Cardiac Muscle Cells Work as Well as Cardiac Progenitor Cells to Repair the Heart


Cell therapies for the heart after a heart attack provide some healing, but the success of these treatments in inconsistent and the majority of the improvements are modest. Whole bone marrow or even bone marrow stem cells can promote the growth of new blood vessels in the heart after a heart attack (Zhou Y, et al., Ann Thorac Surg. 2011 Apr;91(4):1206-12). The treatment of the heart after a heart attack, can also stimulate the regeneration of new heart muscle, but such new muscle comes from endogenous stem cells populations that are induced by the implanted stem cells (Hatzistergos KE, et al., Circ Res. 2010 Oct 1;107(7):913-22).

Nevertheless, the clinical trials with bone marrow cells have produced mixed results. Bone marrow implants work well in some patients and hardly at all in others. The quality of the patient’s bone marrow might be part of the reason for the disparate findings of these trials, but the fact remains, that using cells that can replace dead heart muscle can potentially treat a damaged heart better than bone marrow stem cells.

Pluripotent stem cells, either embryonic stem cells or induced pluripotent stem cells (iPSCs) can efficiently differentiate into heart muscle cells, but a debate remains as to which cell does a better job for healing the heart: Should young heart muscle cells called progenitor cells be used, or can mature heart muscle cells do the job just as well?

Charles Murray from the University of Washington, who has pioneered the use of stem cells to treat the hearts of laboratory animals, and his colleagues tested the ability of heart progenitor cells to repair the heart versus mature heart muscle cells. Both of these cell types were tested against bone marrow stem cells as a control.

Murray and his colleagues used heart muscle cells made from human embryonic stem cells and heart progenitor cells made from the same human embryonic stem cell line to treat the hearts of laboratory rats. These rats were given heart attacks and then the cells were injected directly into the walls of the heart. Injections were given four days after the heart attacks were induced. Each treatment group contained ten rats, including a control group that received injections of cells that are known to possess no healing capabilities.

Measurements of heart function four weeks after treatment showed that both heart progenitor cells and mature heart muscle cells improved the heart equally well and both cells improved heart significantly better than bone marrow stem cells.

Murray said, “There’s no reason to go back to more primitive cells, because they don’t seem to have a practical advantage over more definitive cells types in which the risk for tumor formation is lower.”

In the future, Murry would like to determine if these same cells work in a larger animal model system and then, eventually start clinical trials in human heart attack patients.

Fernandes and Chong et al., Stem Cell Reports, October 2015 DOI: 10.1016/jstemcr.2015.09.011.

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.

Transplanted Human Umbilical Cord Blood Cells Improved Long-Term Heart Muscle Structure and Function in Rats After a Heart Attack


Jianyi Zhang, from the University of Minnesota Health Science Center, in Minneapolis, Minnesota and his co-workers have shown that the transplantation of human umbilical cord blood cells into the rat hearts after a heart attack experience long-term effects that are not observed in the control animals that did not receive the stem cells. Furthermore, none of these laboratory animals required immunosuppressive therapy. The study is scheduled to be published in the journal Cell Transplantation.

“Myocardial infarction induced by coronary artery disease is one of the major causes of heart attack,” said Dr. Zhang. “Because of the loss of viable myocardium after an MI, the heart works under elevated wall stress, which results in progressive myocardial hypertrophy and left ventricular dilation that leads to heart failure. We investigated the long-term effects of stem cell therapy using human non-hematopoietic umbilical cord blood stem cells (nh-UCBCs). These cells have previously exhibited neuro-restorative effects in a rodent model of ischemic brain injury in terms of improved LV function and myocardial fiber structure, the three-dimensional architecture of which make the heart an efficient pump.”

According to Zhang and his co-authors, stem cell researchers have intently examined the ability of stem cells to regenerate and heal damaged heart tissue. Many laboratories all over the world have employed different types of stem cells, different animal models, and distinct modes of stem cell delivery into the heart tissue, and different stem cell doses. All of these studies have produced varying levels of improvement of left ventricular function. Zhang and others also note that, for the most part, the underlying mechanisms by which implanted stem cells improve heart function are “poorly understood and that the overall regeneration of heart muscle cells is modest at best.

In order to investigate the heart’s remodeling processes and to characterize the alterations in cardiac fiber architecture, Zhang’s team used diffusion tensor MRI (DTMRI), which has been previously used to study heart muscle fiber structure in both humans and animals. Most previous studies have concentrated on the short-term effects of umbilical cord blood cells (UCBCs) on damaged heart muscles. Fortunately, this study, which examined the long-term effects of UCBCs, not only demonstrated evidence of significantly improved heart function in treated rats, but also showed evidence of delay and prevention of myocardial fiber structural remodeling. Keep in mind that such alterations in heart muscle fiber structure could have resulted in heart failure.

When compared to the age-matched but untreated rat hearts that had suffered a heart attack, the regional heart muscle function of non-hematopoietic UCBC-treated hearts was significantly improved and the preserved myocardial fiber structure seems to have served as an “underlying mechanism for the observed function improvements.”

“Our data demonstrate that nh-UCBC treatment preserves myocardial fiber structure that supports the improved LV regional and chamber function,” concluded the researchers.

“This study provides evidence that UCBCs could be a potential therapy with long-term benefits for MI” said Dr. Amit N. Patel, director of cardiovascular regenerative medicine at the University of Utah and section editor for Cell Transplantation. “Preservation of the myocardial fiber structure is an important step towards finding an effective therapy for MIs”

See: Chen, Y.; Ye, L.; Zhong, J.; Li, X.; Yan, C.; Chandler, M. P.; Calvin, S.; Xiao, F.; Negia, M.; Low, W. C.; Zhang, J.; Yu, X. The Structural Basis of Functional Improvement in Response to Human Umbilical Cord Blood Stem Cell Transplantation . Cell Transplant. Appeared or available online: December 10, 2013.

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.”

Grafted Stem Cell Derivatives Restore Normal Heart Rhythms in Mice


American researchers, in collaboration with technicians from Fujifilm VisualSonics, Inc., have used advanced ultrasonic software to document microscopic, regenerative improvements to heart muscle that has suffered from previous damage.

High-frequency ultrasound and special cardiac-assessment software was developed by FujiFilm VisualSonics, Inc of Toronto, Canada. Scientists from Mayo Clinic implanted engineered cells into the damaged hearts of mice and then used the special software and ultrasound imaging to observe the regeneration of the heart so that it began to contract with normal cardiac rhythms.

After a heart attack, dead heart tissue is replaced with a cardiac scar that consists of scar tissue that neither contracts nor conducts the signals to contract. Depending of the size of the heart scar, the heart can beat abnormally. An abnormal heart beat is known as arrhythmia. Arrhthymias come in three different categories: a heart that beats too fast (tachycardia), a heart that beats too slowly (bradycardia), and a heart that beats erratically. Arrhythmias after a heart attack can be life-threatening, and restoring normal heart rhythm to the heart after a heart attack is very important.

In this experiment, mice were given heart attacks, and then undifferentiated induced pluripotent stem cells (iPSCs) were implanted into these hearts. Those mice that received induced pluripotent stem cells gradually normalized, their heart beat. The resynchronization of the heart beat of these mice was imaged with high-resolution ultrasound.

Satsuki Yamada, first author of this paper, said, “A high-resolution ultrasound revealed harmonized pumping [of the heart] where iPS cells were introduced to be the previously damaged heart tissue.” Yamada also noted that Induced pluripotent stem cell intervention rescues ventricular wall motion disparity, and achieves resynchronization of the heart beat after a heart attack.

This experiment shows, for the first time that undifferentiated iPSCs have the potential to stabilize a patient’s heart after a heart attack. The healing of the heart was documented by ultrasound imaging and by “speckle-tracking echocardiogram.,” Speckle-tracking echocardiography was designed by VevoStrain Advanced Cardiac Analysis Software, which was manufactured by VisualSonics.

This software package provides advanced imaging and quantification capabilities for studying sensitive movements in heart muscles and it is also the only commercial cardiac-strain package optimized for assessing cardiovascular function preclinical rodent studies.

Yamada and her co-researchers utilized this software during the implantation and observation of the iPSCs within the hearts of mice. This software package the motion of the heart wall both at the regional and global levels and from several different perspectives, measurements of these movements, the changes in dimension in the left ventricle during the heart cycle.

The software definitely showed that homogeneous wall movement was restored in those mice that had received implants of iPSCs.

When iPSCs were implanted into mice that had dysfunctional immune systems, they produced tumors, but in mice with normal immune systems, the implanted iPSCs did not produce tumors. What became of those cells is uncertain, but they clearly helped heal the heart and did not cause tumors.

Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.
Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.

This paper is interesting and suggests that undifferentiated cells can also exert healing effects on the heart.