute, the highest rate tested.

Cardiac cells derived from human stem cells (green) meshed and beat along with primates’ heart cells (red). Credit: Murry Lab/University of Washington.
Cardiac cells derived from human stem cells (green) meshed and beat along with primates’ heart cells (red). Credit: Murry Lab/University of Washington.

All the macaques that received the grafts showed transient arrhythmias or irregular heart rates. However, these subsided by 4 weeks post-transplantation. The animals remained conscious and in no apparent distress during periods of arrhythmia. However, this problem will need to be addressed before this approach can be tested in humans.

“Before this study, it was not known if it is possible to produce sufficient numbers of these cells and successfully use them to remuscularize damaged hearts in a large animal whose heart size and physiology is similar to that of the human heart,” Murry says.

This article shows that despite the obstacles that remain, transplantation of human cardiomyocytes derived from pluripotent stem cells may be feasible for heart patients.

There are a few caveats I would like to mention.  First of all, these animals underwent immunosuppression.  If this procedure were to be used in a human patient, the human patient would need life-long immunosuppression, which has a wide range of side effects and tends to stop working over time.  Therefore, induced pluripotent stem cells are a better choice.  Secondly, the paper admits that the implanted cells underwent “progressive but incomplete maturation over a 3-month period.”  If the implanted cells are not maturing completely, then the risk of arrhythmias still exists, even though they may have subsided in these animals after 4 weeks.  This leads me to my third point.  These animals were watched for 3 months.  How do we know that these results were not transient?  Longer-term experiments are needed to establish that this treatment actually is long-term and not transient.  It is, however, gratifying to see an experiment that was extended to 12 weeks rather than the usual 4 weeks that is usually seen in mice.

Finally, tucked away in the extended data is the statement: “The cell-treated animals showed variable responses, with some having increased function and some having decreased function. Because of small group size, no statistical effects of hESC-CM therapy can be discerned.”  In other words, the treatments worked swimmingly in some animals and not at all in others.  This was a small animal trial and better numbers will be needed if this technology is to come to the clinic.

Making Heart Muscle from Skeletal Muscle Stem Cells


Several experiments in animals and a few clinical trials in human patients have shown that implanting skeletal muscle cells isolated from muscle biopsies into the heart after a heart attack can help the heart to some degree, but the implanted skeletal muscle cells do not integrate into the existing heart muscle mass and the skeletal muscle cells do not differentiate into heart muscle cells.

Experiments like those mentioned above utilized muscle satellite cells. Muscle satellite cells are a resident stem cell population that respond to muscle damage and divide to form skeletal muscle cells form new muscle. Satellite cells are a perfect example of a unipotent stem cell, which is to say a cell that makes one type of terminally differentiated cell type.

Skeletal muscles, however, have another cell population called muscle-derived stem cells or MDSCs. MDSCs express an entirely different set of cell surface proteins than satellite cells, and have the capacity to differentiate into skeletal muscle, smooth muscle, bone, tendon, nerve, endothelial and hematopoietic cells. MDSCs grow well in culture, tolerate low oxygen conditions quite well, and show excellent regenerative potential.

Other laboratories have managed to culture MDSCs in collagen and produce beating heart muscle cells. Others have observed MDSCs forming a proper myocardium under certain conditions. Several studies have established the ability to MDSCs to treat laboratory animals that have suffered a heart attack. The most recent work from Sekiya and others has established that cell sheets made from MDSCs can reduce dilation of the left ventricle, increased capillary density, and promoted recovery without causing erratic heat beat patterns.

Despite their obvious efficacy. MDSCs remain difficult to isolate in high enough numbers to therapeutic purposes. None of the cell surface molecules sported by MDSCs are unique to those cells. Therefore, getting clean cultures of MDSCs remains a challenge. Still, these cells represent some of the best hopes for regenerative medicine in the heart. These cells do form heart muscle cells and heal ailing hearts. They can be grown in bioreactors to high numbers and can also be combined with engineered materials to shore up a damaged heart and mediate its regeneration. While the use of MDSCs is still in its infancy, the promise certainly is there.

Umbilical Cord Stem Cells Preserve Heart Function After a Heart Attack in Mice


A consortium of Portuguese scientists have conducted an extensive examination of the effects of mesenchymal stromal cells from umbilical cord on the heart of mice that have suffered a massive heart attack. Even more remarkable is that these workers used a proprietary technique to harvest, process, and prepare the umbilical cord stem cells in the hopes that this technique would give rise to a commercial product that will be tested in human clinical trials,

Human umbilical cord tissue-derived Mesenchymal Stromal Cells (MSCs) were obtained by means of a proprietary technology that was developed by a biomedical company called ECBio. Their product,, UCX®, consists of clean, high-quality, umbilical cord stem cells that are collected under Good Manufacturing Practices. The use of Good Manufacturing Practice means that UCX is potentially a clinical-grade product. Thus, this paper represents a preclinical evaluation of UCX.

This experiments in this paper used standard methods to give mice heart attacks that were later received injections of UCX into their heart muscle. The same UCX cells were used in experiments with cultured cells to determine their effects under more controlled conditions.

The mice that received the UCX injections into their heart muscles after suffering from a large heart attack showed preservation of heart function. Also, measurements of the numbers of dead cells in the heart muscle of heart-sick mice that did and did not receive injections of umbilical cord cells into their hearts showed that the umbilical cord stem cells preserved heart muscle cells and prevented them from dying. Additionally, the implanted umbilical cord MSCs induced the growth and formation of many small blood vessels in the infarcted area of the heart. This prevented the heart from undergoing remodeling (enlargement), and preserved heart structure and function.

When subjected to a battery of tests on cultured cells, UCX activated cardiac stem cells, which are the resident stem cell population in the heart. Implanted UCX cells activated the proliferation of cardiac stem cells and their differentiation into heart muscle cells. There was no evidence that umbilical cord MSCs differentiated into heart muscle cells and engrafted into the heart. Rather UCX seems to help the heart by means of paracrine mechanisms, which simply means that they secrete healing molecules in the heart and help the heart heal itself.

In conclusion, Diana Santos Nascimento, the lead author of this work, and her colleagues state that, “the method of UCX® extraction and subsequent processing has been recently adapted to advanced therapy medicinal product (ATMP) standards, as defined by the guideline on the minimum quality data for certification of ATMP. Given that our work constitutes a proof-of-principle for the cardioprotective effects UCX® exert in the context of MI, a future clinical usage of this off-the-shelf cellular product can be envisaged.”

Preclinical trials with larger animals should come next, and after that, hopefully, the first human clinical trials will begin.

Pure Heart Muscle Cells from Induced Pluripotent Stem Cells With Molecular Beacons


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.”

Heart Regeneration and the Heart’s Own Stem Cell Population


For years scientists were sure that the heart virtually never regenerated.

Today this view has changed, and researchers at the Max Plank Institute for Heart and Lung Research have identified a stem cell population that is responsible for heart regeneration. Human hearts, as it turns out, do constantly regenerate, but at a very slow rate.

This finding brings the possibility that it might be possible to stimulate and augment this self-healing process, especially in patients with diseases or disorders of the heart, with new treatments.

Some vertebrates have the ability to regenerate large portions of their heart. For example zebrafish and several species of amphibians have the ability to self-heal and constantly maintain the heart at maximum capacity. This situation is quite different for mammals that have a low capacity for heart regeneration. Heart muscle cells in mammals stop dividing soon after birth.

However, mammalian hearts do have a resident stem cell population these cells replace heart muscle cells throughout the life of the organism, In humans, between 1-4% of all heart muscle cells are replaced every year.

Experiments with laboratory mice have identified at heart stem cells called Sca-1 cells that replace adult heart muscle cells and are activated when the heart is damaged. Under such conditions, Sca-1 cells produce significantly more heart muscle.

Unfortunately, the proportion of Sca-1 cells in the heart is very low, and finding them has been likened to searching for a diamond at the bottom of the Pacific Ocean.

Shizuka Uchida, the project leader of this research, said, “We also faced the problem that Sca-1 is no longer available in the cells as a marker protein for stem cells after they have been changed into heart muscle cells. To prove this, we had to be inventive.”

This inventiveness came in the form of a visible protein that was made all the time in the Sca-1 cells that would continue being made even if the cells differentiated into heart muscle.

Uchida put it this way: “In this way, we were able to establish that the proportion of the heart muscle cells originating from Sca-1 stem cells increased continuously in healthy mice. Around five percent of the heart muscle cells regenerated themselves within 18 months.”

When the same measurements were taken in mice with heart disease, the number of heart muscle cells made from Sca-1 stem cells increased three-fold.

“The data show that in principle the mammalian heart is able to trigger regeneration and renewal processes. Under normal circumstances, however, these processes are not enough to ultimately repair cardiac damage,” said Thomas Braun, the principal investigator in whose laboratory this work was done.

The aim is to devise and test strategies to improve the activity and number of these stem cells and, ultimately, to strengthen and augment the heart’s self-healing powers.

A More Efficient Way to Grow Heart Muscle from Stem Cells Could Yield New Regenerative Therapies


An improved method to produce heart muscle from embryonic stem cells or induced pluripotent stem cells could potentially fulfill the demand for heart disease treatments and models of testing new heart drugs. The challenging part of making heart muscle in the laboratory is the production of cells that are all the same. Otherwise their response to drugs or their transplantation into a damaged heart will be unpredictable and unreliable. Fortunately a new study published in the journal STEM CELLS Translational Medicine may provide a way to make large, homogeneous batches of heart muscle cells.

By mixing some small molecules and growth factors together, an international research team led by investigators at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai developed a two-step system that induced embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to efficiently differentiate into ventricular heart muscle cells. This protocol was not only highly efficient but also very reproducible. It also seemed to nicely recapitulate the developmental steps of normal heart development.

“These chemically induced, ventricular-like cardiomyocytes (termed ciVCMs) exhibited the expected cardiac electrophysiological and calcium handling properties as well as the appropriate heart rate responses,” said lead investigator Ioannis Karakikes, Ph.D., of the Stanford University School Of Medicine, Cardiovascular Institute. Other members of this research team consisted of scientists from the Icahn School of Medicine at Mount Sinai, New York, and the Stem Cell & Regenerative Medicine Consortium at the University of Hong Kong.

One of the unusual aspects of this research project was the integrated approach it took. This research group combined computational and experimental systems and by using these techniques, they showed that the use of particular small molecules modulated the Wnt pathway. Signals from the Wnt pathway pass from cell to cell and play a key role in determining whether cells differentiate into an atrial or ventricular muscle cell.

“The further clarification of the molecular mechanism(s) that underlie this kind of subtype specification is essential to improving our understanding of cardiovascular development. We may be able to regulate the commitment, proliferation and differentiation of pluripotent stem cells into heart muscle cells and then harness them for therapeutic purposes,” Dr. Karakikes said.

“Most cases of heart failure are related to a deficiency of heart muscle cells in the lower chambers of the heart,” said Anthony Atala, MD, editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “An efficient, cost-effective and reproducible system for generating ventricular cardiomyocytes would be a valuable resource for cell therapies as well as drug screening.”

Primed Fat-Based Stem Cells Enhance Heart Muscle Proliferation


A Dutch group from the University of Groningen has shown that fat-based stem cells can enhance the proliferation of cultured heart muscle cells. The stem cells used in these experiments were preconditioned and this pretreatment greatly enhanced their ability to activate heart muscle cells.

This paper, by Ewa Przybyt, Guido Krenning, Marja Brinker, and Martin Harmsen was published in the Journal of Translational Medicine. To begin, Przybyt and others extracted human adipose derived stromal cells (ADSC) from fat tissue extracted from human liposuction surgeries. To do this, they digested the fat with enzymes, centrifuged and washed it, and then grew the remaining cells in culture.

Then they used rat neonatal heart muscle cells and infected them with viruses that causes them to glow when certain types of light was shined on them. Then Przybyt and others co-cultured these rat heart cells with human ADSCs.

In the first experiment, the ADSCs were treated with drugs to prevent them from dividing and then they were cultured with rat heart cells in a one-to-one ratio. The heart muscle cells grew faster with the ADSCs than they did without them. To determine if cell-cell contact was required for this stimulation, they used the culture medium from ADSCs and grew the heart cell on this culture medium. Once again, the heart cells grew faster with the ADSC culture medium than without it. These results suggest that the ADSCs stimulate heart cell proliferation by secreting factors that activate heart cell division.

Another experiment subjected the cultured heart cells to the types of conditions they might experience inside the heart after a heart attack. For example, heart cells were subjected to low oxygen tensions (2% oxygen), and inflammation – two conditions found within the heart after a heart attack. These treatments slowed heart cell growth, but this heart cell growth was restored by adding the growth medium of ADSCs. Even more remarkably, when ADSCs were grown in low-oxygen conditions or treated with inflammatory molecules (tumor necrosis factor-alpha or interleukin-1beta), the culture medium increased the fractions of cells that grew. Therefore, ADSCs secrete molecules that increase heart muscle cell proliferation, and increase proliferation even more after the ADSCs are preconditioned by either low oxygen tensions or inflammation.

In the next experiment, Przybyt and others examined the molecules secreted by ADSCs under normal or low-oxygen tensions to ascertain what secreted molecules stimulated heart cell growth. It was clear that the production of a small protein called interleukin-6 was greatly upregulated.

Could interleukin-6 account for the increased proliferation of heart cells? Another experiment showed that the answer was yes. Cultured heart cells treated with interleukin-6 showed increased proliferation, and when antibodies against interleukin-6 were used to prevent interleukin-6 from binding to the heart cells, these antibodies abrogated the effects of interleukin-6.

Przybyt and others then took these results one step further. Since the signaling pathways used by interleukin-6 are well-known, they examined these pathways. Now interleukin-6 signals through pathways, once of which enhances cell survival, and another pathway that stimulated cell proliferation. The cell proliferation pathway uses a protein called “STAT3” and the survival function uses a protein called “Akt.” Both pathways were activated by interleukin-6. Also, the culture medium of ADSCs that were treated with interleukin-6 induced the interleukin-6 receptor proteins (gp80 and gp130) in cultured heart muscle cells. This gives heart muscle cells a greater capacity to respond secreted interleukin-6.

This paper shows that stromal stem cells from fat has the capacity, in culture, to activate the growth of cultured heart muscle cells. Also, if these cells were preconditioned with low oxygen tensions or pro-inflammatory molecules, those fat-based stem cells secreted interleukin-6, which enhanced heart muscle cell survival, and proliferation, even if those heart muscle cells are exposed to low-oxygen tensions or inflammatory molecules.

This suggests that preconditioned stem cells from fat might be able to protect heart muscle cells and augment heart healing after a heart attack. Alternatively, cardiac administration of interleukin-6 after a heart attack might prove even more effective to protect heart muscle cells and stimulate heart muscle cell proliferation. Human trials anyone?

Cardiac Muscle Repair with Molecular Beacons


Pure heart muscle cells that are ready for transplantation. This is one of the Holy Grails of regenerative medicine. Of course when working with pluripotent stem cell lines, isolating nothing but beating heart muscle cells is rather difficult. A new technique makes the isolation of pure cultures of beating heart muscle cells that much easier.

Researchers at Emory and Georgia Tech have developed a method that utilizes molecules called “molecular beacons” to isolate heart muscle cells from pluripotent stem cells. Molecular beacons fluoresce when they come into contact with cells that express certain genes. In this case, the beacons target cells that express heart-specific myosin.

Physicians can use these purified cardiac muscle cells to heal damaged areas of the heart in patient that have suffered a heart attack or are suffering heart failure. This molecular beacon technique might also have applications in other fields of regenerative medicine as well.

“Often, we want to generate a particular cell population from stem cells for introduction into patients,” said Young-sup Yoon, professor of medicine and director for stem cell biology at Emory University School of Medicine. “But the desired cells often lack a readily accessible surface marker, or that marker is not specific enough, as is the case for cardiac muscle cells. This technique could allow us to purify almost any type of cell.”

Gang Bao pioneered he use of molecular beacons and was a co-author of this publication. Yoon and is colleagues and collaborators grew mouse and human embryonic stem cells and induced pluripotent stem cells and differentiated them into heart muscle cells (cardiomyocytes). They then used molecular beacons to label only those cells that expressed messenger RNAs with just the right sequences. These molecular beacons hybridized with the mRNAs and fluoresced. Bao and others then used flow cytometry to sort the fluorescent cells from the non- fluorescent cells. The fluorescent cells have differentiated into heart muscle cells and were isolated from all the other cells.

These purified heart muscle cells could engraft into the heart of a mouse that had suffered a heart attack and they improved heart function and formed no tumors. This proof-of-principle experiment shows that this technique is feasible.

“In previous experiments with injected bare cells, investigators at Emory and elsewhere found that a large proportion of the cells are washed away. We need to engineer the cells into compatible biomaterials to enhance engraftment and retention,” said Yoon,

Do Stem Cells from Bone Outdo Those from the Heart in Regenerating Cardiac Tissue?


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.

Bone cross-section

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.

Transformation of Non-Beating Human Cells into Heart Muscle Cells Lays Foundation for Regenerating Damaged Hearts


After a heart attack, the cells within the damaged part of the heart stop beating and become ensconced in scar tissue. Not only does this region not beat, it does not conduct the signal to beat either and that can not only lead to a slow, sluggish heartbeat, it can also cause irregular heart rates or arrhythmias.

Now, however, scientists have demonstrated that this damage to the heart muscle need not be permanent. Instead there is a way to transform those cells that form the human scar tissue into cells that closely resemble beating heart cells.

Last year, researchers from the laboratory of Deepak Srivastava, MD, the director of Cardiovascular and Stem Cell Research at the Gladstone Institute, transformed scar-forming heart cells (fibroblasts) into beating heart-muscle cells in live mice. Now they report doing the same to human cells in a culture dishes.

“Fibroblasts make up about 50 percent of all cells in the heart and therefore represent a vast pool of cells that could one day be harnessed and reprogrammed to create new muscle,” said Dr. Srivastava, who is also a professor at the University of California, San Francisco. “Our findings here serve as a proof of concept that human fibroblasts can be reprogrammed successfully into beating heart cells.”

In 2012, Srivastava and his team reported that fibroblasts could be reprogrammed into beating heart cells by injecting just three genes (collectively known as GMT, which is short for Gata4, Mef2c, and Tbx5), into the hearts of live mice that had been damaged by a heart attack (Qian L, et al., Nature. 2012 31;485(7400):593-8). From this work, they reasonably concluded that the same three genes could have the same effect on human cells.

“When we injected GMT into each of the three types of human fibroblasts (fetal heart cells, embryonic stem cells and neonatal skin cells) nothing happened—they never transformed—so we went back to the drawing board to look for additional genes that would help initiate the transformation,” said Gladstone staff scientist Ji-dong Fu, Ph.D., the study’s lead author. “We narrowed our search to just 16 potential genes, which we then screened alongside GMT, in the hopes that we could find the right combination.”

The research team began by injecting all candidate genes into the human fibroblasts. They then systematically removed each one to see which were necessary for reprogramming and which were dispensable. In the end, they found that injecting a cocktail of five genes—the 3-gene GMT mix plus the genes ESRRG and MESP1—were sufficient to reprogram the fibroblasts into heart-like cells. They then found that with the addition of two more genes, called MYOCD and ZFPM2, the transformation was even more complete.

To help things along, the team used a growth factor known as Transforming Growth Factor-Beta (TGF-Beta) to induce a signaling pathway during the early stages of reprogramming that further improved reprogramming success rates.

“While almost all the cells in our study exhibited at least a partial transformation, about 20 percent of them were capable of transmitting electrical signals—a key feature of beating heart cells,” said Dr. Fu. “Clearly, there are some yet-to-be-determined barriers preventing a more complete transformation for many of the cells. For example, success rates might be improved by transforming the fibroblasts within living hearts rather than in a dish—something we also observed during our initial experiments in mice.”

The immediate next steps are to test the five-gene cocktail in hearts of larger mammals. Eventually, the team hopes that a combination of small, drug-like molecules could be developed to replace the cocktail, which would offer a safer and easier method of delivery.

This latest study was published online August 22 in Stem Cell Reports.

Overexpression of a Potassium Channel in Heart Muscle Cells Made From Embryonic Stem Cells Decreases Their Arrhythmia Risk


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.

Culture Medium from Endothelial Progenitor Cells Heals Hearts


Endothelial Progenitor Cells or EPCs have the capacity to make new blood vessels but they also produce a cocktail of healing molecules. EPCs typically come from bone marrow, but they can also be isolated from circulating blood, and a few other sources.

The laboratory of Noel Caplice at the Center for Research in Vascular Biology in Dublin, Ireland, has grown EPCs in culture and shown that they make a variety of molecules useful to organ and tissue repair. For example, in 2008 Caplice published a paper in the journal Stem Cells and Development in workers in his lab showed that injection of EPCs into the hearts of pigs after a heart attack increased the mass of the heat muscle and that this increase in heart muscle was due to a molecule secreted by the EPCs called TGF-beta1 (see Doyle B, et al., Stem Cells Dev. 2008 Oct;17(5):941-51).

In other experiments, Caplice and his colleagues showed that the culture medium of EPCs grown in the laboratory contained a growth factor called “insulin-like growth factor-1” or IGF1. IGF1 is known to play an important role in the healing of the heart after a heart attack. Therefore, Caplice and his colleagues tried to determine if IGF1 was one of the main reasons EPCs heal the heart.

To test the efficacy of IGF1 from cultured EPCs, Caplice’s team grew EPCs in the laboratory and took the culture medium and tested the ability of this culture medium to stave off death in oxygen-starved heart muscle cells in culture. Sure enough, the EPC-conditioned culture medium prevented heart muscle cells from dying as a result of a lack of oxygen.

When they checked to see if IGF1 was present in the medium, it certainly was. IGF1 is known to induce the activity of a protein called “Akt” inside cells once they bind IGF1. The heart muscle cells clearly had activated their Akt proteins, thus strongly indicating the presence of IGF1 in the culture medium. Next they used an antibody that specifically binds to IGF1 and prevents it from binding to the surface of the heart muscle cells. When they added this antibody to the conditioned medium, it completely abrogated any effects of IGF1. This definitively demonstrates that IGF1 in the culture medium is responsible for its effects on heart muscle cells.

Will this conditioned medium work in a laboratory animal? The answer is yes. After inducing a heart attack, injection of the conditioned medium into the heart decreased the amount of cell death in the heart and increased the number of heart muscle cells in the infarct zone, and increased heart function when examined eight weeks after the heart attacks were induced. The density of blood vessels in the area of the infarct also increased as a result of injecting IGF1. All of these effects were abrogated by co-injection of the antibody that specifically binds IGF1.

From this study Caplice summarized that very small amounts of IGF1 (picogram quantities in fact) administered into the heart have potent acute and chronic beneficial effects when introduced into the heart after a heart attack.

These data are good enough grounds for proposing clinical studies. Hopefully we will see some in the near future.

Biowire Technology Matures Stem Cell-Derived Heart Cells


Heart research has taken yet another step forward with the invention of a new technique for maturing human heart cells in culture.

Researchers from the University of Toronto have created a fast and reliable method of creating mature human heart muscle patches in a variety of sizes. This technique applies pulsed electric current to the cells that mimics the heart rate of fetal humans.

Milica Radisic, an associate professor at the Institute of Biomaterials and Biomedical Engineering (IBBME), explained the significance of her new discovery: “You cannot obtain human cardiomyocytes (heart cells) from human patients.” However heart cells are vitally important for testing the safety and efficacy of heart drugs, and because human heart muscle cells do not normally divide robustly and form large swaths of heart tissue in culture, finding enough human heart tissue for pharmacological and toxicological test tests has been rather difficult. Tho circumvent this problem, researchers have been using heart muscle cells made from induced pluripotent stem cells (iPSCs). Unfortunately, once these cells are differentiated into heart muscle cells, they form highly immature heart muscle cells that beat too fast to work as a proper model system for adult human heart cells.

As Radisic put it: “The question is, if you want to test drugs or treat adult patients, do you want to use cells that look and function like fetal cardiomyocytes? Can we mature these cells to become more like adult cells?”

Radisic and her co-workers designed the “biowire” culture system for stem cell-derived cardiomyocytes. This system can mature heart muscle cells in culture in a reliable and reproducible manner.

The technique seeds human heart muscle cells along a silk suture, much like the kind used to sew up patients after surgery. The suture directs cells to grow along its length, after which they a treated to cycles of electric pulses. The biowire provides the pulses and acts like a stripped-down pacemaker. The biowire induces the heart muscle cells to increase in size and beat like more mature heart tissue. However the manner in shich the pulses are applied turns out to be very important. Radisic and her team discovered that if the cells were ramped up from zero pulses to 180 pulses per minute to 360 beats per minute, it mimicked the conditions that occur naturally in the developing heart. The fetal heart increases its heart rate prior to birth, and by ramping up the rate at which the pulses were delivered, Radisic and her team exposed the heart cells to the same kind of environment they would have experienced in the fetal heart.

“We found that pushing the cells to their limits over the course of a week derived the best effect,” said Radisic.

Growing the cells on sutures brings an added bonus: They can be sewn directly into a patient, which makes the biowires fully transplantable. Also, the cells can be grown on biodegradable sutures as well, which has practical implications for health care.

“With this discovery we can reduce the costs on the health care system by creating more accurate drug screening.” This discovery brings heart research one step closer to viable heart patches for replacing dead areas of the heart.

The paper’s first author, Sarah Nunes, said this: “One of the greatest challenges of tgransplanting these patches is getting the cells to survive, and for that they need blood vessels. Our next challenge is to put the vascularization together with cardiac cells.” Nunes is a cardiac and a vascular specialist.

Radisic enthusiastically labeled the new technique as a “game changer” in the field of cardiac medicine and it is a sign of how far the field has come in a very short time.

“In 2006 science saw the first derivation of induced pluripotent stem cells from mice. Now we can turn stem cells into cardiac cells and make relatively mature tissue from human samples, without ethical concerns.”

The vascularization part of this should be rather easy, since bone marrow-derived endothelial progenitor cells (EPCs) have been shown to make blood vessels in the heart. Putting these together with the heart patch should provide a winning combination

Inching toward human trials, but definitely making progress!!

Beta Blockers and Cardiac Progenitor Cells


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.

autonomic innervation of the heart

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).

Activation of Beta2 resize bronchial tubes

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.

BMP-2 Treatment Limits Infarct Size in After a Heart Attack in Mice


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.

Coronary arteries

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.

BMP signaling

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.

Recreating an Inherited Heart Condition in the Laboratory


Patients that suffer from an inherited condition known arrhythmogenic right ventricular dysplasia/cardiomyopathy or ARVD/C usually have no idea that they had a heart problem until they are in their 20s. The lack of symptoms at a younger age makes it virtually impossible for researchers to study this condition or to know how it develops. Fortunately, by making induced pluripotent stem cells (iPSCs) from patients with ARVD/C, researchers now have way to solve this very problem.

Small skin biopsies of ARVD/C patients can yield enough cellular material to make iPSCs. These iPSCs can be differentiated into heart muscle cells that are immature. These may not be terribly useful, since the goal is to model a disease that manifest itself during adulthood.

Researchers at Sanford-Burnham Medical Research Institute and Johns Hopkins University have created the first maturation-based “disease in a dish” model for ARVD/C. They created this model system by using iPSCs technology and employing a new method to mimic maturity that makes the metabolism of the hearts muscle cells more like those adult hearts. Thus, this model is likely more relevant to human ARVD/C than other models and therefore better suited for studying the disease and testing new treatments.

Huei-Sheng Vincent Chen, associate professor at Sanford-Burnham and the senior author of this study said, “It’s tough to demonstrate that a disease-in-a-dish model is clinically relevant for an adult-onset disease. But we made a key finding here – we can recapitulate the defects in this disease only when we induce adult-like metabolism. This is an important breakthrough considering that ARVD/C symptoms usually don’t arise until young adulthood. Yet the stem cells we’re working with are embryonic in nature.”

Daniel Judge, associate professor of medicine at Johns Hopkins University School of Medicine, said, “There is currently no treatment to prevent progression of ARVD/C, as rare disorder that preferentially affects athletes. With this new model, we hope we are not on a path to develop better therapies for this life-threatening disease.”

To make this model system, Cheng and his collaborators took skin samples from ARVD/C patients and converted various skin-based cells into iPSCs. After iPSC lines had been established, they differentiated them into heart muscle cells that had the characteristics of embryonic heart muscle cells. Unfortunately, these heart muscle cells showed no signs of ARVD/C, even when grown for over a year in culture.

What was the key? Metabolism. The young heart muscle cells primarily burn sugar for energy, but more mature heart muscle cells burn fat. Therefore, Chen’s group used a cocktail of molecules to get the heart muscle cells to preferentially burn fat.

As it turns out, metabolic malfunction is at the heart of ARVD/C. Chen and his group managed to track down the one piece that would get heart muscle cells made from patient-specific iPSCs to behave like sick ARVD/C heart muscle cells. The answer was the over-stimulation of a protein called PPAR-gamma.

PPARgamma

PPAR-gamma plays an absolutely central role in type II diabetes. It regulates fatty acid storage and sugar metabolism. When PPAR-gamma activates genes, those genes stimulate lipid uptake and the production of fat in fat cells. If mice are made that do not have functional versions of PPAR-gamma, these mice fail to make fat, even when fed a high-fat diet.

PPAR stands for “peroxisome proliferator-activated receptor,” which is a subfamily of nuclear receptors. PPAR proteins bind DNA in combination with retinoid X receptors (RXRs), and these two proteins pair up to regulate transcription of various genes. There are three subtypes of PPARs: PPAR-alpha, PPAR-delta, and PPAR-gamma.

ppar activity

The fact that PPAR-gamma plays such a central role in the pathology of ARVD/C suggests a link between those mechanisms that cause type II diabetes and ARVD/C. According to Chen and Judge, ARVD/C heart muscle cells undergo exaggerated fat production, which leads to cell death. Because PPAR-gamma is a target for a group of drugs known as “glitazones,” perhaps these drugs can play a role in treating ARVD/C.

Glitazones

Induced Pluripotent Stem Cells Used to Define Proper Treatment for Heart Patient


Scientists from Columbia University Medical Center in the laboratory of Robert Kass have used a heart patient’s cells to make induced pluripotent stem cells (iPSCs) that were then differentiated into heart muscle cells. These heart muscle cells were used to test drug strategies to keep the patient’s heart going.

This patient, you see, suffers from Long QT syndrome (LQTS), which is caused by an abnormal ion channel in the heart. LQTS affects the patient’s heart rhythm, which result in fast, chaotic heartbeats. These rapid heartbeats may trigger sudden fainting or a seizure. In some cases, the heart may beat erratically for so long that it can cause sudden death.

Long QT syndrome is treatable by means of decreased physical activity, and certain medicines. Other patents will need surgery or an implantable device.

In this case, the four-year-old patient responded poorly to medicines. Therefore, to find the right combination of drugs, The child had a mutation in the SCN5A gene, which encodes the alpha subunit of the voltage-gated sodium channel. However, this child also had a mutation in the KCNH2 gene, which encodes a potassium channel. This child’s LQTS, therefore, was particularly severe and did not respond to the usual drug regimens.

By using an electrophysiological test called “voltage clamping” on the heart muscle cells made from the patient-specific iPSCs, heart doctors were able to find a drug treatment strategy that eventually stabilized the patient’s heart and saved his small life.

Voltage clamping is a technique used to control the voltage across the membrane of a small or area of a nerve or heart muscle cell by means of an electronic feedback circuit. By sucking a small part of the cell membrane into a micropipette that has a tiny wire in it (yes it sounds hard and yes it is hard), the voltage is increased gradually and the circuit required to hold the voltage at each level is measured. This current is the same as the ionic current that flows across the membrane in response to the applied voltage. This ionic current tells the heart specialist all about what ion channels are present and how well they work in the presence or absence of particular drugs.

Voltage Clamp

These results demonstrate the power of iPSCs in culture as a model system to determine patient-specific therapies.

Making Cardiovascular Progenitor Cells from Induced Pluripotent Stem Cells


In fetal heart, stem cells known as cardiovascular progenitor cell (CPC) differentiates into smooth muscle cells for blood vessels, blood vessel wall cells, and heart muscle cells. Making CPCs from stem cells has proven to be rather difficult because CPCs do not express any known surface molecules that distinguishes them from other cell types. Therefore, if you want to differentiate pluripotent stem cells into CPCs, determining that you have made CPCs is very difficult.

This problem has been addressed by an international research team led by a team from Stuttgart, Germany who have discovered cell surface molecules that allow the identification and isolation of CPCs. With this knowledge, it will be possible to derive CPCs from induced pluripotent stem cells, which can be implanted into damaged hearts, differentiate into heart-specific cell types and integrate into the heart.

Heart attacks are the most frequent cause of death in the developed world. The cause of a heart attack is usually a clogged coronary vessel, which prevents sufficient blood flow through the heart and kills off heart tissue as a result of ischemia. There are some 17 million people who die from cardiovascular disease each.

Heart muscle cells (cardiomyocytes) do not have the ability to regenerate sufficiently after a heart attack. A heart attack causes a huge loss of cells and further impairs blood supply through the heart. This causes the heart to deteriorate further. To fix the heart, new heart muscle cells are required to replace to dead ones.

This now seems to be a distinct possibility. A research team led by Dr. Katja Schenke-Layland from the Frauhofer Institute for Interfacial engineering and Biotechnology IGD in Stuttgart, in collaboration with Dr. Ali Nasar from the University of California and Dr. Robb MacLean from the University of Washington in Seattle have used cultured CPCs to make heart muscle cells.

To identify CPCs, two proteins of the surfaces of CPCs were identified; a receptor called Flt1 and another called Flt4. By exploiting these two surface proteins, scientists can identify and isolate CPCs from a culture of differentiating pluripotent stem cells. To exploit this new finding, these groups, made induced pluripotent stem cells (iPSCs) from a mouse strain that expressed a green fluorescent protein. They then used skin cells from these mice to make iPSCs.

Japanese stem cell researcher Shinya Yamanaka won the Nobel Prize this year for the discovery of iPSCs. To make iPSCs, adult cells are genetically engineered with four different genes and these genes de-differentiate the adult cells to a pluripotent stem cells state.

The iPSCs made from the green fluorescent mice were then differentiated into CPCs. They were able to isolate and identify CPCs by means of capturing all the cells that made Flt1 and Flt4.

According to Schenke-Layland, “Using our newly established cell surface markers, we could detect and isolate the Flt1- and Flt4-positive CPCs in culture. When we cultured the isolated mouse CPCs then in vitro, they actually developed – as well as the embryonic stem cell-derived progenitor cells – into endothelial cells, smooth muscle cells and more interestingly into functional heart muscle cells.”

To determine if these iPSC-derived CPCs could integrate into a living heart, they injected them into the hearts of living mice. 28 days later, the noticed that the injected hearts were loaded with green fluorescent cells that had differentiated into beating heart muscle that were fully integrated into the heart muscle tissue of the heart.

The next step is to determine if these CPCs can help heal a heart after a heart attack. Bone marrow-derived stem cells have been used to help heal the hearts of heart attack patients, and to date, these stem cells are safe, but only seem to help most people just little, even though they seem to help some patients more than others. However, iPSC-derived CPCs could potentially heal the heart to a greater degree.

According to Schenke-Layland, “We are currently focusing on research with human iPS cells. If we can show that cardiovascular progenitor cells can be derived from human iPS cells that have the ability to mature into functional heart muscle, we will have discovered a truly therapeutic solution for heart attack patients.”

See “Characterization and Therapeutic Potential of INduced Pluripotent Stem Cell-Derived Cardiovascular Progenitor Cells;” Ali Nasar et al: PLoS ONE, 2012; 7 (10): e45603 DOI: 10.1371/journal.pone.0045603.

Stem Cell-Derived Exosomes May Prevent Lung Disease in Babies


Now there’s a word you don’t see everyday: Exosome. What on earth is an exosome? They are small, membrane-enclosed vesicles that are released by cells. Exosomes contain proteins and nucleic acids, and they are have surfaces that are decorated with various types of proteins. Exosomes are 40-100 nm in diameter and there are several different cell types that are known to secrete exosomes. Cancer cells use exosomes to mold surrounding cell into structures that the cancer needs (see Huang et al., (2011). Cancer Lett 315, 28-37 and Cho et al., (2012). Int J Oncol. 40(1):130-138). Likewise, exosomes from mesenchymal stem cells seem to delivery proteins and RNA to heart muscle cells that help them heal after a heart attack (Lai et al., Regen. Med. (2011) 6(4), 481–492). Finally, there have been reports that exosomes can protect against tissue injury such as acute kidney damage (see Bruno S, et al. (2009). J. Am. Soc. Nephrol. 20, 1053–1067.

New work from Harvard University’s Boston Children’s Newborn Medicine division in Massachusetts suggests that exosomes from stem cells can protect the fragile lungs of premature babies from serious lung diseases and chronic lung injury. Mesenchymal stem cells (MSCs) can decrease inflammation under several different conditions. They seem to do so by secreting exosomes that hold inflammatory cells at bay. In a mouse model of lung inflammation, infused MSCs can quell inflammation, but the culture medium in which the MSCs were grown can do as good a job and decreasing inflammation as the whole cells. This suggest that the MSCs are making something that assuages inflammation (see Ionescu, L. et al., (2012). Am J Physiol Lung Cell Mol Physiol. doi: 10.​1152/​ajplung.​00144.​2011).

Babies born prematurely have to struggle to get sufficient oxygen into their small, incipient lungs. This causes these poor babies to suffer from chronic oxygen insufficiencies (hypoxia) and they usually need artificial respirators to breathe. The lungs of these babies are susceptible to inflammation and this can lead to chronic lung disease and poor lung function.

Lung inflammation also causes pulmonary hypertension, which simply means that the blood pressure in the artery that carries blood from the heart to the lungs (pulmonary artery) is abnormally high. This causes foaming in the lungs, which reduces gas exchange in the lungs, but the lungs also start to thicken and scar over and that also permanently decreases gas exchange.

Stella Kourembanas is the chair of Boston Medical’s Newborn Medicine Division, and she is heading up investigations into the efficacy of MSC-derived exosomes to stave off inflammation in the lungs of premature babies.

Kourembanas explained, “PH (pulmonary hypertension) is a complex disease fueled by diverse, intertwined cellular and molecular pathways. We have treatments that improve symptoms but no cure, largely because of this complexity. We need to be able to target more than one pathway at a time.”

In 2009, Kourembanas and her colleagues showed that injections of MSCs could prevent PH and chronic lung injury in a newborn mouse model of the disease (Bone marrow stromal cells attenuate lung injury in a murine model of neonatal chronic lung disease (Aslam M, et al., (2009). Am J Respir Crit Care Med. 180(11):1122-30). They also showed that they could achieve the same results by injecting the growth medium that had previously fed the cells.

According to Kourembanas, “We knew, then, that the significant anti-inflammatory and protective effects we saw had to e caused by something released by he MSCs.”

To further their search for factors that heal damaged lungs, Kourembanas and co-workers searched the growth medium of MSCs and they came upon exosomes. By purifying exosomes from the growth medium, Kourembanas’ team showed that the anti-inflammatory effects of the MSCs could be completely recapitulated by simply applying isolated exosomes to the lungs of newborn mice.

Kourembanas said, “We are working to figure out what exactly within the MSC-produced exosomes causes these anti-inflammatory and protective effects. But we know that these exosomes contain microRNAs as well as other nucleic acids. They also induce expression of specific microRNAs in the recipient lung.”

MicroRNAs a small RNA molecules that regulate gene expression. Thousands of microRNAs have been discovered and they are also found in many different biological organisms ranging from plants to worms, to fish, frogs and people.

Kourembanas noted that, “What we may be seeing is the effect of these microRNAs on the expression of multiple genes and the activity of multiple genes and the activity of multiple pathways within the lungs and the immune system all at once.”

Kourembanas hopes that exosome research will someday act alongside stem cell-based therapies. MSC-based exosomes could potentially be used as treatments for premature babies at risk of suffering from chronic lung disease and PH. Also, MSCs are not the only cells that make useful exosomes. Umbilical cord stem cells also secrete exosomes with therapeutic value. Even though many different types of stem cells are recognized by the immune system as foreign, exosomes are not, which gives them an added advantage as therapeutic agents. Kourembanas notes, “they could potentially be collected, banked and given like a drug without the risks of rejection or tumor development that can theoretically come with donor cell or stem cell transplantation.”

Pacemaker Cells Derived from Skin Cells


Dr Oren Caspi, from the Technion-Israel Institute of Technology has done some very interesting work with pluripotent stem cells and the heart. Recently, Caspi and his colleagues made induced pluripotent stem cells (iPSCs) from skin cells extracted from heart patients. These were differentiated into heart muscle cells that had all the characteristics of young, healthy heart tissue from new-born babies.

This has been done before in other labs (for one example, see Ma J., et al., Am J Physiol Heart Circ Physiol 2011 301(5):H2006-17).  What Caspi and others found that was so remarkable was that these reprogrammed cells have the capacity to “reset” the rhythm of any unhealthy heart tissue that surrounds them. Caspi and others in the laboratory of Gepstein Lior think is that patients suffering from irregular or slow heart beats, who normally require a pacemaker, could, instead, be treated with an injection of new heart cells grown from stem cells made from their own cells to create a “biological pacemaker” that could regulate their heartbeat.

At this time, heart attack patients have hearts that pump out of sync or who suffer from irregular heartbeats. Such patients require surgery in order to insert a battery-powered pacemaker that is fitted to control the heart’s rhythm. There are approximately 25,000 pacemakers fitted each year in the United Kingdom alone.

According to Dr. Caspi, “We found that the electrical signal from the heart cells we created synchronized the beat of any surrounding heart tissue. We have seen this happen in dishes in the laboratory and in animal models. When we integrated the cells into the hearts of pigs, they were paced by the cells that were injected. It seems that the cells that beat fastest control the pace, so it could be used to replace artificial pacemakers for people with slow or irregular heartbeats.”

In May, 2012, Caspi and Gepstein became the first scientists in the world to make heart muscle cells from iPSCs that were made from heart patients. They reverted adult skin cells into iPSCs and then used special culture conditions to convert those cells into fully functioning heart cells. These cells integrated into the hearts of rats, and researchers believe that it will be possible to use skin cells from patients to create injectable biological pacemakers. This will reduce the risk of them being rejected by the patient’s body. They are now working with clinical heart specialists in a bid to design a human clinical trial that will evaluate the efficacy of such a treatment in human patients.

According to Caspi: “We are working with clinicians to take some of our data to the clinic, but it is still a very new technology so there is still a lot of research to be done before any treatments will emerge.”