Heart Function Improved by Injecting Discarded Surgery Fat


Many patients with heart problems – such as heart disease or angina – may need to undergo cardiac surgery in order to restore or improve blood flow. But a new study suggests that the procedure may offer so much more; stem cells in fat discarded during cardiac surgery could be injected back into the patient’s heart to further improve its function.

A research team led by senior author Canadian cardiologist Dr. Ganghong Tian will present their findings at the Frontiers in Cardiovascular Biology meeting in Barcelona, Spain.

Previous work by this group has shown that subcutaneous fat (adipose tissue) contains stem cells that can reduce the severity of heart attacks, improve cardiac function, and augment blood vessel regeneration in laboratory animals with experimentally induced heart attacks. These fat-based stem cells can be easily obtained through liposuction. However, Tian noted, “But obtaining these from a patient undergoing cardiac surgery requires pre-surgery to collect adipose tissue from the subcutaneous region.”

Is there a better way? According to Tian, during cardiac surgery, the surgeon often removes fat tissue that resides around the heart (so-called mediastinal fat) in order to properly expose the heart. Tian wondered if this fat contain stem cells that could be re-introduced to the heart to improve its function after heart surgery

In order to test this hypothesis, Tian and others collected mediastinal fat tissue from 24 patients who had undergone cardiac surgery. Then Tian’s group injected rats with mediastinal fat stem cells. The rats injected with stem cells from mediastinal fat showed greater ventricular movement in their hearts and no reduction in left ventricular ejection fraction.

Closer examination of the stem cells from mediastinal fat showed that mediastinal fat housed a rather robust number of stem cells, and that these stem cells could differentiate into fat and bone cells. Also, these stem cells expressed genes that are often found in heart muscle cells.

With this pre-clinical information in hand, Tian and others examined the use of mediastinal fat-based stem cells in 13 rats with congestive heart failure. These stem cells were directly injected into the hearts of eight rats, and five were injected with a saline solution.

After 6 weeks, all the rats underwent magnetic resonance imaging (MRI). When the five control rats were compared with those who those rats that received injections of mediastinal fat-based stem cells, the stem cell-injected rats demonstrated greater ventricular movement in their hearts and no reduction in left ventricular ejection fraction (ejection fraction measures how much blood is being pumped out of the left ventricle of the heart).

Commenting on the team’s findings, Dr. Tian says: “This is the first evidence that stem cells collected from the mediastinal fat region are cardioprotective. They displayed the same cardioprotective capacity we found in our previous research on stem cells from subcutaneous fat tissue. This raises the exciting possibility of using a patient’s own stem cells, isolated from waste tissue during cardiac surgery, to improve their heart function.”

Tian noted that there are currently some issues with this procedure that need to be addressed with further research. Techniques must be developed to quickly isolate stem cells from mediastinal fat so they can be injected back into a patient’s heart during cardiac surgery. Tian said, “It currently takes several hours to purify the cells and we are looking for collaborators to help us devise a more efficient method.”

Tina and others would also like to examine the ability of these stem cells to improve cardiac function long-term, beyond the 6 weeks monitored in this study. Furthermore, Tian and his group would like to induce the stem cells into functional heart muscle cells that display electrical pulses and beating.

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.

Three New Clinical Trials Examine Bone Marrow-Based Stem Cells To Treat Heart Failure


In April of 2013, the results of three clinical trials that examined the effects of bone marrow-derived stem cell treatments in patients with acute myocardial infarction (translation – a recent heart attack) or chronic heart failure. These trials were the SWISS-AMI trial, the CELLWAVE trial, and the C-CURE trial.

The SWISS-AMI trial (Circulation. 2013;127:1968-1979), which stands for the Swiss Multicenter Intracoronary Stem Cells Study in Acute Myocardial Infarction trial, was designed to examine the optimal time of stem cell administration at 2 different time points: early or 5 to 7 days versus late or 3 to 4 weeks after a heart attack. This trial is an extension of the large REPAIR-AMI, which showed that patients who tended to receive bone marrow stem cell treatments later rather than earlier had more pronounced therapeutic effects from the stem cell treatments.

SWISS-AMI examined 60 patients who received standard cardiological care after a heart attack, 58 who received bone marrow stem cells 5-7 days after a heart attack, and 49 patients who received bone marrow stem cells 3-4 weeks after their heart attacks. All stem cells were delivered through the coronary arteries by means of the same technology used to deliver a stent.

When the heart function of all three groups were analyzed, no significant differences between the three groups were observed. Those who received stem cell 5-7 days after a heart attack showed a 1.8% increase in their ejection fractions (the percentage of blood that is ejected from the ventricle with each beat) versus an average decrease of 0.4% in those who received standard care, and a 0.8% increase in those who received their stem cells 3-4 weeks after a heart attack. If these results sound underwhelming it is because they are. The standard deviations of each group so massive that these three groups essentially overlap each other. The differences are not significant from a statistical perspective. Thus the results of this study were definitely negative.

The second study, CELLWAVE (JAMA, April 17, 2013—Vol 309, No. 15, 1622-1631), was a double-blinded, placebo-controlled study conducted among heart attack patients between 2005 and 2011 at Goethe University Frankfurt, Germany. In this study, the damaged area of heart was pretreated with low-energy ultrasound shock waves, after which patients in each group were treated with either low dose stem cells, high-dose stem cells, or placebo. Patients also received either shock wave treatment or placebo shock wave treatment. Thus this was a very well-controlled study. Stem cells were administered through the coronary arteries, just as in the case of the SWISS-AMI study.

The results were clearly positive in this study. The stem cell + shock wave treatment groups showed definite increases in heart function above the placebo groups, and showed fewer adverse effects. The shock wave treatments seem to prime the heart tissue to receive the stem cells. The shock waves induce the release of cardiac stromal-derived factor-1, which is a potent chemoattractor of stem cells.  This is an intriguing procedure that deserves more study.

The third study, C-CURE, is definitely the most interesting of the three (Bartunek et al. JACC Vol. 61, No. 23, June 11, 2013:2329–38). In this trial, mesenchymal stromal cells (MSCs) were isolated from bone marrow and primed with a cocktail of chemicals that pushed the stem cells towards a heart muscle fate. Then the cells were transplanted into the heart by direct injection into the heart muscle as guided by NOGA three-dimensional imaging of the heart.

After initially screening 320 patients with chronic heart failure, 15 were treated with standard care and the other 32 received the stem cell treatment. After a two-year follow-up, the results were remarkable: those who received the stem cell treatment showed an average 7% increase in ejection fraction versus 0.2% for receiving standard care, an almost 25 milliliter reduction in end systolic volume (measures degree of dilation of ventricle – not a good thing and the fact that it decreased is a very good thing) versus a 9 milliliter decrease for those receiving standard care, and were able to walk 62 meters further in 6 minutes as opposed to standard care group who walked 18 meters less in 6 minutes.

While these studies do not provide definitive answers to the bone marrow/heart treatment debate, they do extend the debate. Clearly bone marrow stem cells help some patients and do not help others. The difference between these two groups of patients continues to elude researchers. Also, how the bone marrow is processed is definitely important. When the cells are administered also seems to be important, but the exact time slot is not clear in human patients. It is also possible that some patients have poor quality bone marrow in the first place, and might be better served by allogeneic (someone else’s stem cells) treatments rather than autologous (the patient’s own stem cells) stem cell treatments.

Also, stem cell treatments for heart patients will probably need to be more sophisticated if they are to provide greater levels of healing. Heart muscle cells are required, but so are blood vessels to feed the new heart muscle. If mesenchymal stem cells work by activating resident heart stem cells, then maybe mesenchymal transplants should be accompanied by endothelial progenitor cell transplants (CD117+, CD45+ CD31+ cells from bone marrow) to provide the blood vessels necessary to replace the clogged blood vessels and the new heart muscle that is grown.

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.

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.

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.

Reducing the Heart Scar After a Heart Attack


After a heart attack, inflammation in the heart kills off heart muscle cells and fibroblasts in the heart make a protein called collagen, which forms a heart scar. The heart scar does not contract and does not conduct electrochemical signals. The scar will contract over time, but its presence can lead to abnormal heart rhythms, also known as arrhythmias. Arrythmias can be fatal, since they can cause a heart attack. To prevent a heart attack, physicians will treat heart attack patients with a group of drugs called beta-blockers that slow down the heart rate and protect the heart from the deleterious effects of norepinephrine (secreted by the sympathetic nerve inputs to the heart). An alternative treatment is digoxin or digitalis, which is a chemical found in foxglove. Digitalis inhibits ion pumps in heart muscle cells and slows the heart and the force of its contractions. Digitalis, however, interacts with a whole shoe box fill of drugs, has a very long half-life, and is hard to dose. Therefore it is not the first choice.

Given all this, helping the heart to make a smaller heart scar is a better strategy for treating a heart after a heart attack. To accomplish this, you need to inhibit the heart fibroblasts that make the heart scar in the first place. Secondly, you must move something into the place of the dead cells. Otherwise, the heart could burst or scar tissue will move into the area anyway.

To that end, Yigang Wang and his colleagues at the University of Cincinnati Medical Center in Ohio have published an ingenious paper in which they tried two different strategies to reduce the size of the heart scar, which concomitantly increased the colonization of the heart by induced pluripotent stem cells engineered to express a sodium-calcium exchange pump.

Previously, Wang and his colleagues used a patch to heal the heart after a heart attack. The patch consisted of endothelial cells, which make blood vessels, induced pluripotent stem cells engineered to make a sodium-calcium exchange pump called NCX1, and embryonic fibroblasts. This so-called tri-cell patch makes new blood vessels, establishes new heart muscle, and the foundational matrix molecules to form a platform for beating heart muscle.

In order to get these cells to spread throughout the injured heart, Wang and others used a reagent that specifically inhibits heart fibroblasts. They used a small non-coding RNA molecule. A group of microRNAs called miR-29 family are downregulated after a heart attack. As it turns out, these microRNAs inhibit a group of genes that involved in collagen deposition. Therefore, by overexpressing miR-29 microRNAs, they could prevent collagen deposition and reduce scar formation.

The experimental design in this paper is rather complex. Therefore, I will go through it slowly. First, they tried to overexpress miR-29 microRNAs in cultured heart fibroblasts and sure enough, they inhibited collagen synthesis. Cells overexpressing miR-29 made less than a third of the collagen of their normal counterparts. When they placed these fibroblasts into the heart and induced heart attacks, again, they made significantly less collagen when they were expressing miR-29.

Then they used their miR-29 RNAs by injecting them directly into the heart before inducing a heart attack, and then after the heart attack, they applied the tri-patch. Their results were significant. The scar size was smaller (almost one-third the size of the controls), and the density of blood vessels was much higher in the tri-patched hearts treated with miR-29. The induced pluripotent stem cells differentiated into heart muscle cells and spread throughout the heart. Heart function measures also consistently went up too.  The echiocardiograph before more normal, the ejection fraction went up, the % shortening of the heart muscle fibers was increased, and the relaxation phase of the heart (diastole) also was not so puffy (see graphs and figures below).

(A): M-mode echocardiograph data in three groups. (B): Quantification analysis for heart function. Quantitative data for LVDd (B-1), LVDs (B-2), EF (B-3), and FS (B-4) 4 weeks after Tri-P implantation. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. Anti-29b+MI+Tri-P group. LVDd, left ventricular enddiastolic diameters; LVDs, left ventricular end-systolic diameters; EF, ejection fraction index; FS, fractional shortening. All values expressed as mean 6 SEM. n = 6 for each group. (C): Two-D mode echocardiograph data in three groups, analyzed by long-axis and short-axis views. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. miR-29b+MI+Tri-P group. Ctrl, control mimic pretreatd rat with Tri-cell patch graft; miR-29b, miR- 29b mimic pretreated rat with Tri-cell patch graft; Anti-29b, miR-29b inhibitor pretreated rat with Tri-cell patch graft. White dotted lines indicate endocardium and epicardium.
(A): M-mode echocardiograph data in three groups. (B): Quantification analysis for heart function. Quantitative data for LVDd (B-1), LVDs (B-2), EF (B-3), and FS (B-4) 4 weeks after Tri-P implantation. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. Anti-29b+MI+Tri-P group. LVDd, left ventricular enddiastolic diameters; LVDs, left ventricular end-systolic diameters; EF, ejection fraction index; FS, fractional shortening. All values expressed as mean 6 SEM. n = 6 for each group. (C): Two-D mode echocardiograph data in three groups, analyzed by long-axis and short-axis views. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. miR-29b+MI+Tri-P group. Ctrl, control mimic pretreatd rat with Tri-cell patch graft; miR-29b, miR-29b mimic pretreated rat with Tri-cell patch graft; Anti-29b, miR-29b inhibitor pretreated rat with Tri-cell patch graft. White dotted lines indicate endocardium and epicardium.

There is a cautionary note to this study. Inhibiting collagen formation after a heart attack could create soft fragile regions of the heart that are subject to rupture should the vascular systolic pressure increase. While that threat was not observed in this study, human hearts, which are much larger, would be much more susceptible to such a mishap. Therefore, while this study is interesting and suggest a strategy in humans, it requires more testing and refinement before anyone can even think about applying it to humans.

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.

For Treating Heart Attacks, Satellite Cells Lacking MyoD are Superior to Those With MyoD


Atsushi Asakura and his colleagues at the University of Minnesota Stem Cell Institute have extended some of their earlier findings in a paper that appeared in PLoS One last year. This paper is almost a year old by now, but its results are fascinating and are definitely worth examining.

In 2007, Asakura published a paper with the Canadian researcher Michael A. Rudnicki in the Proceedings of the National Academy of Sciences. In this paper, Asakura and his colleagues examined the ability of muscle satellite cells from MyoD- mice to integrate into injured muscle. I realize that last sentence just sounded like gobbledygook, to some of you, but I will try to put the cookies on a lower shelf.

Satellite cells constitute a stem cell population within skeletal muscle. They are a small population of muscle-making stem cells found in skeletal muscle and they express a whole host of muscle-specific genes (e.g., desmin, Pax7, MyoD, Myf5, and M-cadherin). Satellite cells are responsible for muscle repair, but previous work has shown that there are at least two populations of satellite cells in skeletal muscle. One population rapidly contributes to muscle repair, whereas the other population is more stem cell-like and remains longer in an undifferentiated state in the recipient muscle (see Beauchamp JR , et al (1999) J Cell Biol 144:1113–1122; Kuang S , et al (2007) Cell 129:999–1010). Presently, it is not clear which population is more efficient in repairing continuously degenerating muscle.

MyoD is a gene that encodes a protein that binds to DNA and activates the expression of particular genes. It plays a vital role in regulating muscle differentiation, and belongs to a family of proteins known as myogenic regulatory factors or MRFs. All MRFs are bHLH or basic helix loop helix transcription factors, and they act sequentially in muscle differentiation. MRF family members include MyoD, Myf5, myogenin, and MRF4 (Myf6). MyoD is one of the earliest genes that indicates a cell has committed to become a muscle cell. MyoD is expressed in activated satellite cells, but not in quiescent (sleeping) satellite cells. Strangely, even though MyoD marks myoblast commitment, muscle development is not dramatically prevented in mouse mutants that lack the MyoD gene. However, this is likely to result from functional redundancy from Myf5. Nevertheless, the combination of MyoD and Myf5 is vital to the success of muscle production.

MyoD
MyoD

Therefore, Asakura and his crew decided to isolated muscle satellite cells from mice that lacked functional copies of the MyoD gene. Making such mice is labor intensive, but doable with mouse embryonic stem cell technology. When such MyoD- mice were made, Asakura and others isolated the satellite cells from these mice and characterized them. They discovered in their 2007 paper, that the satellite cells from the MyoD- mice were much more stem cell-like than satellite cells from MyoD+ mice. The MyoD- satellite cells grew better in culture, integrated into injured muscles better and survived better than their MyoD+ counterparts.

Why is this important? Because when it comes to treating degenerative muscle diseases like muscular dystrophy, finding the best cell is crucial. MyoD+ satellite cells have been used, but they are limited in the amount of muscle repair they provide. MyoD- cells might be a better option for treating a disease like muscular dystrophy.

Or for that matter, what about the heart? Finding the right cell to treat the heart after a heart attack has proven difficult. There are some things bone marrow cells do well, and other things they do not do well when it comes to regenerating the heart. Likewise, there are some things mesenchymal do well and other things they do not do well when placed in a damaged heart. Can MyoD- satellite cells do a better job than either of these types of stem cells?

That was the question addressed in the 2012 Nakamura paper that was published in PLoS One. Clinical trials that have treated heart attack patients with injections of MyoD+ satellite cells into the heart have shown that such treatments can improve heart function, but usually only transiently. They also prevent remodeling of the heart after a heart attack. However, two larger studies failed to produce significant improvements in heart function compared to the placebo, and patients who received the satellite cell transplants were also susceptible to very fast heart beats (tachycardia). Because of these downsides, the excitement for transplanting muscle satellite cells into the heart has waned.

So how did MyoD- satellite cells do? All the laboratory animals used in this experiment (BALB/c mice) were given heart attacks, and injected with either MyoD+ or MyoD- satellite cells. The hearts of animals injected with MyoD- satellite cells were compared with animals whose hearts were injected with MyoD+ satellite cells.

In culture, the MyoD- satellite cells grew better than the MyoD+ cells. When injected into the heart, the MyoD- cells integrated into the heart muscle and spread throughout the heart muscle much more robustly than the MyoD+ cells. The MyoD- cells were also much less susceptible to cell death and survived better than their MyoD+ counterparts.

(A) These panels show MI induced by left coronary artery ligation. Wild-type and MyoD−/− myoblasts were directly injected into the peri-infarct regions of LV. After 1 week, X-gal staining of whole heart indicated that more MyoD−/− myoblasts engrafted than wild-type myoblasts (arrows). Arrowheads indicate left coronary artery ligation points. X-gal staining of cross sections indicated that more MyoD−/− myoblasts than wild-type myoblasts engrafted in both injured and uninjured areas of the heart. Arrows indicate engrafted lacZ+ wild-type and MyoD−/− myoblasts. Scale bars = 1 mm.
(A) These panels show MI induced by left coronary artery ligation. Wild-type and MyoD−/− myoblasts were directly injected into the peri-infarct regions of LV. After 1 week, X-gal staining of whole heart indicated that more MyoD−/− myoblasts engrafted than wild-type myoblasts (arrows). Arrowheads indicate left coronary artery ligation points. X-gal staining of cross sections indicated that more MyoD−/− myoblasts than wild-type myoblasts engrafted in both injured and uninjured areas of the heart. Arrows indicate engrafted lacZ+ wild-type and MyoD−/− myoblasts. Scale bars = 1 mm.

Functionally speaking for the heart, animals that had received transplantations of MyoD- satellite cells had higher ejection fractions, small areas of dead heart tissue, lower end systolic and end diastolic volumes, and more normal echocardiograms. Even though MyoD- cells differentiated into skeletal muscle and not heart muscle (no surprise there), the MyoD- cells induced a very substantial quantity of new blood vessels to sprout in the scar area.

(A) Two weeks post-transplantation, immunofluorescence staining of heart cross sections showed that the progeny of lacZ+ wild-type and MyoD−/− myoblasts formed nestin+ multinucleated skeletal myotubes. Laminin (red) indicates cardiomyocytes and skeletal myotubes. Arrows indicate lacZ+ donor cell-derived nuclei in nestin+ myotubes. (B) Comparison of the relative numbers of lacZ+/nestin+ myotubes for wild-type and MyoD−/− myoblasts 2 weeks after injection (n = 3).
(A) Two weeks post-transplantation, immunofluorescence staining of heart cross sections showed that the progeny of lacZ+ wild-type and MyoD−/− myoblasts formed nestin+ multinucleated skeletal myotubes. Laminin (red) indicates cardiomyocytes and skeletal myotubes. Arrows indicate lacZ+ donor cell-derived nuclei in nestin+ myotubes. (B) Comparison of the relative numbers of lacZ+/nestin+ myotubes for wild-type and MyoD−/− myoblasts 2 weeks after injection (n = 3).

From these experiments, it seems that the MyoD- satellite cells are superior to the MyoD+ satellite cells for treating heart after a heart attack. These cells secrete a whole host of factors that aid the heart in healing and also structurally support the heart and prevent remodeling.

Might it be possible to use such cells in human trials? Asakura notes that engineering MyoD- satellite cells would be impractical for human clinical purposes, but it might be possible to downregulate MyoD expression with drugs (bromodeoxyuridine) or other reagents (RNAi or Id protein transformation).

This work shows that there is a better way to use muscle satellite cells for heart treatments. It simply requires you to remove MyoD function, and the cells will grow and spread throughout the heart better, and more robustly augment heart function and healing.

See NakamuraY, et al PLoS One 7(7) 2012:e41736.

Treating the Heart with Mesenchymal Stem Cells: Timing and Dosage


Stephen Worthley from the Cardiovascular Investigation Unit at the Royal Adelaide Hospital in Adelaide, Australia and his colleagues have conducted a timely experiment with rodents that examines the effects of dosage and timing on stem cell treatments in the heart after a heart attack.

Mesenchymal stem cells from bone marrow and other sources have been used to treat the heart of laboratory animals and humans after a heart attack. The optimal timing for such a treatment remains uncertain despite a respectable amount of work on this topic. Early intervention (one week) seems offer the best hope for preserving cardiac function, but the heart at this stage is highly inflamed and cell survival is poor. If treatment is delayed (2-3 weeks after the heart attack), the prospects for cell survival are better, but the heart at this time is undergoing remodeling and scar formation. Therefore, stem cell therapy at this time seems unlikely to work. Human clinical trials seem to suggest that mesenchymal stem cell treatment 2-3 weeks after a heart attack does no good (see Traverse JH, et al JAMA 2011;306:2110-9). The efficacy of the delivering mesenchymal stem cells to the heart at these different times has also not been compared.

If that degree of uncertainty is not enough, dosage is also a mystery. Rodent studies have used doses of one million cells, but studies have not established a linear relationship between efficacy and dose, and higher dosages seem to plateau in effectiveness (see Dixon JA, et al Circulation 2009;120(11 Suppl):S220-9). High doses might even be deleterious.

So what is the best time to administer after a heart attack, and how much should be administered? These are not trivial questions. Therefore a systematic study is required and laboratory animals such as rodents are required.

In this study, five groups of rats were given heart attacks by ligation of the left anterior descending artery, and two groups of rats received bone marrow-derived mesenchymal stem cells immediately after the heart attack. The first group received a low dose (one million cells) and the second group received twice as many cells. The three other groups received their treatments one week after the heart attack. The third group received the low dose of stem cells received the low dose of cells (one million cells), and the fourth group received the higher dose (two million cells). The fifth group received no such cell treatment.

All mesenchymal stem cells were conditioned before injection by growing them under low oxygen conditions. Such pretreatments increase the viability of the stem cells in the heart.

The results were interesting to say the least. when assayed four weeks after the heart attacks, the hearts of the control animals showed a left ventricular function that tanked. The ejection fraction fell to 1/3rd the original ejection fraction (~60% to ~20%) and stayed there. The early high dose animals showed the lowest decrease in ejection fraction (-8%). The early low dose group showed a greater decrease in ejection fraction. Clearly dose made a difference in the early-treated animals with a higher dose working better than a lower dose.

In the later-treated animals, dose made little difference and the recovery was better than the early low dose animals. when ejection fraction alone was considered. However, when other measures were considered, the picture becomes much more complex. End diastolic and end systolic volumes were all least increased in the early high dose animals, but all four groups show significantly lower increases than the controls. The mass of the heart, however, was highest in the late high-dose animals as was ventricular wall thickness.

When the movement of the heart walls were considered, the early-treated animals showed the best repair of those territories of the heart near the site of injection, but the later-treated animals showed better repair at a distance from the site of injection. The same held for blood vessel density: higher density in the injected area in the early-treated animals, and higher blood vessel density in those areas further from the site of injection in the later-treated animals.

The size of the heart scar clearly favored the early injected animals, which the lower amount of scarring in the early high dose animals. Finally when migration of the mesenchymal stem cells throughout the heart was determined by using green fluorescent protein-labeled mesenchymal stem cells, the later injected mesenchymal stem cells were much more numerous at remote locations from the site of injection, and the early treated animals only had mesenchymal stem cells at the site of injection and close to it.

These results show that the later doses of mesenchymal stem cells improve the myocardium further from the site of the infarction and the early treatment improve the myocardium at the site of the infraction. Cell dosage is important in the early treatments favoring a higher dose, but not nearly as important in the later treatments, where, if anything, the data favors a lower dose of cells.

Mesenchymal stem cells affect the heart muscle by secreting growth factors and other molecules that aids and abets healing and decreases inflammation. However, research on these cells pretty clearly shows that they modulate their secretions under different environmental conditions (see for example, Thangarajah H et al Stem Cells 2009;27:266-74). Therefore, the cells almost certainly secrete different molecules under these conditions.

In order to confirm these results, similar experiments in larger animals are warranted, since the rodent heart is a relatively poor model for the human heart as it beats much faster than human hearts.

See James Richardson, et al Journal of Cardiac Failure 2013;19(5):342-53.

Stem Cell Fixes for the Heart


Two recent papers have provided very good evidence that pluripotent stem cells can help heal a heart that has experienced a heart attack. One of these papers used induced pluripotent stem cells from rats, and the other used embryonic stem cells.

The first paper comes from the laboratory of Yoshiki Sawa, who is a professor in the Department of Surgery at the Osaka University Graduate School of Medicine in Osaka, Japan. In this paper, Sawa’s group made induced pluripotent stem cells (iPSCs) from mice and cultured them under conditions known to induce differentiation into heart muscle cells. Beating cells were detected and grown on gelatin-coated plates with Delbecco’s medium. When these cells were tested for gene expression, they made all the same genes as those found in a mouse heart.

To get the cells to form sheets of heart muscle cells, Sawa and his team plated his iPSCs on UpCell plates that are coated with a chemical that causes the cells to adhere to it at normal temperatures, but when the temperature is dropped, the cells detach from the plate. Sawa used another innovation with this culture system; he grew cell without any sugar. This caused all the non-heart cells to die off. The result was a sheet of heart muscle cells that contracted in unison.

Next, the Sawa team took induced heart attacks in a Japanese rat strain. 2 weeks after suffering the heart attack, the sheet of heart muscle cells were placed on the heart scar in half of the rats and the other half received no implants.

Four weeks after implantation of the heart muscle sheet, the differences in heart function were stark. The ejection fraction in the hearts of the animals that had received the iPSC-derived heart muscle sheets increased almost 10%. The fractional shortening, which is the degree to which the heart muscle shortens when it contracts, also increased more than 5%. Also, the amount of stretching during pumping decreased, which indicates that the heart is pumping more efficiently.

When the heart muscle from the implants were examined, they were also filled with molecules associated with the production of new blood vessels. Thus the implanted heart muscle sheets also helped heal the heart by inducing the formation of new blood vessels.

A danger of using iPSC-derived heart muscle cells is the tendency to miss undifferentiated cells and have undifferentiated cells that cause tumors. In this experiment, they noticed tumors if they only grew the cells in the sugar-free medium for a little while. However, if they grew the iPSC-heart muscle cells in sugar-free media for at least three days, all the tumor-causing cells died and implants from these sheets never formed any tumors.

This paper demonstrated the efficacy and plausibility of using patient-specific iPSCs to treat a heart that has had a heart attack some time ago.

The second paper comes from the laboratory of Marisa Jaconi in Geneva, Switzerland. In this paper, Jaconi and her gang of stem cell scientists at the Geneva University Hospitals and the Ecole Polytechnique Fédérale de Lausanne used a “cardiopatch” seeded with cardiac-committed embryonic stem cells to treat a heart attack in rats.

Because the injection of stem cells can induce arrhythmias (irregular heart beats), narrowing of blood vessels, blood vessel obstruction, and other types of damage, these two papers tried to use sheets of cells or cells embedded in biodegradable patches to treat the heart. In this paper, Jacobi and others used a hydrogel made from fibrin, which is the same material found in blood clots. Into that fibrin hydrogel, they placed mouse embryonic stem cells that had been treated with a protein called BMP-2, which drives pluripotent stem cells toward a heart cell fate.

To use these cardiopatches, Jacobi and her group induced heart attacks in a French rat strain and then applied the patch to the heart. They had two groups of rats; those that had been given heart attacks and those that had not. The sham group received either a patch with cells, a patch with iron particles (for detection with MRI) or not patch. The heart attack group received the same.

The results are a little hard to interpret, but the patch + cells definitely improved heart function. First, the hearts that had received patches with cells showed in increase in small blood vessels and blood vessel-making (CD31+) cells. Therefore the patches + cells improved heart circulation. Second, the hearts with the patch + cells showed the presence of new heart muscle cells and much mess thinning of the walls of the heart. Third, the heart functional parameters were better preserved in the patch + cells hearts. The ejection fraction decreased substantially in the hearts that did not receive cells, but in the hearts that received patch + cells, the amount of blood left in the heart after pumping and at rest did not increase nearly as much as in the other groups. These parameters are in indication of the efficiency with which the heart is pumping. The fact that the heart + cells hearts did not decrease in efficiency nearly as precipitously as the others shows that the stem cells are healing the heart.

While these results may not seem terribly robust, we must remember that the cardiopatch was only placed over a small portion of the heart. Therefore, we would not expect to see large increased in function. The fact that we do see new heart muscle cells, new blood vessels, and an arrest in the functional free fall of the heart is significant, given the small area of the heart that was cover with the cells.

The cardiopatch is a new technology and this experiment showed that the patch biodegrades quickly and without incident. It also showed that embedding cells in the patch is feasible, and that the patch is a plausible vehicle to deliver cells to the heart. This procedure also induced the formation of new heart muscle cells in the heart scar and new blood vessels too. Perhaps even more encouraging is the absence of tumors reported in this paper. Even though the ESCs were not differentiated completely into heart muscle cells, the cardiac-directed cells were differentiated enough to form either blood vessels, smooth muscle, or heart muscle. This seems to be enough to prevent the cells from forming tumors. Also, the fibrin scaffold was not deleterious to the heart, even though some studies have used other scaffolds that are damaging to the heart.

Thus cardiopatches and cardiac muscle sheets are perfectly good strategies for treating heart with stem cells. More work needs to be done, but the results are encouraging.

Major Clinical Trial Finds Bone Marrow Stem Cell Treatments Provide No Benefits After a Heart Attack


A large and very well designed and carefully controlled clinical trial known as TIME has failed to demonstrate any benefit for infusions of bone marrow stem cells into the heart 3-7 days after a heart attack.  This study comes on the heals of a similar clinical study known as LateTIME, which stands for Late Timing In Myocardial infarction Evaluation, and tested the effects of bone marrow stem cells infusions into the heart of heat attack patients 2-3 weeks after a heart attack.

LateTIME enrolled 87 heart attack patients, and harvested their bone marrow stem cells.  The stem cells were delivered into the hearts through the coronary arteries, but some received a placebo.  All patients had their ejection fractions measured, their heart wall motions in the damaged areas of the heart and outside the damaged areas and the size of their infarcts.  There were no significant changes in any of these characteristics after six months. Because another large clinical study known as the REPAIR-AMI study showed significant differences between heart attack patients that had received the placebo and those that had received bone marrow stem cells 3-7 days after a heart attack, this research group, known as the Cardiovascular Cell Therapy Research Network (CCTRN), sponsored by the National Institutes of Health, decided the test their bone marrow infusions at this same time frame.

TIME was similar in design to LateTIME.  This study enrolled 120 patients that had suffered a heart attack and all patients received either an infusion of 150 million bone marrow stem cells or a placebo within 12 hours of bone marrow aspiration and cell processing either 3 days after the heart attack to 7 days.  The researchers examined the changes in ejection fraction, movement of the heart wall, and the number of major adverse cardiovascular events plus the changes in the infarct size.

The results were resoundingly negative.  At 6 months after stem cell infusion, there was no significant increase in ejection fractions versus the placebo and no significant treatment effect on the function of the left ventricle in either the infarct or the border zones.  These findings were the same for those patients that received bone marrow stem cell infusions 3 days after their heart attack or 7 days after their heart attacks.  Fortunately, the incidence of major adverse events were rare among all treatment groups.

Despite the negative results for these clinical trials, there are a few silver linings.  First of all, the highly controlled nature of this trial sets a standard for all clinical trials to come.  A constant number of stem cells were delivered in every patient, and because the stem cells were delivered soon after they were harvested, there were no potential issues about bone marrow storage.

Jay Traverse, the lead author of this study, made this point about this trial:  “With this baseline now set, we can start to adjust some of the components of the protocol to grow and administer stem cell [sic] to find cases where the procedure may improve function.  For example, this therapy may work better in different population groups, or we might need to use new cell types or new methods of delivery.”

When one examines the data for this study, it is clear that some patients definitely improved dramatically, whereas others did not.  Below is a figure from the Traverse et al paper that shows individual patient’s heart function data 6 months after the stem cell infusions.

BMC indicates bone marrow mononuclear cell; MI, myocardial infarction.

From examining these data even cursorily, it is clear that some patients improved dramatically while others tanked.  Traverse is convinced that bone marrow stem cell infusions help some people, but not others (just like any other treatment).  He is convinced that by mining these data, he can begin to understand who these patients are who are helped by bone marrow stem cell transplants and who are not.  Also, the stem cells of these patients have been stored.  Hopefully, further work with them will help Traverse and his colleagues clarify what, if anything, about the bone marrow of these patients makes them more likely to help their patients and so on.

There are some possible explanations for these negative results.  Whereas the positive REPAIR-AMI used the rather labor-intensive Ficoll gradient protocols for isolating mononculear cells from bone marrow aspirates, the TIME trials used and automated system for collecting the bone marrow mononuclear cells.  Cells isolated by the automated system have neither been tested in an animal model of heart attacks, nor established as efficacious in a human study of heart disease.  Therefore, it is possible that the bone marrow used in this study was largely dead.  Secondly, the cell products were kept in a solution that had a heparin concentration that is known to inhibit the migratory properties of mononuclear cells (See Seeger et al., Circ Res 2012 111(7): 1385-94).  Therefore, there is a possibility that the bone marrow used in this study was no good.  Until the bone marrow stem cells collected by this method are confirmed to be efficacious, judgment must be suspended.

CADUCEUS Clinical Trial Shows that Cardiosphere-Derived Stem Cells Can Regrow Heart Muscle After a Heart Attack


Cedars-Sinai Heart Institute is home to the CADUCEUS clinical trial. CADUCEUS stands for cardiosphere-derived autologous stem cells to reverse ventricular dysfunction. In this clinical trial, patients who had experienced a heart attack (and had left ventricular ejection fractions between 25% – 45%) were split into two groups. One group was given standard medical care for a heart attack patient and the other group was given standard care plus heart-based stem cells known as CDCs, which is short for “Cardiosphere-Derived Cells.” Patients assigned to receive CDC infusions of 12-25 million cells into the infarct-related artery 1.5 – 3 months after the heart attack.

The results 6 months after the stem cell infusion revealed that none of the patients in either group had died, developed tumors in their hearts or had experienced any major adverse heart-related event. Also Magnetic Resonance Imaging analysis of patients from both groups showed that those patients treated with CDCs displayed reductions in the mass of the heart scar, and increases in living, heart muscle mass. Additionally, the ability of the region of the heart that had experienced the heart attack contracted better in those patients who had received the CDC infusion. Also, the thickness of the wall of the heart was thicker in those patients who had received CDC infusions. Unfortunately, changes in other heart-specific functions such as EDV (end-diastolic volume), ESV (end-systolic volume), and LVEF (left ventricular ejection fraction) did not differ between the two groups by 6 months, which is difficult to reconcile with the structural changes in the hearts. .

Inventor of the procedures and technology used in this study, Eduardo Marbán, MD, PhD, who is also the director of the Cedars-Sinai Heart Institute, noted, “While the primary goal of our study was to verify safety, we also looked for evidence that the treatment might dissolve scar and regrow lost heart muscle. This has never been accomplished before, despite a decade of cell therapy trials for patients with heart attacks. Now we have done it. The effects are substantial, and surprisingly larger in humans than they were in animal tests.”

Shlomo Melmed, MD, dean of the Cedars-Sinai medical faculty and the Helene A. and Philip E. Hixon Chair in Investigative Medicine added, “These results signal an approaching paradigm shift in the care of heart attack patients. In the past, all we could do was to try to minimize heart damage by promptly opening up an occluded artery. Now, this study shows there is a regenerative therapy that may actually reverse the damage caused by a heart attack.”

An initial part of this study was conducted in 2009. In that study, Marbán and his colleagues used a patient’s own heart tissue to grow specialized heart stem cells. These specialized stem cells were injected back into the patient’s heart in an effort to repair and re-grow healthy muscle in a heart that had been injured by a heart attack. This experiment, at that time, was the first of its kind.

The results of that initial study were quite encouraging. The 25 patients, who participated in the study, had an average age of 57 and had suffered heart attacks that left them with damaged heart muscle. Each patient underwent extensive imaging scans to precisely locate the exact location and severity of the scars generated by the heart attack. Patients were treated at Cedars-Sinai Heart Institute and at Johns Hopkins Hospital in Baltimore.

Of these patients, eight received conventional medical care for heart attack survivors (prescription medicine, exercise recommendations and dietary advice) and were the control patients in this study. The remaining 17 patients were randomized to receive the stem cells underwent a minimally invasive heart biopsy, under local anesthesia that utilized a catheter inserted through a vein in the patient’s neck. From this catheter, doctors removed small pieces of heart tissue, about half the size of a raisin, that were taken to Marbán’s laboratory at Cedars-Sinai, where they were subjected to culture methods invented by Marbán to grow and expand the heart-based stem cells.

During a second, minimally invasive [catheter] procedure, the expanded heart-derived cells were reintroduced into the patient’s coronary arteries. Patients who received stem cell treatments experienced an average of 50 percent reduction in their heart attack scars 12 months after infusion while patients who received standard medical management did not experience shrinkage in the damaged tissue.

Marbán explained, “This discovery challenges the conventional wisdom that, once established, scar is permanent and that, once lost, healthy heart muscle cannot be restored.”

This phase I study definitely shows that the CDC infusion procedure is safe, which warrants the expansion of this procedure to a phase 2 study.