Caduceus Clinical Trial One-Year Update


The CADUCEUS clinical trial, which stands for CArdiosphere-Derived aUtologous stem CElls, to reverse ventricUlar dySfunction) was the brainchild of Cedar-Sinai cardiologist Eduardo Marbán and his colleagues. 

This CADUCEUS trial used a heart-specific stem cell called CDCs or cardiosphere-derived cells to treat patients who had recently suffered a heart attack.  CDCs are extracted from the patient’s own heart and they can be grown in culture, expanded, and then implanted back into the patient’s heart. The initial assessments of those patients who had received the stem cell treatments was published in 2012 in the Journal Lancet (R.R. Makkar, R.R. Smith, K. Cheng et al. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet, 379 (2012), pp. 895–904). The initial assessments of these patients showed shrinkage of their heart scars.  However, these patients showed regional improvements in heart function but no significant differences in global heart function.  Despite these caveats, the initial results were hopeful. 

Now the one-year follow-up of these patients has been published in the Journal of the American College of Cardiology.  The results of this examination are even more exciting.

CDCs were extracted from patients by means of heart biopsies of the inner part of the heart muscle (myocardium). After the cells were grown in culture to larger numbers, they were reintroduced to the hearts of the patients by means of “stop-flow” technique. This procedure utilizes the same technology as stents in that an over-the-wire balloon angioplasty catheter that was positioned in the blood vessels on the heart that were blocked. The figure below shows the cultured cardiospheres.

Specimen processing for human cardiosphere growth and CDC expansion. a, Schematic depicts the steps involved in specimen processing. b, Endomyocardial biopsy fragment on day 1. c, Explant 3 days after plating. d, Edge of explant 13 days after plating showing stromal-like and phase-bright cells. e, Cardiosphere-forming cells collected from the explant after 13 days and plated on poly-d-lysine for 2 days. f, Fully formed cardiospheres on day 25, 12 days after collection of cardiosphere-forming cells. g, CDCs during passage 2, plated on fibronectin for expansion. h and i, Cell growth is expressed as number of population doublings from the time of the first harvest for specimens from nontransplant patients (h) and specimens from transplant patients (i).
Specimen processing for human cardiosphere growth and CDC expansion. a, Schematic depicts the steps involved in specimen processing. b, Endomyocardial biopsy fragment on day 1. c, Explant 3 days after plating. d, Edge of explant 13 days after plating showing stromal-like and phase-bright cells. e, Cardiosphere-forming cells collected from the explant after 13 days and plated on poly-d-lysine for 2 days. f, Fully formed cardiospheres on day 25, 12 days after collection of cardiosphere-forming cells. g, CDCs during passage 2, plated on fibronectin for expansion. h and i, Cell growth is expressed as number of population doublings from the time of the first harvest for specimens from nontransplant patients (h) and specimens from transplant patients (i).

The initial assessment of these patients showed shrinkage of the heart scar and regional improvements in heart function. However in the one-year follow-up the scar showed even more drastic shrinkage (-11.9 grams or -11.1% of the left ventricle). Also, several of the indicators of global heart function showed substantial improvements (end-diastolic volume – -12.7 mls and end-systolic volume – -13.2 mls).

When it come to the all-important ejection fraction, which is the percentage of blood pumped from the left ventricle, the results are a little more complicated. When the ejection factions of each patient was compared with the size of their heart scars, there was a tight correlation between the increase in ejection fraction and the shrinkage of the heart scar. See the figure below for a scatter plot of ejection fraction versus heart scar size.

(A) Scatterplot showing the natural relationship between scar size and left ventricular ejection fraction ∼5 months post-myocardial infarction (circles). Each cross symbol represents the mean values (at the intersection of the vertical and horizontal bars [obtained from all patients with magnetic resonance imaging measurements]), whereas the width of each bar equals ±SEM of scar size and left ventricular ejection fraction of CADUCEUS patients at baseline, 6 months, and 1 year; the crosses are superimposed onto the scatterplot showing prior data from post-myocardial infarction patients with variable scar sizes. The changes in left ventricular ejection fraction in CDC-treated subjects are consistent with the natural relationship between scar size and ejection fraction in convalescent myocardial infarction, whereas the changes in left ventricular ejection fraction in controls fall within the margins of variability. (B) Changes in end-diastolic volume from baseline to 1 year. (C) Changes in end-systolic volume from baseline to 1 year. CDCs = cardiosphere-derived cells; EDV = end-diastolic volume; EF = ejection fraction; ESV = end-systolic volume; LV = left ventricle.
(A) Scatterplot showing the natural relationship between scar size and left ventricular ejection fraction ∼5 months post-myocardial infarction (circles). Each cross symbol represents the mean values (at the intersection of the vertical and horizontal bars [obtained from all patients with magnetic resonance imaging measurements]), whereas the width of each bar equals ±SEM of scar size and left ventricular ejection fraction of CADUCEUS patients at baseline, 6 months, and 1 year; the crosses are superimposed onto the scatterplot showing prior data from post-myocardial infarction patients with variable scar sizes. The changes in left ventricular ejection fraction in CDC-treated subjects are consistent with the natural relationship between scar size and ejection fraction in convalescent myocardial infarction, whereas the changes in left ventricular ejection fraction in controls fall within the margins of variability. (B) Changes in end-diastolic volume from baseline to 1 year. (C) Changes in end-systolic volume from baseline to 1 year. CDCs = cardiosphere-derived cells; EDV = end-diastolic volume; EF = ejection fraction; ESV = end-systolic volume; LV = left ventricle.

Other observations included safety assessments. When the number of adverse events between the control group and CDC-receiving group were measured, there were no differences between the two groups. The patients in the CDC-receiving group were more likely to be hospitalized and had transient cases of fast heartbeats, and there was also one death in this group. However the incidence of these events were not statistically different from the control group.

From these assessments, it is clear that the CDC treatments are safe, and decreased the scar size and regional function of infarcted heart muscle. From these results, the researchers state that “These findings motivate the further exploration of CDCs in future clinical studies.

Anti-Diabetes Drug Acts With Stem Cells to Repair Heart After Heart Attack


Exenatide is the generic name of a antidiabetes drug whose market name is Byetta. Made by Eli Lilly and Company, Exenatide binds to a receptor on the surface of insulin-secreting pancreatic beta cells and stimulates the insulin response of those cells. Insulin is the main hormone that tells the cells in our bodies to take up sugar and use it. Type 2 diabetics, however, have cells that have become de-sensitized to insulin and they require more insulin to signal to the cells to metabolize sugar, Once a type 2 diabetic injects himself with Exenatide, the body secretes more insulin than it might normally secrete, Thus, type 2 diabetics are able to more effectively control their blood sugar levels with this drug.

A) The molecular structure of human GLP-1. B) The molecular structure of exenatide (gray colors indicate differences in structure from human GLP-1. C) The molecular structure of liraglutide (gray colors indicate changes in structure from human GLP-1).
A) The molecular structure of human GLP-1. B) The molecular structure of exenatide (gray colors indicate differences in structure from human GLP-1. C) The molecular structure of liraglutide (gray colors indicate changes in structure from human GLP-1).

Normally, these receptors are bound by a small protein that is made by cells in the small intestine called GLP-1 (glucagon-like peptide-1). The small intestine makes GLP-1 when it is exposed to sugar, and it is the combination of high levels of sugar in the blood, plus the presence of GLP-1 that causes the pancreatic beta cells to release insulin. In type 2 diabetics, the beta cells release insulin, but the body is de-sensitized to it. Therefore, more insulin is needed to control the blood sugar levels. Exenatide does just that by acting like GLP-1.

What does this have to do with stem cells and regenerative medicine? It turns out that the heart also has GLP-1 receptors on the surfaces of its cells and the binding of GLP-1 to these receptors decreases inflammation in the heart, prevents the death of heart muscle cells, protects blood vessels, and protects against damage from reactive oxygen species (also known as free radicals). A fascinating paper has appeared in the Journal of Cellular and Molecular Medicine from the Chinese PLA Hospital in Beijing, China.  In this publication, Chinese cardiologists used a compound that is closely related to Exenatide called Exendin-4 in combination with stem cells to determine if the activation of the GLP-1 receptor influences the healing qualities of stem cells after a heart attack.

In this paper, Yundai Chen and colleagues extracted stem cells from the fat tissue of rats.  This fat tissue was minced and then the stem cells were isolated on the basis of cell surface proteins that are common only to stem cells in fat.  Then Chen and his co-workers gave heart attacks to 120 rats and divided the rats into five groups, with 30 animals each.  The first group received injections of buffer into their hearts immediately after the induction of a heart attack.  The second group received injections of Extendin-4 three days prior to the heart attack and seven days after.  The third group received injections of fat-derived stem cells into the heart tissue bordering the infarcted tissue.  The fourth group received the stem cell injections plus the treatments with Extendin-4.

Exendin-4 is one of the compounds extracted from the salivary glands of the Gila monster, a colorful lizard (shown below)  found in the deserts of California.  Exenatide is the acetate salt of Extendin-4, and both compounds bind the GLP-1 receptor and elicits a biological response.

Gila Monster

 

The results of these experiments were as follows:  the animals that received buffer injections showed respectable amounts of cell death and oxidative damage in their hearts.  However, those animals that received Extendin-4 injections showed less oxidative damage and significantly less cell death.  The same could be said for those animals that received the fat-derived stem cell treatments.  However, those animals that were treated with Extendin-4 plus the stem cells showed substantially less oxidative damage and cell death than all the other groups.

The heart function tests show similar trends.  The ejection fraction, which measures the percentage of the blood that comes into that heart that is pumped out, was in the cellar in the buffer-injected animals, about 10% higher in the Extendin-4 and stem cell-treated animals, and almost 20% higher in the animals treated with both Extendin-4 and the stem cells.   The degree to which the heart muscle contracted (reported as % of shortening or fractional shortening) was over double that in the dually treated animals.  Also the size of the heart scar in the dually treated animals was half the size observed in the animals treated with buffer.

Further examinations of the heart of the dually treated animals showed that the fat-derived stem cells expressed genes normally found in blood vessels cells and heart muscle cells.  This is not definitive evidence that these cells differentiated into heart-specific cell types, but they clearly are surviving and doing something beneficial to the heart.

In culture, the fat-derived stem cells made a whole host of healing molecules when they are treated with Extendin-4.  Also, Extendin-4 treatment protected with stem cells from being damaged by noxious chemicals (e.g., hydrogen peroxide).  Biochemical studies showed that the stem cells that had been treated with Extendin-4 had activated the STAT3 pathway.  Why is this significant?  Because the STAT3 is normally activated by cells when they are stressed.  It is a “I want to survive” kind of pathway.  Extendin-4 seems to cause the stem cells to kick into high gear, survive better, and heal better.

A scheme illustrating the potential cardioprotective signalling pathways through which exenatide may reduce myocardial infarct size and protect the heart against lethal myocardial reperfusion injury. The actual mechanism underlying the cardioprotective effects elicited by exenatide remains to be elucidated, although it is assumed that many of the beneficial effects are mediated through the activation of the glucagon-like peptide-1 (GLP-1) receptor on the cardiomyocytes. The activation of this receptor then recruits pro-survival signalling cascades such as the phosphatidylinositol 3-kinase (PI3K)–Akt and adenylate cyclase (AC)–cAMP–protein kinase A (PKA) pathways which protect the heart against acute ischaemia–reperfusion injury through a number of potential mechanisms including: the inhibition of the mitochondrial permeability transition pore (mPTP), the activation of AKAPs (protein kinase A-anchoring proteins), increased myocardial glucose uptake (possibly via p38 mitogen-activated protein kinase and iNOS), reduced apoptotic cell death, and the transcription of cardioprotective factors (such as PPAR-β/δ, Nrf-2, and HO-1). eNOS, endothelial nitric oxide synthase; GSK, glycogen synthase kinase; HO-1, haem oxygenase 1; iNOS, inducible nitric oxide synthase; NO, nitric oxide; Nrf-2, nuclear respiratory factor 2; PKC, protein kinase; PKG, protein kinase G; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species.
A scheme illustrating the potential cardioprotective signalling pathways through which exenatide may reduce myocardial infarct size and protect the heart against lethal myocardial reperfusion injury. The actual mechanism underlying the cardioprotective effects elicited by exenatide remains to be elucidated, although it is assumed that many of the beneficial effects are mediated through the activation of the glucagon-like peptide-1 (GLP-1) receptor on the cardiomyocytes. The activation of this receptor then recruits pro-survival signalling cascades such as the phosphatidylinositol 3-kinase (PI3K)–Akt and adenylate cyclase (AC)–cAMP–protein kinase A (PKA) pathways which protect the heart against acute ischaemia–reperfusion injury through a number of potential mechanisms including: the inhibition of the mitochondrial permeability transition pore (mPTP), the activation of AKAPs (protein kinase A-anchoring proteins), increased myocardial glucose uptake (possibly via p38 mitogen-activated protein kinase and iNOS), reduced apoptotic cell death, and the transcription of cardioprotective factors (such as PPAR-β/δ, Nrf-2, and HO-1). eNOS, endothelial nitric oxide synthase; GSK, glycogen synthase kinase; HO-1, haem oxygenase 1; iNOS, inducible nitric oxide synthase; NO, nitric oxide; Nrf-2, nuclear respiratory factor 2; PKC, protein kinase; PKG, protein kinase G; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species.

Now, these experiments were performed in rodents.  Therefore, it remains to be seen if the FDA-approved Exenatide will improve stem cell survival as well as the unapproved Extendin-4.  While they should have the same biological activity, different preparations of the similar molecules can elicit different responses.  Also, it is unclear if this strategy would work in humans.  Do human fat-derived or even bone marrow-derived stem cells respond in a similar fashion to Extendin-4 or Exenatide?  Would such an experiment work in humans?  Do the risks associated with Exenatide administration outweigh the potential benefits of administering it?  Could the stem cells simply be pre-treated with Exenatide before being administered?

This paper has truly opened up a can of worms that should keep scientists busy for many years to come.

Heart Muscle in Young Children May Be Capable of Regeneration


The heart of young children might possess untapped potential for regeneration, according to new research. For decades, scientists believed that after a child’s first few days of life, cardiac muscle cells did not divide. Heart growth was thought to occur by means of enlargement of muscle cells.

This view, however, has been seriously challenged in the last few years. New findings in mice that have recently been published in the journal Cell seriously question this dogma. These results have serious implications for the treatment of congenital heart disorders in humans.

Researchers at Emory University School of Medicine have discovered that in 15-day old mice, cardiac muscle cells undergo a precisely timed spurt of cell division that lasts about a day. The total number of cardiac muscle cells in the heart increases by about 40% during this time when the child’s body is growing rapidly. To give you some perspective, a 15-day-old mouse is roughly comparable to a child in kindergarten, and puberty occurs at day 30-35 in mice.

This burst of cell division is driven by a surge of thyroid hormone. This suggests that thyroid hormone might be able to aid in the treatment of children with congenital heart defects. Small trials have even tested thyroid hormone in children with congenital heart defects.

These findings also have broader hints for researchers who are developing regenerative heart therapies. Activating the regenerative potential of the muscle cells themselves is a strategy that is an alternative to focusing on the heart’s stem cells, according to senior author Ahsan Husain, PhD, professor of medicine (cardiology) at Emory University School of Medicine.

“It’s not as dramatic as in fish or amphibians, but we can show that in young mice, the entire heart is capable of regeneration, not just the stem cells,” he says.

This Emory group collaborated with Robert Graham, MD, executive director of the Victor Change Cardiac Research Institute in Australia.

One test conducted by these groups was to determine how well 15-day old mice can recover from the blockage of a coronary artery. Consistent with previous research, newborn (day 2) mice showed a high level of repair after such an injury, but at day 21, endogenous heart repair was quite poor. The 15-day old mice recovered better than the day 21 mice, indicating that some repair is still possible at day 15.

This discovery was an almost accidental finding while Naqvi and Husain were investigating the role of the c-kit gene. The c-kit gene is an important marker for stem cells in cardiac muscle growth. Adult mice that lack a functional c-kit gene in the heart have more cardiac muscle cells. When do these differences appear?

“We started counting the cardiomyocyte cell numbers from birth until puberty,” Naqvi says. “It was a fascinating thing, to see the numbers increasing so sharply on one day.” According to Naqvi, c-kit-deficient and wild-type mice both have a spurt of proliferation early in life but the differences in cardiac muscle cells between the c-kit+ and c-kit- mice appear later.

“Probably, previous investigators did not see this burst of growth because they were not looking for it,” Husain says. “It occurs during a very limited time period.” Even if in humans, the proliferation of cardiac muscle cells does not take place in such a tight time period as it does in mice, the finding is still relevant for human medicine, he says. “Cardiomyocyte proliferation is happening long after the immediate postnatal period,” Husain says. “And cells that were once thought incapable of dividing are the ones doing it.”

Naqvi and Husain plan to continue to investigate the relationships between thyroid hormone, nutrition during early life, and cardiac muscle growth.

Reference: N. Naqvi et al. A Proliferative Burst during Preadolescence Establishes the Final Cardiomyocyte Number. Cell 157, 795-807, 2014.

Modified RNA Induces Vascular Regeneration After a Heart Attack


Regenerating the heart after a heart attack remains one of the Holy Grails of regenerative medicine. It is a daunting task. Even though text books may say, “the heart is just a pump,” this pump has a lot of tricks up its sleeve.

Stem cell treatments can certainly improve the structure and function of the heart after a heart attack, but getting the heart back to where it was before the heart attack is a whole different ball game. To truly regenerate, the heart, the organ or parts of it need to be reprogrammed to a time when the heart could regenerate itself. If that sounds difficult, it’s because it is. But some recent work suggests that it might at least partially possible.

Kenneth Chien and his colleagues from the Department of Stem Cell Biology and Regenerative Medicine at Harvard University have published a terrific paper in the journal Nature Biotechnology that tries to turn back to clock of the heart to augment its regenerative capabilities.

The outermost layer of the heart that surrounds the heart muscle is a layer called the “epicardium.”

epicardiumIn the epicardium are epicardial heart progenitors and these cells are activated within 48 hours after a heart attack in the mouse.  In the fetal heart, epicardial heart progenitors migrate into the heart and differentiate into heart muscle, blood vessels and smooth muscles.  In adults, these cells remain on the surface of the heart and differentiate largely into fibroblasts.  When it comes to regenerative medicine, can we take adult epicardial cells and reprogram them to act like fetal epicardial heart progenitors?

A few experiments have suggested that we can.  In 2011, Smart and others used a small peptide called thymosin β4 to reprogram epicardial cells in mice to form heart muscle and other heart-specific tissues.  Even though the reprogramming was not terribly robust, Smart and others convincingly showed that it was real (Nature 474,640–644).

The Chien group used modified RNA molecules made with unusual nucleotides that encoded the protein vascular endothelial growth factor-A (VEGF-A) to reprogram the epicardium of mice.  VEGF-A is very good and reprogramming the epicardium, and this modified RNA technique does not induce and immune response the way injecting DNA does and the RNA causes bursts of VEGF-A activity that efficiently reprograms the epicardium.

After giving mice heart attacks, Chien and others injected the VEGF-A modified RNAs into the border of the infarcted area of the heart. The modified RNAs induced new gene expression that is normally seen during the establishment of blood vessels.  VEGF-A expression was elevated for up to 6 days after the injections, and animals that had their hearts injected with modified VEGF-A RNA had smaller scars in their hearts, less cell death, and greater tissue volume in their hearts than animals that received either injections of VEGF-A DNA, buffer, or modified RNA that expressed a glowing protein.  Also, the effects of the modified VEGF-A RNA could be abrogated with co-administrating the drug Avastin, which is an antagonist of VEGF-A

Tests with cultured heart cells showed that VEGF-A modified RNA induced blood vessel-specific genes.  These inductions were sensitive to drugs that blocked the VEGF-A receptor, which shows that it is indeed the VEGF-A protein that is inducing these trends.  Finally, a heart muscle gene, Tnnt2 is also induced by the modified VEGF-A RNA.  When the efficacy of the modified VEGF-A RNA was tested in living animals, if was clear that the most numerous cells induced by the modified VEGF-A RNA was endothelial cells, which line blood vessels, followed by smooth muscle cells, and then by heart muscle cells.

Thus, the growth factor VEGF-A can signal to epicardial heart progenitor cells to heal the heart after a heart attack in mice.  It works through the VEGF-A receptor (KDR), and it induces epicardial derived cells (EPDCs) to differentiate into blood vessels, heart muscle cells, and smooth muscle cells, all of which are required to heal the heart.  If VEGF-A signaling can be used to augment heart healing after a heart attack, it might provide a new strategy for healing the heart after a heart attack in a manner that helps the heart heal itself from the inside rather than placing something from the outside into it.

Induced Pluripotent Stem Cells Slow to Grow Tumors in Monkeys


One of the major concerns that dogs the use of pluripotent stem cells in human clinical trials is the risk of tumor formation. Embryonic stem cells and induced pluripotent stem cells have an inherent ability to form special tumors known as teratomas. Teratomas are a rather strange group of tumors that develop from cells early in the developmental program of cells, before they have become committed to mature, adult cell types. Therefore, they contain a mixture of cell types organized to a greater or lesser extent into recognizable structures such as muscles or nerve tissue. In bizarre cases partial teeth may be found.

Embryonic stem cells and their derivatives have a distinct disadvantage in that they are rejected by the immune system of the patient. However, induced pluripotent stem cells (iPSCs), which are made from the patient’s own mature, adult cells, possess the same array of cell surface proteins as the patient’s own cells. Therefore, they are not rejected by the patient’s immune systems. Unfortunately, iPSCs can harbor cancer-causing mutations that were induced during the reprogramming process, and these mutations can seriously compromise their clinical usefulness and safety. Having, not all iPSC lines are the same. Some appear to be safer than others and screening methods that have been developed by stem cell scientists seem to be able to detect unsafe iPSC lines over others.

Now, a new study has shown that it takes a lot of effort to get iPSCs to form tumors after transplantation into a monkey. These findings will bolster the prospects of one day using iPSCs human patients.

Making iPSCs from an animal’s own skin cells and then transplanting them back into the creature also does not trigger an inflammatory response as long as the cells have first been differentiated into a more mature, specialized cell type.

“It’s important because the field is very controversial right now,” says Ashleigh Boyd, a stem-cell researcher at University College London, who was not involved in the work. “It is showing that the weight of evidence is pointing towards the fact that the cells won’t be rejected.”

Pluripotent stem cells have the ability to differentiate into many specialized cell types in culture. Therefore, they have been held out as potential sources of treatments for regenerative therapies for diseases such as Parkinson’s and some forms of diabetes and blindness. iPSCs, which are made by reprogramming adult cells, have an extra advantage because transplants made from them could be genetically matched to the recipient. Also, iPSC derivation is cheaper than cloning procedures and does not destroy a young embryo.

Globally, stem cells researchers are pursuing a variety of iPSCs-based therapies. For example, a group in Japan began enrolling patients for an iPSC-based human clinical trial last year. Experiments in mice from 2011 suggested that even genetically matched iPSCs can elicit an immune response, and pluripotent stem cells can also form slow-growing tumors. Both of these results have elicited deep safety concerns.

A stem-cell scientist from the National Institutes of Health in Bethesda, Maryland, named Cynthia Dunbar led this new study. She decided to evaluate both of these above-mentioned concerns in healthy rhesus macaques. The ability of pluripotent stem cells to form teratomas in laboratory mice is normally a test of their pluripotency. However, to prevent the immune systems of the mice from attacking and destroying these implanted stem cells, mice that lack the cell-mediated arm of the immune response are used. Such mice are called “nude” mice because they do have any hair.

Dunbar said, “We really wanted to set up a model that was closer to human. It was somewhat reassuring that in a normal monkey with a normal immune system you had to give a whole lot of immature cells to get any kind of tumor to grow, and they were very slow-growing.”

Dunbar and her team made iPSCs from skin and white blood cells from two rhesus macaques, and transplanted them back into the monkeys. She and her coworkers were careful to make sure that each monkey was injected with those iPSCs that had been derived from their own cells. For example, if monkey A provided cells that were used to derive iPSC cell line A1, then monkey A was only injected with iPSC line A1 cells and so on. Dunbar and others found that tumor formation required 20 times as many iPSCs as those needed for form a tumor in a nude mouse. These data are invaluable for safety assessments of potential iPSC-dependent therapies. Additionally, even though the injected iPSCs did trigger a mild immune response (white blood cells were attracted to the site of injection, which caused local but not systemic inflammation), when iPSCs were differentiated to a more mature cell types caused no such response.

Dunbar’s study is the first to examine the effects of transplanting undifferentiated iPSCs into the monkey they came from. However it is not the first primate study what happens when cells differentiated from iPSCs are transplanted into non-human primates. Scientists at Kyoto University in Japan transplanted monkey iPSCs that had been differentiated into dopaminergic neurons (the type of neuron that dies in Parkinson’s disease) into the brains of other monkeys and notes that these cells survived for months without forming tumors. Researchers at RIKEN in Kobe, Japan, observed similar results when they transplanted iPSCs that had been differentiated into retinal pigment epithelial cells, which support the photoreceptors at the back of the eye. In neither study did the implanted cells form tumors nor were they immunologically rejected when animals received their own cells. However, in both cases, the transplantation sites that were chosen tend to have a weak capacity to trigger immune responses.

In contrast, Dunbar differentiated iPSCs into bone precursor cells and placed them into small scaffolds just under the skin. Such a location can potentially elicit a robust immune response. However, the transplants did not cause irritation or inflammation, since the differentiated cells do not express embryonic proteins that are normally absent in mature tissues. By eight weeks, new bone had formed, and almost a year later no tumors had formed, and bone formation persisted.

The caveat to these studies is that some work has suggested that bone precursor cells can suppress the immune response against them. To circumvent this problem, Dunbar hopes to repeat these studies using iPSCs that have been differentiated into heart and liver cells.

Doubts About Cardiac Stem Cells


Within the heart resides a cell population called “c-kit cells,” which have the ability to proliferate when the heart is damaged. Several experiments and clinical trials from several labs have provided some evidence that these cells are the resident stem cell population in the heart that can repair the heart after an episode of cardiac injury.

Unfortunately, a few new studies, and in particular, one that was recently published in the journal Nature, seem to cast doubt on these results. Jeff Molkentin of Cincinnati Children’s Hospital Medical Center and his co-workers have used rather precise cell lineage tracing studies in mice to follow c-kit cells and their behavior after a heart attack. His results strongly suggest that c-kit cells rarely produce heart muscle cells, but they do readily differentiate into cardiac endothelium, which lines blood vessels.

“The conclusion I am led to from this is that the c-kit cell is not a cardiac stem cell, at least in term of its normal, in vivo role,” said Charles Murry, a heart regeneration researcher at the University of Washington who was not involved in this study.

Molkentin’s study is what some stem cells researchers are calling the nail in the coffin for c-kit cells. In fact the Molkentin paper is simply the latest in a series of papers that were unable to reproduce the results of others when it comes to c-kit cells. Worse still, one of the leading laboratories in the c-kit work, Piero Anversa at Harvard Medical School, has had to retract on of this papers and there is also some concern about his publication regarding the SCIPIO trial. Eduardo Marbán, an author of the new study and a cardiologist at the Cedars-Sinai Heart Institute in Los Angeles, said, “There’s been a tidal wave in the last few weeks of rising skepticism,” Nevertheless, the present dispute is not yet settled, and many scientists still regard the regenerative powers of c-kit cells as a firmly established fact.

In his laboratory, Piero Anversa and his colleagues and collaborators have shown that c-kit cells—cardiac progenitor cells expressing the cell surface protein c-kit—can produce new heart muscle cells (cardiomyocytes). Anversa and others also helped usher these cells, which are also known as CPCs or cardiac progenitor cells, into clinical trials to test whether they might help repair damaged cardiac tissue. This culminated in the SCIPIO trial, which showed that patients treated with their own CPCs showed long-lasting and remarkable improves in heart function.

Follow-up work by other research teams, however, has not been able to confirm these studies, and their work has raised doubts about the potential of c-kit cells to actually build new heart muscle. In his contribution to the c-kit controversy, Molkentin and his colleagues genetically engineered mouse strains in which any c-kit-expressing cells and their progeny would glow green. To do this, they inserted a green fluorescent protein gene next to the c-Kit locus. Therefore any c-kit-expressing cells in the heart would not only glow green, but whatever cell type they differentiated into would also glow green. After inducing heart attacks in these mice, Molkentin and others discovered that only 0.027 percent of the heart muscle cells in the mouse heart originated from c-kit cells. “C-kit cells in the heart don’t like to make myocytes,” Molkentin told The Scientist. “We’re not saying anything that’s different” from groups that have not had success with c-kit cells in the past,” Molkentin continued, “we’re just saying we did it in a way that’s unequivocal.”

Molkentin’s study did not address why there’s a discrepancy between his results and those of Anversa and another leader in the c-kit field, Bernardo Nadal-Ginard, an honorary professor at King’s College London. Last year in a paper published in the journal Cell, Nadal-Ginard and his colleagues showed that heart regeneration in rodents relies on c-kit positive cells and that depleting these cells abolishes the regenerative capacity of the heart.

In an email to a popular science news publication known as The Scientist, Nadal-Ginard suggested that technical issues with Molkentin’s mouse model could have affected his results, causing too few c-kit cells to be labeled. Additionally, “the work presented by Molkentin used none of our experimental approaches; therefore, it is not possible to compare the results,” Nadal-Ginard said in an e-mail.

Anversa said his lab is working with the same mouse model Molkentin used, “but our data are too preliminary to make any specific comment. Time will tell.”

Molkentin’s paper seems point to further problems with Aversa’s work with c-kit cells.  Last month, one of the papers Anvera and his group had published in the journal Circulation had to be retracted because the data used the write that paper were “sufficiently compromised.”  Then a few days later, the paper describing the results of the SCIPIO study that appeared the journal The Lancet expressed concern about supplemental data that was included with the published results.  These data came from the human clinical trial that treated heart patients with their own c-kit cells.  Harvard Medical School and Brigham and Women’s Hospital are investigating what went wrong with this study and the publication itself.

Regardless of Anversa’s present tribulations, Marbán is advancing another type of heart-specific stem cell, called cardiosphere-derived cells or CDCs.  Marbán and his colleagues have already used CDCs in a human clinical trial known as the CADUCEUS trial.  In this trial, heart attack patients treated with CDCs saw their heart scars shrink.  Marbán said he had been a true believer in c-kit cells, until the data started mounting against them. “The totality of the evidence now says the c-kit cell is no longer a cardiomyocyte progenitor,” he said.

Now even if c-kit cells do not make new heart muscle, it is possible that they heal the heart through other means.  The patients in the SCIPIO trial saw real, genuine improvements in their heart function and these results cannot be so cavalierly dismissed.  In fact, Murry said that just because the mechanistic basis for the human study remains in doubt, promising clinical results should not be dismissed. “Those results can be considered independent,” he said.  Molkentin also added that it’s possible that c-kit cells work in unknown ways to repair heart tissue.  Since clinical treatments involves high levels of c-kit cells that have been immersed in culture conditions, “Perhaps these cells act a little different,” Molkentin said.

Nadal-Ginard also noted that discrepancies do exist between his data and those of others, and that these discrepancies should not be papered over, but should be robustly debated and addressed.  He said he’d be willing to work with Molkentin to get to the bottom of it. “The concept under dispute is too important for the field of regenerative medicine—and regenerative cardiology, in particular—to turn into a philosophical/dogmatic argument instead of settling it in a proper scientific manner.”  Here here.

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.