Stem Cells Embedded in a Fibrin Patch Help Hearts Recover After a Heart Attack

If a patient has a heart failure, there is little you can do for them. Medications can take some of the stress off the failing heart, and in extreme cases, a heart transplant is warranted. However, organ transplants are hampered by both the limited number of organ donors and the potential for the patient’s body to reject the new heart.

A new study from the journal STEM CELLS Translational Medicine has shown that heart tissue can be regenerated if engineered patches made up of a mixture of fibrin and mesenchymal stem cells (MSCs) derived from human umbilical cord blood are applied to the heart.

Previous studies show the potential of MSCs to repair damage generated by a heart attack. In these clinical studies, the MSCs were delivered through injections into the heart muscle or intravenously. “While feasible and safe, the treatments exhibited only modest benefits,” said Antoni Bayes-Genis, M.D., Ph.D., member of the ICREC (Heart Failure and Cardiac Regeneration) Research Program, Germans Trias i Pujol Health Science Research Institute (IGTP) and professor at Universitat Auto`noma de Barcelona. Dr. Bayes-Genis is a lead investigator on this study.

“The survival rate of the implanted stem cells was generally low and about 90 percent of them either died or migrated away from the implantation site, generally to the liver,” added the study’s first author, Santiago Roura, Ph.D., also a member of the ICREC Research Program and IGTP. “These limited effects are probably due to the adverse mechanical stress and hypoxic conditions present in the myocardium after the heart attack.”

Now could a better way to deliver the MSCs to the injured site yield more efficient results? Synthetic scaffolds (or patches) in which the cells are embedded in matrices constructed of biological and/or synthetic materials and supplemented with growth or differentiation factors can generate so-called “bioimplants.” Bioimplants are a promising way to potentially apply stem cells to the heart in a way that will allow them to survive, grow and thrive. Unfortunately, none of the current materials being tested for heart patches, whether synthetic or natural has been shown to provide optimal properties for cardiac tissue repair.

Dr. Bayes-Genis and his colleagues examined how a fibrin patch filled with human umbilical cord blood-derived MSCs might serve to repair a damaged heart. Fibrin is widely used in medical applications, since it can act as a bio-compatible glue that holds cells in place and stimulating the production of new blood vessels (angiogenesis). Bayes-Genis and others hypothesized that fibrin scaffolds might offer a nurturing environment for the growth and proliferation of MSCs at the site of the heart injury. There, the cells could induce the repair of damaged heart tissue.

Bayes-Genis and coworkers mixed MSCs and fibrin to form the patches that were then applied to the hearts of mice that had undergone heart attacks. Three weeks later, they compared the recovery of these animals to a control group of mice that were treated with fibrin alone without embedded stem cells, and a third group that received no treatment at all. The results showed that the patches adhered well to the hearts and the MSCs grew and differentiated. The patch cells also participated in the formation of new, functional blood vessels that connected the patch to both the heart tissue directly beneath it and the mouse’s endogenous circulatory system, too.

“As a result, the heart function in this group of mice was better than that of the animals in either of the other control groups,” Dr. Bayes-Genis said. “Thus, this study provides promising findings for the use of umbilical cord-blood MSCs and fibrin patches in cardiac repair.”

“This is an interesting study that suggests a news strategy for using stem cells to repair injured heart tissue, without the drawbacks that cell injections have shown,” said Anthony Atala, M.D., Editor-in-Chief of STEM CELLS Translational Medicine and Director of the Wake Forest Institute for Regenerative Medicine.

Genetically Engineered Bone Marrow Stem Cells on a Fibrin Patch Repairs Damaged Heart

Regenerative therapies for the heart have come a long way from the first clinical trials and injected bone marrow cells directly into the heart muscle. Despite the modest improvements shown in those earlier studies, it became clear that the vast majority of cells that were implanted into the heart died soon after their introduction. This single fact left researchers looking for a better way to deliver cells into the damaged heart.

Several laboratories have tried to condition the stem cells before their injection in order to “toughen them up” so that they do not tend to die so easily. While these experiments have worked well in laboratory animals, no clinical trials have been conducted to date with conditioned stem cells. Another strategy is to place the cells on a patch that is then applied to the dead heart tissue in order to promote healing of the heart.

The patch strategy was employed by Hao Lai and Christopher Wang and their co-workers at the Shanghai Institute of Cardiovascular Disease in Shanghai, China. Lai and others extracted bone marrow stem cells from the bones of Shanghai white pigs. These cells were cultured, and genetically engineered to expressed IGF-1 (insulin-like growth factor-1). Once IGF-1 expression was confirmed, the cells were loaded onto a fibrin patch and placed over the hearts of Shanghai white pigs that had just experienced laboratory-induced heart attacks. There were four groups of pigs: 1) those treated with fibrin patches with bone marrow stem cells that were not genetically engineered; 2) another group treated with fibrin patches that contained genetically engineered bone marrow stem cells that did not express IGF-1; 3) fibrin patches containing bone marrow stem cells that had been engineered to express IGF-1; and 4) a control group that was not treated with any cells or patches.

In culture, the IGF-1 engineered cells did not differentiate into heart muscle cells, and they did induce proliferation in Human Umbilical Vein Endothelial cells, which suggests that these engineered cells would induce the formation of new blood vessels.

When transplanted into heart injured pigs, the IGF-1-expressing cells on a fibrin patch significantly reduced the size of the infarct in the hearts, and increased the thickening of the walls of the heart. Gene expression studies showed that the IGF-1-expressing cells on the fibrin patch induced anti-cell death genes that promote cell survival. These cells also induced the growth of many new blood vessels and seemed to promote the growth of new heart muscle, but the cells on the patch are almost certainly not the source of these new cells, but resident stem cell populations in the heart probably were.  The increase in heart mass suggests that the implanted cells induced the resident stem cell populations in the heart to divide and differentiate into heart muscle cells.

This new technique proved safe and effective. It prevented remodeling (enlargement) of the heart and promoted cell survival. It is a technique that shows promise, especially since the fibrin patch is biodegradable and the bone marrow stem cells will not last indefinitely in the heart. These cells simply work by serving as a platform for the secretion of IGF-1 and perhaps other healing molecules.

Another caveat of this experiment is that the bone marrow stem cells were genetically engineered with lentivirus vectors. Because of the tendency for these vectors to insert genes willy-nilly into the genome, this is almost certainly not the safest way to genetically modify cells Finally, the improvements in these animals was significant albeit modest. In order for this technique to come to the clinic, it will have to induce better improvements in heart function. There were modest, albeit insignificant increases in ejection fraction. The ejection fraction will need to be increases for this technique to have a fighting chance to come to clinical trials. Nevertheless, this is a fine start to what might become a new strategy to treat patients with ailing hearts.

Heart Muscle Cells Produced from Induced Pluripotent Stem Cells Repair Heart Attacks in Pigs

When heart muscle cells are made from embryonic stem cells, they integrate into the heart and form proper connections with other heart muscle cells. Such experiments have been conducted in mice, guinea pigs, and nonhuman primates (i.e. monkeys). Chong and others earlier this year (Nature (2014) 510, 273-277) implanted heart muscle cells produced from embryonic stem cells into the hearts of nonhuman primates that had suffered from heart attacks. There was extensive evidence of engraftment of these cells, remuscularization of the heart, and electrical synchronization 2 to 7 weeks after transplantation. However, despite these successes, the hearts of some of these animals also showed abnormal heart beat patterns (known as arrhythmias). Such a problem has also been observed in other laboratory animals as well (see my book The Stem Cell Epistles), and this problem has to be addressed before derivatives of pluripotent stem cells can be used to treat damaged hearts (pluripotent means capable of differentiating into all the mature adult cell types).

Jianyi Zhang and his colleagues at the University of Minnesota have used induced pluripotent stem cells made from human skin cells to produce heart muscle cells that were used to treat pigs that had suffered from induced heart attacks.  Their results differed slightly from those of Chong and others.

Zhang and others noted that implanted heart muscle cells typically survive better if they are implanted with blood vessel cells (endothelial cells or ECs).  This was first shown in culture by Xiong and others in 2012 (Circulation Research 111, 455-468), but other work has confirmed this.  That is, Zhang’s coworkers in his laboratory co-transplanted heart muscle cells made from induced pluripotent stem cells with endothelial cells and smooth muscle cells (which are also a part of blood vessels), and saw that the co-transplanted cells survived much better than heart muscle cells that were transplanted without these other cell types.

On the basis of these experiments, Zhang and his crew decided that implanted heart muscle cells would do much better if they were implanted into pig hearts if they were implanted with endothelial and smooth muscle cells.  This was the hypothesis that Zhang and others wanted to test in this paper (which was published in Cell Stem Cell, Dec 4, 2014, 750-761).

Skin biopsies from human volunteers were used as a source of skin cells that were then genetically engineered and then cultured to form human induced pluripotent stem cells (hiPSCs).  These cultured hiPSCs were differentiated into heart muscle cells by means of the “Sandwich method,” which yielded beating heart muscle cells in about 30 days.  Additionally, their hiPSC lines were differentiated into smooth muscle and endothelial cells as well.

Next, Zhang and his colleagues and collaborators used 92 pigs and subjected them to experimentally-induced heart attacks.  Why pigs?  Pigs are a larger animal than rodents, and their hearts are larger and beat much slower than the hearts of rats and mice.  Therefore, they are a more expensive, but better experimental model system for the human heart.  Nevertheless, these pigs were divided into six different groups (3 pigs died from the procedure, so there were 89 pigs involved in this experiment).  Animals in the first group or SHAM group underwent the surgery to induce a heart attack, but no heart attack was induced.  The second group was called the MI group and this group received no other interventions after surgery.  The Patch group received a fibrin patch over the site of injury, but no cells.  The CM + EC + SMC group received injections of 2 million heart muscle cells, two million endothelial cells, and two million smooth muscle cells directly into the injured portion of the heart.  The Cell + Patch group received all three cell types in a fibrin patched that was imbued with a growth factor called Insulin-like growth Factor-1 (IGF-1) that had been loaded into microspheres.  This causes the growth factor to be released gradually and exert its effects over a much greater period of time.

That’s a lot of information so let’s review – six groups: 1) SHAM (no heart attack; 2) MI (heart attack and no treatment); 3) Patch (just the fibrin patch); 4) Cells + Patch (fibrin patch with the three cell types); 5) Cells (cells, but no patch), and a final group cells Patch + CM (just heart muscle cells in the patch).

Animals were evaluated one week after the heart attack and four weeks after their heart attacks. I am uncertain how soon after the heart attack the treatments were given, but in the paper it reads to me as though the treatments were given right after the heart attacks had been induced.  Because all implanted cells were engineered to glow in the dark, the number of surviving cells could be counted and tracked.

Only 4.2% of the cell survived in the Cells group, up to 9% of the cells in the Cell + Patch group survived.  32% of the cells in the CM + Patch group survived.  Thus, it seemed as though the presence of the other cell types did increase the survival of the heart muscle cells and the patch also increased cell survival rates.  Secondly, the heart function of all the treated groups was better than the MI group, but the hearts treated with Cells + Patch were clearly superior to all the others, with the exception of the SHAM group.  The hiPSC-derived heart muscle cells also clearly engrafted into the hearts of the pigs, but the big surprise in this paper is that THERE WERE NO INDICATIONS OF ARRHYTHMIAS!!!  Apparently the manner in which these hiPSC-derived heart muscle cells integrated and adapted to the native heart in such as way as to preclude irregular electrical activity.  Another indicator measured was ratio of phosphocreatine to ATP.  If that sounds like a language from outer space, it simply means a measurement of the efficiency of muscle mitochondria (the part of the cell that makes all the energy).  Again the Cells + Patch hearts had significantly more efficient mitochondria, and, hence, better energy production than the other hearts.  Damage to mitochondria also tends cause cells to up and die, which means that these cells were in better health that those from the MI group.

This paper shows that an ingenious tissue engineering innovation that uses a fibrin patch and a a combination of cells, not just heart muscle cells can significantly increase the healing after a heart attack.  Also, even though neither embryonic stem cell-derived cells nor iPSC-derived cells are ready for clinical trials, this paper shows that iPSCs are not as far behind iPSCs as some authors have suggested.  Furthermore, because iPSCs would not be subject to immunological rejection, they have an inherent superiority over embryonic stem cells.  The problem comes with the time required to make iPSCs and then derived heart muscle cells from them, which might put it outside the time window for treat of an acute heart attack.