Combining Umbilical Cord Cells with Hyaluronic Acid Improves Heart Repair After a Heart Attack


Umbilical cord blood cells have an advantage over bone marrow or peripheral blood cells in that aging, systemic inflammation, and stress or damage caused by cell processing procedures can potentially compromise and diminish the regenerative capability of these cells. This problem is particularly acute in the case of treating patients who have recently suffered a heart attack, since transplanted cells experience a rather hostile environment that kills off most cells. Additionally, blood flow through the heart tends to wash out infused cells, which further decreases any regenerative activities the cells might have otherwise exerted.

With this in mind, Patrick Hsieh and his colleagues at the Academia Sinica, in Taipei, Taiwan tested if ability of human cord blood mononuclear cells (CB-MNCs) injected into the heart in combination with a hyaluronan (HA) hydrogel could extend the regenerative abilities of these cells in a pig model. HA is a common component of connective tissue, and, in general, it is very well tolerated by patients and implanted cells. Furthermore, it has the added bonus of shielding cells from a hostile environment and preventing them from being washed out of the heart.

Hsieh used a total of 34 minipigs and divided them into five different groups. One group was the sham operation group in which minipigs received surgical incisions but no heart attack was induced. The second group had heart attacks surgically induced and received infusions of normal saline solutions. The third group of minipigs also experienced heart attacks, and had HA injected into the heart walls. The fourth group also suffered heart attacks and received injections of human umbilical cord stem cells into their heart walls. The fifth group experienced heart attacks and received injections of both HA and human umbilical cord blood cells. The animals were kept and examined two months after surgery.

Two months after the surgery, the minipigs that received injections of human umbilical cord blood cells plus HA showed the highest left ventricle ejection fraction (51.32% ± 0.81%). This is significant when compared to 42.87% ± 0.97%, for the group that received injections of normal saline, 44.2% ± 0.63% for the group that received injections of HA alone, and 46.17% ± 0.39% for the group that received injections of umbilical cord blood cells only. Additionally, hearts from minipigs that received cord blood cells plus HA improved the systolic and diastolic function significantly better than the other experimental groups. Injections of either cord blood cells alone or in combination with HA significantly decreased the scar area and promoted the formation of new blood vessels in the infarcted region. In general, this study suggests that combined infusion of umbilical cord blood cells and HA improves the function of the heart after a heart attack and might prove to be a promising treatment option of heart attack patients.

This is a preclinical study, but it is a preclinical study in a larger animal model system. Umbilical cord blood cells have a demonstrated ability to induce healing in the heart after a heart attack. However, the combination of these cells with HA almost certainly significantly increases cell retention in the heart, thereby significantly improving cardiac performance, and preventing cardiac remodeling. Therefore, using healthy cells donated from another source to replace damaged or moribund cells may be a better option to treat a heart patient and repair their sick heart.

This work appeared in Stem Cells Trans Med November 2015, doi: 10.5966/sctm.2015-0092

Umbilical Cord Stem Cells Improve Heart Function after a Heart Attack


The umbilical cord connects the baby to the placenta and contains umbilical arteries, umbilical veins, and a gooey material between the umbilical vessels called Warton’s jelly. Warton’s Jelly (WJ), besides being rich in extracellular matrix molecules also contains a mesenchymal stem cell population that is rather primitive. These WJ mesenchymal stem cells or WJMSCs have excellent potential for therapeutic strategies.

Lian Gao and her colleagues from the Navy General Hospital in Beijing, China, in collaboration with coworkers from the Shenzhen Beike Cell Engineering Research Institute in Shenzhen, China conducted a clinical trial that examined the use of these WJMSCs in human patients who had suffered a heart attack.  The results are as interesting as they are suggestive and were published in the journal BMC Medicine.

First we must consider the design of the study. Gao and others recruited 160 heart attack patients who were no younger than 18 and no older than 80-years old. All patients had to be free of liver or kidney disease, cancer or some other terminal illness. They were admitted to 11 hospitals in China between February 2011 and January 2012 and had suffered from a documented heart attack as defined by symptoms and their EKG (ST elevation). All patients has also been treated with the implantation of a stent within 12 hours of their heart attack and still retained a respectable amount of movement of the heart wall in the left ventricle. If patients were outside these parameters, they were excluded from the study.

Of the 160 patients who were recruited for the study, 44 were excluded, either because they did not fit within the exclusion criteria, did not wish to participate in the trial, or opted out for undisclosed reasons. This left 116 patients who were randomly assigned to the placebo group or the experimental group (58 in each group). Of these two groups, the placebo group had one patient discontinue the study because of a bout with stomach cancer. The experimental group had one patient die ten days into the trial, another was lost because they moved and a third patients withdrew because of leukemia. This left 57 subjects for the placebo group and 55 for the experiment group who went through all 18 months of follow-up after their respective procedures.

There were two end points for this clinical trial after patients were observed for 18 months after the procedure. The first was safety and this was measured by examining the number of adverse effects (AEs) within these 18 months. Such AEs include things like death, hospitalization for worsening heart function, severe arrhythmias, repeated coronary intervention, blood clots forming in the stents (stent thrombosis), coronary artery obstruction, and the growth of extra tissue in the heart that does not belong there, disorders of the immune system and so on. The second end pointy was efficacy of the implanted cells. To ascertain this, the function of the heart was measured using positron emission computer tomography (PET), and single-photo-emission computer tomography or SPECT. These imaging procedures allow cardiologists to take very precise snapshots of the heart and determine with a good deal of accuracy the performance of the heart.

The WJMSCs were acquired from umbilical cords that were donated from healthy mothers who had delivered healthy babies by means of Caesarian section. 21 of these umbilical cords had their blood vessels removed and then the gelatinous tissue surrounding the vessels was removed, sliced up, and cultured. The MSCs in the gelatinous tissue, which is Warton’s Jelly, migrated from the WJ to the culture dishes. After three passages in the culture dishes, he cells were harvested, concentrated, and tested for viruses, toxins, and cell viability. All cells were negative for viruses and toxins and other contaminants, and were also clearly MSCs, based on the ensemble of cell surface proteins that presented on their membranes, and showed high degrees of viability.

In infuse the cells into the hearts of the patients, six million WJMSCs were delivered into the coronary arteries using the usual over-the-wire techniques that are used to place stents, except that instead of placing stents, WJMSCs were slowly released into the coronary arteries. The cells will home to the damaged heart tissue and are able to pass through the blood vessels into the area of the infarct. Patients receiving the placebo, only received infusions of physiological saline solution, which was used to resuspend the WJMSCs.

The results are very encouraging. With respect to safety, the number of AEs was approximately the same for both groups. In the words of the study, “The groups did not differ in occurrences of MACEs (major adverse cardiac events), including death, recurrences of AMIs (acute myocardial infarctions) and re-hospitalization due to heart failure, during the course of treatment and the 18-month follow-up period.” There were no indications of cancer or the increase in tumor-specific molecules in the blood of the patients from either group. No biochemical or immune abnormalities were observed in any pf the patients either. The stomach cancer in one patient in the placebo group and leukemia in a patient from the experimental group were shown to be unrelated to the procedures. Therefore, at 18 months after the procedure, the infusion of these cells appears to be safe.

As to the efficacy of the procedure, there were significant improvements in the heart function of patients who had received the WJMSCs over those who had received placebo. First of all, the baseline heart function of patients in both groups was approximately the same on the average, except that the patients in the experimental group had slightly better heart parameter than those in the placebo group. Therefore, the efficacy of this procedure was determined by measuring the change in heart performance after the procedure. Patients who had received the placebo had about a 3% increase in the uptake of the F18-labeled sugar molecule after 4 months. The uptake of this marker indicates the presence of live cells. An increase in uptake of the modified sugar molecule shows that some new heart tissue has been produced, probably by the resident stem cell population in the heart. The experimental group, however, after 4 months showed an approximate 7% increase in PET signal intensity. This shows that a good deal more heart cells are being formed in the WJMSC-treated hearts that in the placebo-treated hearts. The SPECT imaging assays the “perfusion” of the heart tissue or the degree to which the heart tissue is being fed by blood vessels. After a heart attack, the dead area of the heart lacks blood vessels and its poor perfusion can affect nearby areas. The placebo-treated patients had a roughly 4% increase in SPECT signal, whereas the WJMSC-treated group had a 7% increase. Thus, the WJMSC-treated hearts had more blood vessels to feed the blood, oxygen and nutrients to the heart muscle and therefore, better perfusion.

Finally, the percentage of blood ejected by the heart during each contraction increase about 3% in the placebo group, but increase by about 8% in the WJMSC-treated group after 18 months. This parameter of heart function, the ejection fraction, is a very important measure of heart function and the fact that it significantly increased in the WJMSC-treated patients over the placebo-treated patients is an important finding.

This was a double-blinded, placebo-controlled study that determined the safety and efficacy of infusions of WJMSCs into the hearts of patients who had recently suffered from a heart attack. In animal experiments, these cells have been shown to increase heart function, increase blood vessel density in the hearts of animals, and increase resident heart-specific stem cell activity in the heart (see Lupu and others, Cell Physiol Biochem 2011; 28:63-76; Gao and others, Cell Transplant 2013; 22:1883-1900; Lopez Y, and others, Current Stem Cell Res Ther 2013;8:46-59). This clinical trial suggests that those benefits documented in laboratory animals might translate to human patients.

This is not a perfect study. These patients will need to be followed for several years to establish that these benefits are long-term and not short-term. Also, there is no indication that patients were given a 6-minute walking test to determine if the improvements in cardiac function translated to improvements in basic activities. However, it is an interesting study and it suggests that banking WJMSCs in addition to cord blood might be a good idea for use in trials like this one and maybe, someday for treatments of heart attack patients.

Culture Medium from Human Amniotic Membrane Mesenchymal Stem Cells Promotes Cell Survival and Blood Vessel Production in Damaged Rat Hearts


The laboratory of Massimiliono Gnecchi at the Fondazione IRCCS Policlinico San Matteo in Pavia, Italy has used the products of amniotic mesenchymal stem cells to treat heart attacks in laboratory rodents. The results are rather interesting.

In a paper published in the May 2015 edition of the journal Stem Cells Translational Medicine, Gnecchi and his colleagues grew human amniotic mesenchymal stem cells derived from amniotic membrane (hAMCs) in cell culture.

These cells were isolated from amniotic membrane donated by mothers who were undergoing Caesarian sections. The membranes were removed, and grown in standard culture media under standard conditions. Once the cells grew out, they were collected and grow in a medium known as DMEM (Dulbecco’s modified Eagle Medium). After the cells had grown for 36 hours, they culture medium was filtered, concentrated, and readied for use.

The first experiments included the use of this conditioned culture medium to treat H9c2 embryonic heart muscle cells with in culture and then expose the heart muscle cells to low oxygen conditions. Normally, low oxygen conditions kill heart muscle cells. However, the cells pre-treated with conditioned medium from hAMCs showed much more robust survival in low-oxygen conditions. This shows that molecules secreted by hAMCs had promote the survival of heart muscle cells.

Next, Gnecchi and his team used their conditioned medium to treat laboratory rats that had suffered heart attacks. Some of the rats were treated with conditioned culture medium from cultured skin cells and others with sterile saline. The culture medium was injected directly into the heart muscle.  The rats treated with conditioned medium from hAMCs showed far less cell death than the other rats. The rats treated with the hAMC-treated culture medium also had vastly denser concentrations of new blood vessels.

It is well-known that mesenchymal stem cells from many sources are filled with small vesicles known as exosomes that are loaded with healing molecules. Mesenchymal stem cells release these exosomes when they home to damaged tissues. The culture medium from the hAMCs were almost certainly filled with exosomes. The molecules released by these cells helped promote heart muscle cell survival in the oxygen-depleted heart, and induced the recruited large numbers of EPCs (endothelial progenitor cells), which established large numbers of new blood vessels. These new blood vessels gave oxygen to formerly depleted heart tissue and promoted heart healing. The size of the heart scar was smaller in the rats treated with hAMC-conditioned medium.

Unfortunately there were no measurement of cardiac function so we are not told if this treatment affected ejection fraction, or other physiological parameters. Nevertheless, this paper does show that exosomes from hAMCs do promote the production of blood vessels and cell survival.

Dead Heart Muscle Regrown in Rodents


If you cut a piece of tissue from the heart of a salamander or zebrafish, they wild simply grow new heart tissue. Unfortunately, humans are unable to easily regenerate heart cells, and this males it difficult to recover from the permanent damage caused by heart attacks.

Fortunately, life scientists from the Weizmann Institute of Science in Israel and the Victor Chang Institute in Sydney have discovered a way to stimulate heart muscle cells in mammals to grow. This finding could have major implications for future heart attack sufferers.

Even though human blood, hair and skin cells renew themselves throughout life, cell division in the heart comes to a virtual standstill shortly after birth, according to Prof. Richard Harvey, from the Victor Chang cardiac research institute, and one of the authors of this research. Harvey said, “So there’s always been an intense interest in the mechanism salamanders and fish use which makes them capable of heart regeneration, and one thing they do is send their cardiomyocytes, or muscle cells, into a dormant state, which they then come out of to go into a proliferative state, which means they start dividing rapidly and replacing lost cardiomyocytes.”

Harvey continued: “There are various theories why the human heart can not do that, one being that our more sophisticated immune system has come at a cost, and because human cardiomyocytes are in a deeper state of quiescence, that has made it very difficult to stimulate them to divide.”

Today, for the first time in history, more people in developing countries die from strokes and heart attacks than infectious diseases. Fortunately there are cost-effective ways to save lives

By studying mice, Harvey and his colleagues found a way to overcome that regenerative barrier – at least in the rodents.

Harvey and others found that by stimulating a cell signaling pathway in the heart that is driven by a hormone called neuregulin, heart muscle cells divided in a spectacular way in both adolescent and adult mice. In humans, neuregulin expression is usually muted about one week after birth, and by about 20 weeks after birth in mice.

By triggering of the neuregulin pathway following a heart attack in mice, Harvey and others induced the replacement of lost muscle, which repaired the heart to a level close to that prior to the heart attack. Harvey said that he and other scientists should be able to determine with in the next five years if it is possible to replicate these results in humans.

“This is such a significant finding that it will harness research activities in many labs around the world, and there will be much more attention now on how this neuregulin-response could be maximised,” Harvey said.

“We will now examine what else we can use, other than genes, to activate that pathway, and it could be that there are already drugs out there, used for other conditions and regarded as safe, that can trigger this response in humans.”

When one of the blood vessels that provide blood to the heart muscle becomes blocked, the patient suffers a heart attack. Heart attacks or “myocardial infractions” cause billions of cardiomyocytes to die. Even if you survive a heart attack, you usually experience diminished quality of life because of it.

“The dream is that one day we will be able to regenerate damaged heart tissue, much like a salamander can regrow a new limb if it is bitten off by a predator,” Harvey said.

Molecular biologist Gabriele D’Uva lead this research, which was published in the scientific journal Nature Cell Biology.

Nonhematopoietic Stem Cells from Umbilical Cord Blood Improve Heart Function After a Heart Attack


Xin Yu, who has dual appointments at Case Western Reserve University, in Cleveland, Ohio and the University of Minnesota Medical School in Minneapolis, Minnesota, has published a remarkable paper in the journal Cell Transplantation that describes the use of a stem cell population from umbilical cord blood to treat mice that had suffered heart attacks. The non-invasive way in which these cells were administered and the tremendous healing qualities of these cells makes paper unique.

In 2005, Water Low at the University of Minnesota Medical School described the isolation and characterization of a unique stem cell population from umbilical cord blood that he called nonhematopoietic umbilical cord blood stem cells or nh-UCBSCs. These cells were used to treat animals with strokes and they induced the growth of new brain cells in the brains of treated mice (see J Xiao, Z Nan, Y Motooka, and WC Low, Stem Cells Dev. 2005 Dec;14(6):722-33).

Yu used these cells to treat male Lewis rats that had suffered heart attacks. In all cases, the rats were subjected to open-heart surgery and the left anterior descending artery was tied off to induce a heart attack. One group of rats were operated on but no heart attacks were induced. A second group was given heart attacks, and then two days later were given intravenous saline infusions. The third group was given a heart attack and then two days later, were injected with one million nonhematopoietic umbilical cord stem cells into their tail veins.

Ten months after the surgery, the heart structure and function of animals from all three groups was assessed with tensor diffusion magnetic imaging, and a pressure‐volume conductance catheter. The hearts were also extirpated from the animals and structurally assessed by means of staining and 3-D imaging.

The stem cell-treated animals were compared with the sham-operated animals and the saline-treated animals. In almost all categories, the stem cell-treated animals had better function. Also, the overall structure of the heart was preserved and looked more like the normal heart than the saline-treated hearts. For example, in the saline-treated group, the heart wall thickness in the infarct zone was reduced by 50% compared to the control rats, and wall thickness at the border zone was also significantly
decreased. However, there were no statistical difference in wall thickness between the stem cell-treated group and the control group.

Additional finds were that the stem cell-treated group had significantly smaller areas of dead cells, more blood vessels, and better heart muscle fiber structure that contracted better.

These data show that the long-term effects of nh-UCBSC administration was to preserve the structure, and, consequently, the function of the heart after a heart attack.

However, the added bonus to this work is that the animals were injected with these cells into the tail vein. The animals did not have to have their chests cracked, or have over-the-wire stent technology to implant these cells; they merely introduced them intravenously. Apparently, the nh-UCBSCs homed to the damaged heart and mediated its healing. If such healing can be translated to human patients, this could truly be a revolutionary find.

Five-Year Follow-up of REPAIR-AMI Clinical Trial


The REPAIR-AMI clinical trial was a double-blind placebo-controlled trial in which 204 recent heart attach patients received either an infusion of bone marrow stem cells or a placebo. The results of this clinical trial have been published in three different papers (Schächinger, et al., N Engl J Med 2006 355: 1210-1221; Schächinger, et al., Eur Heart Journal 2006 27: 2775-2783; Schächinger, et al., Nat Clin Pract Cardvasc Med 2006 3(Suppl 1): 523-528).

This clinical trial showed that the bone marrow-treated group showed significant functional improvements over the placebo group. However, a long-term follow-up of these patients was required to demonstrate that the benefits conferred by the stem cell treatments were long-lasting and not merely transient.

Upon 5-year examination, the stem cell-treated group showed lower rates of a second heart attack, hospitalization, strokes, cancer, surgical interventions to open blocked vessels and death. Thus, the stem cell-treated group fared better in almost all the major categories.

There was, however, an additional experiment that gave a truly remarkable result. After each patient had their bone marrow extracted, the stem cells were subjected to individual tests, one of which were mobility tests. When this research group examined the stem cell motility data and correlated it to the five-year follow-up, they discovered a very tight association between the motility of the bone marrow stem cells and the absence of cardiac events. More active bone marrow cells provided greater recovery and fewer post-procedural events.

These data show that the quality of the bone marrow is a significant factor in the success of the stem cell treatment.

This also brings up another question: Can be beef up the quality of the bone marrow some how? Culturing stem cells can expand them, but it can also significantly change them. Therefore, this remains a fertile field for research and development, and the bone marrow quality may also explain why bone marrow transplants into the heart work so well or some patients and not at all for others.

New US Phase IIa Trial and Phase III Trial in Kazakhstan Examine CardioCell’s itMSC Therapy to Treat Heart Attack Patients


The regenerative medicine company CardioCell LLC has announced two new clinical trials in two different countries that utilize its allogeneic stem-cell therapy to treat subjects with acute myocardial infarction (AMI), which is a problem that faces more than 1.26 million Americans annually. The United States-based trial is a Phase IIa AMI clinical trial that is designed to evaluate the clinical safety and efficacy of the CardioCell Ischemia-Tolerant Mesenchymal Stem Cells or itMSCs. The second clinical trial in collaboration with the Ministry of Health in Kazakhstan is a Phase III AMI clinical trial on the intravenous administration of CardioCell’s itMSCs. This clinical trial is proceeding on the strength of the efficacy and safety of itMSCs showed in previous Phase II clinical trials.

CardioCell’s itMSCs are exclusively licensed from CardioCell’s parent company Stemedica Cell Technologies Inc. Normally, when mesenchymal stem cells from fat, bone marrow, or some other tissue source are grown in the laboratory, the cells are provided with normal concentrations of oxygen. However, CardioCell itMSCs are grown under low oxygen or hypoxic conditions. Such growth conditions more closely mimic the environment in which these stem cells normally live in the body. By growing these MSCs under these low-oxygen conditions, the cells become tolerant to low-oxygen conditions (ischemia-tolerant), and if transplanted into other low-oxygen environments, they will flourish rather than die.

Another advantage of itMSCs for regenerative treatments over other types of MSCs is that itMSCs secrete higher levels of growth factors that induce the formation of new blood vessels and promote tissue healing. These clinical trials have been designed to help determine if CardioCell’s itMSC-based therapies stimulate a regenerative response in acute heart attack patients.

“CardioCell’s new Phase IIa AMI study is built on the excellent safety data reported in previous Phase I clinical trials using our unique, hypoxically grown stem cells,” says Dr. Sergey Sikora, Ph.D., CardioCell’s president and CEO. “We are also pleased to report that the Ministry of Health in Kazakhstan is proceeding with a Phase III CardioCell-therapy study following its Phase II study that was highly promising in terms of efficacy and safety. Our studies target AMI patients who have depressed left ventricular ejection fraction (LVEF), which makes them prone to developing extensive scarring and therefore to the development of chronic heart failure. CardioCell hopes our itMSC therapies will inhibit the development of extensive scarring and, thus, the occurrence of chronic heart failure in these patients.”

The United States-based Phase IIa clinical trial will take place at Emory University, Sanford Health and Mercy Gilbert Medical Center. The CardioCell Phase IIa AMI trial is a double-blinded, multicenter, randomized study designed to assess the safety, tolerability and preliminary clinical efficacy of a single, intravenous dose of allogeneic mesenchymal bone-marrow cells infused into subjects with ST segment-elevation myocardial infarction (STEMI).

“While stem-cell therapy for cardiovascular disease is nothing new, CardioCell is bringing to the field a new, unique type of stem-cell technology that has the possibility of being more effective than other AMI treatments,” says MedStar Heart Institute’s Director of Translational and Vascular Biology Research and CardioCell’s Scientific Advisory Board Chair Dr. Stephen Epstein. “Evidence exists demonstrating that MSCs grown under hypoxic conditions express higher levels of molecules associated with angiogenesis and healing processes. There is also evidence indicating they migrate with greater avidity to various cytokines and growth factors and, most importantly, home more robustly to ischemic tissue. Studies like those underway using CardioCell’s technology are designed to determine if we can evoke a more potent healing response that will reduce the extent of myocardial cell death occurring during AMI and thereby decrease the amount of scar tissue resulting from the infarct. A therapy that could achieve this would have a major beneficial impact in reducing the occurrence of chronic heart failure.”

Kazakhstan’s National Scientific Medical Center is conducting a Phase III AMI clinical trial using CardioCell’s itMSCs, which are sponsored by local licensee Altaco. This clinical trial is entitled, “Intravenous Administration of itMSCs for AMI Patients,” and is proceeding based on a completed Phase II efficacy and safety study. However, the results of this previous Phase II study are preliminary because the sample group was so small. Despite these limitations, the findings demonstrated statistically significant elevation (more than 12 percent over the control group) in the ejection fraction of the left ventricle of the heart in patients who had received itMSCs. Also, a significant reduction in inflammation was also observed, as ascertained by lower CRP (C-reactive protein) levels in the blood of treated patients in comparison to control groups. Thus, Dr. Daniyar Jumaniyazov, M.D., Ph.D., principal investigator in Kazakhstan clinical trials states: “In our clinical Phase II trial for patients with AMI, treatment using itMSCs improved global and local myocardial function and normalized systolic and diastolic left ventricular filling, as compared to the control group. We are encouraged by these results and look forward to confirming them in a Phase III study.”

CardioCell’s treatment is the first to apply itMSC therapies for cardiovascular indications like AMI, chronic heart failure and peripheral artery disease. Manufactured by CardioCell’s parent company Stemedica and approved for use in clinical trials, itMSCs are manufactured under Stemedica’s patented, continuous-low-oxygen conditions and proprietary media, which provide itMSCs’ unique benefits: increased potency, safety and scalability. itMSCs differ from competing MSCs in two key areas. itMSCs demonstrate increased migratory ability towards the place of injury, and they show increased secretion of growth and transcription factors (e.g., VEGF, FGF and HIF-1), as demonstrated in a peer-reviewed publication (Vertelov et al., 2013). This can potentially lead to improved regenerative abilities of itMSCs. In addition, itMSCs have significantly fewer HLA-DR receptors on the cell surface than normal MSCs, which might reduce the propensity to cause immune responses. As another benefit, itMSCs are highly scalable. A single donor specimen can currently yield about 1 million patient treatments, and this number is expected to grow to 10 million once full robotization of Stemedica’s facility is complete.

Taiwanese Group Identifies Stem Cell-Based Drug to Rejuvenate Aged Hearts


A southern Taiwan-based National Cheng Kung University research team led by Patrick Ching-Ho Hsieh has discovered that a molecule called prostaglandin E2 can regenerate aged hearts in rodents.

This discovery provides a useful new perspective on heart regeneration and presents an effective option for heart disease patients other than heart transplant.

According to Hsieh, congestive heart disease and other cardiovascular diseases are a leading cause of morbidity and mortality throughout the world. There are some six million patients with congestive heart failure in the US alone and some 400,000 in Taiwan. Despite intensive drug, surgical and other medical interventions, 80 percent of all heart patients die within 8 years of diagnosis.

Even though several experiments and clinical trials have established that heart regeneration can take place, the means by which the heart regenerates is still not completely clear, and there are also no drugs to stimulate heart regeneration by the resident stem cell population in the heart.

Now, after seven years of hard work, Hsieh’s team has identified the critical time period and the essential player that directs heart repair.

Hsieh and his colleagues used genetically engineered mice that Hsieh had developed as a postdoctoral research fellow at Harvard Medical School. By using this transgenic mouse strain, Hsieh and others showed that the self-repair process of the heart begins 7 days after injury and peaks at 10 days after injury.

The “director” of this self-repair process is the molecule PGE2. PGE2 regulates heart-specific stem cell activities.

PGE2

“More importantly, both young and old mice have significant improvements for cardiac remodeling if you treat both of them [with] PGE2,” said Hsieh.

Hsieh’s team also established that PGE2 decreases expression of a gene associated with aging, TGF-beta1. PGE2 also rejuvenates the micro-environment of the aged cells, according to Hsieh.

When Is the Best Time to Treat Heart Attack Patients With Stem Cells?


Several preclinical trials in laboratory animals and clinical trials have definitively demonstrated the efficacy of stem cell treatments after a heart attack. However, these same studies have left several question largely unresolved. For example, when is the best time to treat acute heart attack patients? What is the appropriate stem cell dose? What is the best way to administer these stem cells? Is it better to use a patient’s own stem cells or stem cells from someone else?

A recent clinical trial from Soochow University in Suzhou, China has addressed the question of when to treat heart attack patients. Published in the Life Sciences section of the journal Science China, Yi Huan Chen and Xiao Mei Teng and their colleagues in the laboratory of Zen Ya Shen administered bone marrow-derived mesenchymal stromal cells at different times after a heart attack. Their study also examined the effects of mesenchymal stem cells transplants at different times after a heart attack in Taihu Meishan pigs. This combination of preclinical and clinical studies makes this paper a very powerful piece of research indeed.

The results of the clinical trial came from 42 heart attack patients who were treated 3 hours after suffering a heart attack, or 1 day, 3 days, 2 weeks or 4 weeks after a heart attack. The patients were evaluated with echocardiogram to ascertain heart function and magnetic resonance imaging of the heart to determine the size of the heart scar, the thickness of the heart wall, and the amount of blood pumped per heart beat (stroke volume).

When the data were complied and analyzed, patients who received their stem cell transplants 2-4 weeks after their heart attacks fared better than the other groups. The heart function improved substantially and the size of the infarct shrank the most. 4 weeks was better than 2 weeks,

The animal studies showed very similar results.

Eight patients were selected to receive additional stem cell transplants. These patients showed even greater improvements in heart function (ejection fraction improved to an average of 51.9% s opposed to 39.3% for the controls).

These results show that 2-4 weeks constitutes the optimal window for stem cell transplantation. If the transplant is given too early, then the environment of he heart is simply too hostile to support the survival of the stem cells. However, if the transplant is performed too late, the heart has already experiences a large amount of cell death, and a stem cell treatment might be superfluous. Instead 2-4 weeks appears to be the “sweet spot” when the heart is hospitable enough to support the survival of the transplanted stem cells and benefit from their healing properties. Also, this paper shows that multiple stem cell transplants a two different times to convey additional benefits, and should be considered under certain conditions.

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.

New Tool for Stem Cell Transplantation into the Heart


Researchers from the famed Mayo Clinic, in collaboration with scientists at a biopharmaceutical biotechnology company in Belgium have invented a specialized catheter for transplanting stem cells into a beating heart.

This new device contains a curved needle with graded openings along the shaft of the needle. The cells are released into the needle and out through the openings in the side of the needle shaft. This results in maximum retention of implanted stem cells to repair the heart.

“Although biotherapies are increasingly more sophisticated, the tools for delivering regenerative therapies demonstrate a limited capacity in achieving high cell retention in the heart,” said Atta Behfar, the lead author of this study and a cardiologist. “Retention of cells is, of course, crucial to an effective, practical therapy.”

Researchers from the Mayo Clinic Center for Regenerative Medicine in Rochester, MN and Cardio3 Biosciences in Mont-Saint-Guibert, Belgium, collaborated to develop the device. Development of this technology began by modeling the dynamic motions of the heart in a computer model. Once the Belgium group had refined this computer model, the model was tested in North America for safety and retention efficiency.

These experiments showed that the new, curved design of the catheter eliminates backflow and minimizes cell loss. The graded holes that go from small to large diameters decrease the pressures in the heart and this helps properly target the cells. This new design works well in healthy and damaged hearts.

Clinical trials are already testing this new catheter. In Europe, the CHART-1 clinical trial is presently underway, and this is the first phase 3 trial to examine the regeneration of heart muscle in heart attack patients.

These particular studies are the culmination of years of basic science research at Mayo Clinic and earlier clinical studies with Cardio3 BioSciences and Cardiovascular Centre in Aalst, Belgium, which were conducted between 2009 and 2010.  This study, the C-CURE or Cardiopoietic stem Cell therapy in heart failURE study examined 47 patients, (15 control and 32 experimental) who received injections of bone marrow-derived mesenchymal stem cells from their own bone marrow into their heart muscle.  Control patients only received standard care.  After six months, those patients who received the stem cell treatment showed an increase in heart function and the distance they could walk in six minutes.   No adverse effects were observed in the stem cell recipients.

This study established the efficacy of mesenchymal stem cell treatments in heart attack patients.  However, other animal and computer studies established the efficacy of this new catheter for injecting heart muscle with stem cells.  Hopefully, the results of the CHART-1 study will be available soon.

Postscript:  The CHART-2 clinical trial is also starting.  See this video about it.

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.”

Nanotubules Link Damaged Heart Cells With Mesenchymal Stem Cells to Both of Their Benefit


Mesenchymal stem cells are found throughout the body in bone marrow, fat, tendons, muscle, skin, umbilical cord, and many other tissues. These cells have the capacity to readily differentiate into bone, fat, and cartilage, and can also form smooth muscles under particular conditions.

Several animal studies and clinical trials have demonstrated that mesenchymal stem cells can help heal the heart after a heart attack. Mesenchymal stem cells (MSCs) tend to help the heart by secreting a variety of particular molecules that stimulate heart muscle survival, proliferation, and healing.

Given these mechanisms of healing, is there a better way to get these healing molecules to the heart muscle cells?

A research group from INSERM in Creteil, France has examined the use of tunneling nanotubes to connect MSCs with heart muscle cells. These experiments have revealed something remarkable about MSCs.

Florence Figeac and her colleagues in the laboratory of Ann-Marie Rodriguez used a culture system that grew fat-derived MSCs and with mouse heart muscle cells. They induced damage in the heart muscle cells and then used tunneling nanotubes to connect the fat-based MSCs.

They discovered two things. First of all, the MSCs secreted a variety of healing molecules regardless of their culture situation. However, when the MSCs were co-cultured with damaged heart muscle cells with tunneling nanotubes, the secretion of healing molecules increased. The tunneling nanotubes somehow passed signals from the damaged heart muscle cells to the MSCs and these signals jacked up secretion of healing molecules by the MSCs.

The authors referred to this as “crosstalk” between the fat-derived MSCs and heart muscle cells through the tunneling nanotubes and it altered the secretion of heart protective soluble factors (e.g., VEGF, HGF, SDF-1α, and MCP-3). The increased secretion of these molecules also maximized the ability of these stem cells to promote the growth and formation of new blood vessels and recruit bone marrow stem cells.

After these experiments in cell culture, Figeac and her colleagues used these cells in a living animal. They discovered that the fat-based MSCs did a better job at healing the heart if they were previously co-cultured with heart muscle cells.

Exposure of the MSCs to damaged heart muscle cells jacked up the expression of healing molecules, and therefore, these previous exposures made these MSCs better at healing hearts in comparison to naive MSCs that were not previously exposed to damaged heart muscle.

Thus, these experiments show that crosstalk between MSCs and heart muscle cells, mediated by nanotubes, can optimize heart-based stem cells therapies.

Phase 2 Clinical Trial that Tests Stem Cell Treatment for Heart Attack Patients to be Funded by California Institute for Regenerative Medicine


A new stem cell therapy that treats heart attack patients with cells from a donor has been approved to begin a Phase 2 clinical trial.

Capricor Therapeutics Inc. a regenerative medicine company, has developed this treatment, which extracts donor stem cells from the heart called “cardiosphere-derived cells,” and then infuses them into the heart of the heart attack patient by means of a heart catheter procedure, which is quite safe. These stem cells are introduced into the heart to reduce scarring in the heart and potentially replace dead heart muscle cells. One clinical trial called the CADUCEUS trial has already shown that cardiosphere-derived cells can reduce the size of the heart scar.

In a previous phase I study (phase I studies typically only ascertain the safety of a treatment), cardiosphere-derived cells were infused into the hearts of 14 heart attack patients. No major safety issues were observed with these treatments, and therefore, phase 2 studies were warranted.

Alan Trounson, Ph.D., president of the California Institute for Regenerative Medicine (CIRM), which is funding the trial, said this about the phase 2 trial approval: “This is really encouraging news and marks a potential milestone for the use of stem cells to treat heart disease. Funding this type of work is precisely what our Disease Team Awards were designed to do, to give promising treatments up to $20 million dollars to develop new treatments for some of the deadliest diseases in America.”

Capricor was given approval by the National Heart Lung and Blood Institute (NHLBI) Gene and Cell Therapy (GST) to move into the next phase of clinical trials after these regulatory bodies had thoroughly reviewed the safety data from the phase 1 study. After NHLBI and GST determined that the phase 1 study met all the required goals, CIRM also independently reviewed the safety data from the Phase 1 and other aspects of the Phase 2 clinical trial design and operations. Upon successful completion of the independent review, Capricor was given approval to move forward into the CIRM-funded Phase 2 component of the study

Capricor CEO Linda Marbán, Ph.D., said, “Meeting the safety endpoints in the Phase 1 portion of the trial is a giant leap forward for the field and for Capricor Therapeutics. By moving into the Phase 2 portion of this trial, we can now attempt to replicate the results in a larger population.”

For the next phase, an estimated 300 patients who have had heart attacks will be evaluated in a double-blind, randomized, placebo-controlled trial. One group of heart-attack patients will include people 30 to 90 days following the heart attack, and a second group will follow patients 91 days to one year after the incident. Other patients will receive placebos and neither the patients nor the treating physicians know who will receive what.  This clinical trial should definitely determine if an “off-the-shelf” stem cell product can improve the function of a heart attack patient’s heart.

The California Institute for Regenerative Medicine (CIRM) is funding this clinical trial, and for this CIRM should be lauded.  However, when CIRM was brought into existence through the passage of proposition 71, it sold itself as a state-funded entity that would deliver embryonic stem cell-based cures.  Now I know that director Alan Trounson has denied that, but Wesley Smith at the National Review “Human Exceptionalism” blog and the LA times blogger Michael Hiltzik have both documented that Trounson and others said exactly that.  Isn’t ironic that one of the promises intimated by means of embryo-destroying research is now being fulfilled by means of non-embryo-destroying procedures?  If taxpayer money is going to fund research like this, then I’m all for it, but CIRM has to first clean up its administrative act before they deserve a another penny of taxpayer money.

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.

Encapsulation of Cardiac Stem Cells and Their Effect on the Heart


Earlier I blogged about an experiment that encapsulated mesenchymal stem cells into alginate hydrogels and implanted them into the hearts of rodents after a heart attack. The encapsulated mesenchymal stem cells showed much better retention in the heart and survival and elicited better healing and recovery of cardiac function than their non-encapsulated counterparts.

This idea seems to be catching on because another paper reports doing the same thing with cardiac stem cells extracted from heart biopsies. Audrey Mayfield and colleagues in the laboratory of Darryl Davis at the University of Ottawa Heart Institute and in collaboration with Duncan Steward and his colleagues from the Ottawa Hospital Research Institute used cardiac stem cells extracted from human patients that were encased in agarose hydrogels to treat mice that had suffered heart attacks. These experiments were reported in the journal Biomaterials (2013).

Cardiac stem cells (CSCs) were extracted from human patients who were already undergoing open heart procedures. Small biopsies were taken from the “atrial appendages” and cultured in cardiac explants medium for seven days.

atrial appendage

Migrating cells in the culture were harvested and encased in low melt agarose supplemented with human fibrinogen. To form a proper hydrogel, the cells/agarose mixture was added drop-wise to dimethylpolysiloxane (say that fast five times) and filtered. Filtration guaranteed that only small spheres (100 microns) were left. All the larger spheres were not used.

Those CSCs that were not encased in hydrogels were used for gene profiling studies. These studies showed that cultured CSCs expressed a series of cell adhesion molecules known as “integrins.” Integrins are 2-part proteins that are embedded in the cell membrane and consist of an “alpha” and “beta” subunit. Integrin subunits, however, come in many forms, and there are multiple alpha subunits and multiple beta subunits.

integrin-actin2

This mixing and matching of integrin subunits allows integrins to bind many different types of substrates. Consequently it is possible to know what kinds of molecules these cells will stick to based on the types of integrins they express. The gene prolifing experiments showed that CSC expressed integrin alpha-5 and the beta 1 and 3 subunits, which shows that CSC can adhere to fibronectin and fibrinogen.

fibronectin

fibrinogen-cleave

When encapsulated CSCs were supplemented with fibrinogen and fibronectin, CSCs showed better survival than their unencapsulated counterparts, and grew just as fast ans unencapsulated CSCs. Other experiments showed that the encapsulated CSCs made just as many healing molecules as the unencapsulated CSCs, and were able to attract circulating angiogenic (blood vessel making) cells. Also, the culture medium of the encapsulated cells was also just as potent as culture medium from suspended CSCs.

With these laboratory successes, encapsulated CSCs were used to treat non-obese diabetic mice with dysfunctional immune systems that had suffered a heart attack. The CSCs were injected into the heart, and some mice received encapsulated CSCs, other non-encapsulated CSCs, and others only buffer.

The encapsulated CSCs showed better retention in the heart; 2.5 times as many encapsulated CSCs were retained in the heart in comparison to the non-encapsulated CSCs. Also, the ejection fraction of the hearts that received the encapsulated CSCs increased from about 35% to almost 50%. Those hearts that had received the non-encapsulated CSCs showed an ejection fraction that increased from around 33% to about 39-40%. Those mice that had received buffer only showed deterioration of heart function (ejection fraction decreased from 36% to 28%). Also, the heart scar was much smaller in the hearts that had received encapsulated CSCs. Less than 10% of the heart tissue was scarred in those mice that received encapsulated CSCs, but 16% of the heart was scarred in the mice that received free CSCs. Those mice that received buffer had 20% of their hearts scarred.

Finally, did encapsulated CSCs engraft into the heart muscle? CSCs have been shown to differentiate into heart-specific tissues such as heart muscle, blood vessels, and heart connective tissue. Encapsulation might prevent CSCs from differentiating into heart-specific cell types and connecting to other heart tissues and integrating into the existing tissues. However, at this point, w have a problem with this paper. The text states that “encapsulated CSCs provided a two-fold increase in the number of engrafted human CSCs as compared transplant of non-encapsulated CSCs.” The problem is that the bar graft shown in the paper shows that the non-encapsulated CSCs have twice the engraftment of the capsulated CSCs. I think the reviewers might have missed this one. Nevertheless, the other data seem to show that encapsulation did not affect engraftment of the CSCs.

The conclusion of this paper is that “CSC capsulation provides an easy, fast and non-toxic way to treat the cells prior to injection through a clinically acceptable process.”

Hopefully large-animal tests will come next. If these are successful, then maybe human trials should be on the menu.

The Use of Synthetic Messenger RNAs Augment Heart Regeneration and Healing After a Heart Attack


A collaborative effect between researchers at Harvard University and Karolinska Institutet has shown that the application of particular factors to the heart after a heart attack can heal the heart and induce the production of new heart muscle.

Kenneth Chien, who has a dual appointment at the medical university Karolinska Institutet and Harvard University, led this research teams said this about this work: “This is the beginning of using the heart as a factory to produce growth factors for specific families of cardiovascular stem cells, and suggests that it may be possible to generate new heart parts without delivering any new cells to the heart itself.”

This study builds upon previous work by Chien and his colleagues in which the growth factor VEGFA, which is known to activate the growth of endothelial cells in the adult heart (endothelial cells line blood vessels), also serves as a switch that converts heart stem cells away from making heart muscle to forming coronary vessels in the fetal heart.

To drive the expression of VEGFA in the heart, Chien and others made synthetic messenger RNAs that encoded VEGFA and injected them into the heart cells. Injections of these synthetic VEGFA messenger RNAs produced a short burst of VEGFA.

Chien induced a heart attack in mice and then administered the synthetic VEGFA messenger RNAs to some mice and buffer to others 48 hours after the heart attacks. Chien and his crew was sure to inject the synthetic VEGFA mRNAs into the regions of the heart known to harbor the resident cardiac stem cell populations.

Not only did the VEGFA-mRNA-injected mice survive better than the other mice, but their hearts had smaller heart scars, and had clear signs of the growth of new heart muscle that had been made by the resident cardiac stem cell populations. One pulse of VEGFA had long-term benefits and those cells that would have normally made the heart scar ended up making heart muscle instead as a result of one pulse of VEGFA.

Chien said of this experiment, “This moves us very close to clinical studies to regenerate cardiovascular tissue with a single chemical agent without the need for injecting any additional cells into the heart.”

At the same time, Chien also noted that this technology is in the early stages of development. Even though these mice had their chests cracked open and their hearts injected, for human patients, the challenge is to adapt heart catheter technologies to the delivery of synthetic messenger RNAs. Also, to demonstrate the safety and efficacy of this technology to humans, Chien and others will need to repeat these experiments in larger animals that serve as a better model system for the human heart than rodents. Chien’s laboratory is presently in the process of doing that.

To adapt catheter technology to deliver these reagents, Chien had co-founded a company called Moderna Therapeutics to research this problem and develop the proper platform technology for clinical use. Chien is also collaborating with the biotechnology company AstraZeneca to help expedite moving the synthetic RNA technology into a clinical setting.

Remote Ischemic Conditioning Enhances Stem Cell Retention in the Heart


Stem cell administration to the heart after a heart attack is a difficult venture. Direct injection into the heart muscle is definitely the most sure-fire way to get stem cells into the heart tissue. However, direct injection requires that the physician crack the patient’s chest (thoracotomy), which is exquisitely unpleasant for the patient. Alternatively, there are devices that an deliver stem cell injections into the heart through the large veins in the legs, but these procedures require special equipment and lots of skills that your average cardiologist does not have. Another way is to administer stem cells through angioplasty. Using the same procedure as stent implantation, a delivery device is replaced at the site of heart damage through over-the-wire angioplasty technology, and the stem cells are delivered slowly and gradually through the coronary blood vessels. This does not require fancy equipment, and your average cardiologist could perform this technique pretty easily.

Problems exist with both procedures. Direct injection places cells and fluid into the heart wall and there is a risk of rupture. Likewise, with over-the-wire delivery of stem cells, there is the risk of clogging the coronary artery.

With both techniques, many stem cells end up in places other than the heart. In fact, the majority of the stem cells end up somewhere else – the lungs and liver mostly. Is there a better way?

Intravenous administration would be sweet, but that has been tried and the bottom line is that it bombed (Barbash et al., Circulation. 2003 19;108(7):863-8; Freyman et al., Eur Heart J. 2006 May;27(9):1114-22).

Well, some very enterprising scientists from China had an idea to get the intravenously administered stem cells to go to the heart and stay there. Bone marrow stem cells respond to a molecule called SDF1alpha (stromal cell derived factor-1alpha). On their cell surfaces, bone marrow cells have a receptor called CXCR4 which binds the SDF1alpha and bone marrow cells move towards higher and higher concentrations of SDF1alpha. Therefore, can you get the heart to make more SDF1alpha?

Sure. You can genetically engineer it to make more SDF1alpha. If you do that, the stem cells will pour out of the bone marrow and go to the heart and help fix it (Sundararaman S et al., Gene Ther. 2011 18(9):867-73). However, is there another way to get more SDF1alpha in the heart?

Yes there is. Let me introduce Remote Ischemic Conditioning or RIC. RIC increases the protection against injury that results from loss of blood flow to an organ. The way RIC works is that the blood supply to another organ is clamped so that this other organ is deprived of oxygen long enough to sound the alarm, but not long enough to do it serious damage. This deprivation of oxygen induces a flash of SDF1alpha production, which brings stem cells from bone marrow to the bloodstream and to the damaged organ.

Qin Jiang and colleagues from the Peking Union Medical College in Beijing, China used RIC in animals that had undergone a heart attack to determine if RIC could recruit more stem cells to the heart. Also, they administered bone marrow stem cells intravenously to see if RIC increased stem cell retention in the heart.

Jiang and others broke their laboratory rats into three groups (it gets a little complicated).

The first group was given heart attacks and then split into two subgroups. One subgroup received RIC and the second subgroup received surgery but no RIC.

The second group was given a heart attack and then split into six subgroups. Once subgroup was given RIC and intravenous bone marrow mesenchymal stem cells. the second received bone marrow mesenchymal stem cells by no RIC, only the incision, the third subgroup only received intravenous mesenchymal stem cells, the fourth group received RIC and intravenous saline, the fifth subgroup received no RIC, only an incision and intravenous saline, and the sixth subgroup received only intravenous saline.

The third group was given heart attacks and then split into two groups, one of which received RIC, intravenous mesenchymal stem cells and intravenous antibodies against CXCR4, and the other of which received RIC, mesenchymal stem cells and an antibody against nothing in particular.

The results showed that RIC GREATLY increased the amount of SDF1alpha in the heart. There was simply no getting around this. At 1 hour after RIC, SDF1alpha and VEGF (vascular endothelial growth factor) levels were up, but these levels decreased by 3 hours and back to normal by 6 hours after RIC.

Did these increased SDF1alpha levels increase stem cell retention? Oh yes!! The RIC-treated rats had almost twice the number of stem cells in their hearts than the animals that did not have RIC. Did this make a functional difference? Again, yes! The RIC-treated animals had hearts that functioned more normally (relatively speaking) than hearts from the non-RIC-treated animals.

The third experiment was even more informative, since the co-administration of the CXCR4 antibody abrogated the response induced by RIC. This demonstrates that effects of RIC are mediated by the SDF1alpha/CXCR4 axis and blocking this signaling axis prevented any benefits from RIC.

This paper is short, but very informative. It suggests that a relatively simple procedure like RIC could potentially improve the clinical efficacy of stem cell treatments. If this can be shown to work in larger animals, then clinical trials might be warranted. In fact clinical trials are presently underway in which SDF1alpha is being engineered into the heart to treat heart attack patients (see Hajjar RJ, Hulot JS. Circ Res. 2013 Mar 1;112(5):746-7).

Mesenchymal Stem Cells from Umbilical Cord Promote Repair After Heart Attacks in Minipigs


The umbilical cord contains a major umbilical vein and an umbilical artery, but these blood vessels are embedded in a gel-like matrix called “Wharton’s jelly.” Wharton’s jelly is home to a population of mesenchymal stem cells that have peculiar properties.

You might first say, “what on earth is a mesenchymal stem cell?” Fair enough. Mesenchymal stem cells were first discovered in bone marrow. In bone marrow, mesenchymal stem cells (MSCs) do not make blood cells; that;’s the job of the hematopoietic stem cells (HSCs). MSCs in bone marrow serve an important support role for HSCs in bone marrow. Traditionally, MSCs have the capacity to differentiate into fat cells, bone cells, and cartilage cells. However, further has shown that MSCs can also form a variety of other cell types as well if manipulated in the laboratory. MSCs also express are characteristic cadre of cell surface proteins (CD10, CD13, CD29, CD44, CD90, and CD105 for those who are interested).

MSCs, however, are found in more places that just bone marrow. As it turns out, MSCs have been found in fat, muscle, liver, tendons, synovial membrane (the membranes that surround joints, skin, and so on. Some scientists think that every organ in the body may harbor a MSC population. Furthermore, these MSC populations differ in the genes they express, their capability to differentiate into different cell types, and their cell surface proteins (see this article on this website for a rather exhaustive foray into this topic).

Now that you are more savvy about MSCs, Wharton’s jelly contains a MSC population, but this population seems to have a younger profile than MSCs from other parts of the body.  They are more plastic and more invisible to the immune system than other types of MSCs.  For that reason,  they might be good candidates for treating a sick heart after a heart attack.  A recent paper by Wei Zhang and others from the TEDA International Cardiovascular Hospital and the Tianjin Medical Cardiovascular Clinical College examined the ability of MSCs from the Wharton’s jelly of human umbilical cords to heal the hearts of minipigs after a heart attack.  Oh, before I forget – this paper was published in the journal Coronary Artery Disease.

Twenty-three minipigs were subjected to open-heart surgery and given heart attacks.  Then the pigs were divided into three groups, a control group, a group that received injections of saline into their hearts, and a third group that received injections of 40 million human Wharton’s jelly derived MSCs into the region of the infarct.  The animals were sewn up and given antibiotics to prevent infection.

Six weeks after surgery, each animal was examined by means of Technetium-sestamibi myocardial perfusion imaging, and electrocardiography. For those who do not know what Technetium-sestamibi myocardial perfusion imaging is for, it works like this.  Cardiolite is the trade name of a large, fat-soluble molecule that flows through the heart in a fashion proportion to the blood flow through the heart muscle.  Single photon emission computed tomography or SPECT is used to detect the Cardiolite.    Areas of the heart without blood flow are the regions damaged during the heart attack.  Therefore, this technique is extremely useful to determine the area of damage in the heart.

Cardiolite
Cardiolite

After the animals were examined, they were put down and their hearts were extracted, sectioned, and stained for areas or cell death, and the areas where the injected stem cells resided.  All injected stem cells were labeled before injection so that they were easily detectable.

The results were clear.  The heart injected with MSCs from umbilical cord did not show any decrease in ejection fraction, whereas the other two groups showed an average reduction in injection fraction of around 10%.  In fact the stem cell-injected hearts showed an average 1 % increase in ejection fraction.  The blood flow in the hearts was even more different.  blood flow is measured as a ratio of dead heart tissue to total heart tissue.  The control of saline-injected hearts had an average ratio of about 4%, whereas the stem cell-injected hearts had a slightly negative percentage.  This is a significant difference.   Echocardiography confirmed that the wall thickness of the stem cell-injected hearts was significantly thicker than the walls of the control or the saline-injected hearts; some 14 times thicker!!

When the dissected hearts were examined, the MSC-injected hearts had lots of stem cells still in them.  The cells not only survived, but, according to Zhang and his colleagues, differentiated into heart muscle cells.  Their rationale for this conclusion is three-fold – the cells had the same shape and form or native heart muscle cells, they expressed heart specific Troponin T and vWF proteins, and electrically coupled with other heart muscle cells by expressing connexin.  Connexin is a protein that traverses the membranes of two closely apposed cells and forms small pores between two cells that allows the exchange of SMALL molecules such as ions, ATP, and things like that.  These connexin constructed pores are called “gap junctions” and they are the reason heart muscle cells work as a single unit, since any electrochemical change in one cell immediately spreads to all other nearby, connected cells.

gap junctions

As much as I would like to believe Zhang and his colleagues, I remain skeptical that these cells differentiated into heart muscle cells.  I say this because MSCs can be differentiated in culture to form cells that look and act like heart muscle cells.  These cells will even express some heart-specific genes.  However, they lack the calcium handling machinery of true heart muscle cells and do not function as true heart muscle.  To convince that these Wharton jelly MSCs truly are heart muscle cells, they will need to show that they contain heart specific calcium handling proteins (see Shake JG, Gruber PJ, Baumgartner WA et al. Ann Thorac Surg 2002;73:1919–1925; Davani S, Marandin A, Mersin N et al.  Circulation 2003;108(suppl 1):II253–258; Hou M, Yang KM, Zhang H et al. Int J Cardiol 2007;115:220 –228).  If they can show this, then I will believe them.

However, there are two findings of this paper that are not in doubt.  The number of blood vessels in the hearts of the MSC-treated animals far exceeded the number found in the control or the saline-treated hearts (3-4 times the number of blood vessels).  Therefore, the Wharton’s jelly MSCs induced lots and lots of blood vessels.  Many of these blood vessels contained labeled cells, which shows that the MSCs differentiated into endothelial and smooth muscle cells, Also, the Wharton’s jelly MSCs clearly induced resident cardiac stem cell (CSC) populations in the hearts of the minipigs, since several cells that expressed CSC surface molecules were found in the heart muscle tissue.  Previous work by Hatzistergos and others showed that MSCs induce the endogenous CSC population and this is one of the ways that MSCs help heal ailing hearts (Circulation Research 2010 107:913-22).

Zhang’s paper is interesting and it shows that Wharton’s jelly MSCs are safe and efficacious for treating the heart after a heart attack.  Also, none of the minipigs in this experiment were treated with drugs to suppress the immune system.  No immune response against the cells was reported.  Therefore, the invisibility of these cells to the immune system seems to last, at least in this experiment.