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.

Growth Factor Delivery Stimulates Endogenous Heart Repair After Heart Attacks in Pigs

Steven Chamulean and his colleagues at the University Medical Center Utrecht in Holland have examined the use of growth factors to induce healing in the heart after a heart attack. Because simply applying growth factors to the heart will cause them to simply be washed out, Chamulean and his coworkers embedded the growth factors in a material called hydrogel. They were able to measure how long the implanted growth factors lasted. As it turns out, when the growth factors were embedded in the hydrogel, they lasted for four days, and the hydrogel caused the growth factors to spread out into heart tissue with a gradient with the highest concentration at the site of injection (see Bastings, et al., Advanced Healthcare Materials 2013 doi: 10.1002/adhm.201300076).

In his new publication in the Journal of Cardiovascular Translational Research, Chamulean and his group used a new hydrogen called UPy to into which they embedded their growth factors. UPy stands for ureido-pyrimidinone end-capped poly(ethylene glycol) polymer. At the pH of our bodies, UPy hydrogels form a gel-like material made of fibers. When the pH changes, the gel becomes liquid. They embedded the growth factors insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF).

The experimental design of this paper used pigs that were given heart attacks and then reperfused 75 minutes later. One month later, the animals were broken into three groups: just hydrogel, hydrogel with growth factors embedded in it, and growth factors injected into other heart without hydrogel. One month later, the animals were examined for their heart function, and then the animals were sacrificed to examine their heart tissue.

In every case, the hearts treated with only the hydrogel did the poorest of the three groups. The animals injected with gel-less growth factors did better than the controls, but those animals treated with growth factors embedded in UPy hydrogel did the best. The physiological indicators of the hearts from the animals treated with UPy embedded with IGF-1 and HGF improved significantly more than the controls that were treated with only UPy hydrogel. The hearts from animals treated with IGF and HGF without hydrogels improved over controls, by not nearly as well as those treated with growth factor-embedded UPy hydrogels.

When the hearts were examined even more surprises were observed. The animals with hearts that had been treated with UPy + growth factors did not show the enlargement observed in the control hearts. This is significant, because enlargement of the heart is a side effect for a heart attack and is the sign of heart failure. The UPy + growth factor hearts also displayed many signs of dividing cells; far more than hearts from the other two groups. Since the heart has its own resident stem cell population, these growth factors stimulated these stem cells to divide and form new heart muscle, and new blood vessels. Blood vessel density was much higher in the UPy + growth factor group and the pressure against which blood flowed in these hearts was substantially less in this groups, demonstrating that not only was the blood vessel density higher, but blood flow through these vessel networks was much more efficient. There was also plentiful evidence of the formation of new muscle in the UPy + growth factor group. When these hearts were also stained for c-kit, which is a cell surface marker for cardiac stem cells, the UPy + growth factor hearts had lots of them – much more than the other two groups.

This paper reports significant findings because the resident stem cell population in the heart was actively mobilized without having to extract them by means of a biopsy. There is also evidence from Torella and others that IGF-1 and HGF can reactivate the sleeping cardiac stem cells of aged laboratory animals (Circulation Research 2004 94: 514-524). The UP{y hydrogels are well tolerated and are biodegradable. They provide a medium that stays in place and releases embedded growth factors in a sustained manner. The results in this paper provide the rationale to develop growth factor therapy for human patients.

“In Body” Muscle Regeneration

Researchers at Wake Forest Baptist Medical Center’s Institute for Regenerative Medicine have hit upon a new strategy for tissue healing: mobilizing the body’s stem cells to the site of injury. Thus harnessing the body’s natural healing powers might make “in body” regeneration of muscle tissue is a possibility.

Sang Jin Lee, assistant professor of Medicine at Wake Forest, and his colleagues implanted small bits of biomaterial scaffolds into the legs of rats and mice. When they embedded these scaffolds with proteins that mobilize muscle stem cells (like insulin-like growth factor-1 or IGF-1), the stem cells migrated from the muscles to the bioscaffolds and formed muscle tissue.

“Working to leverage the body’s own regenerative properties, we designed a muscle-specific scaffolding system that can actively participate in functional tissue regeneration,” said Lee. “This is a proof-of-concept study that we hope can one day be applied to human patients.”

If patients have large sections of muscle removed because of infections, tumors or accidents, muscle grafts from other parts of the body are typically used to restore at least some of the missing muscle. Several laboratories are trying the grow muscle in the laboratory from muscle biopsies that can be then transplanted back into the patient. Growing muscle on scaffolds fashioned from biomaterials have also proven successful.

Lee’s technique overcomes some of the short-comings of these aforementioned procedures. As Lee put it, “Our aim was to bypass the challenges of both of these techniques and to demonstrate the mobilization of muscle cells to a target-specific site for muscle regeneration.”

Most tissues in our bodies contain a resident stem cell population that serves to regenerate the tissue as needed. Lee and his colleagues wanted to determine if these resident stem cells could be coaxed to move from the tissue or origin, muscle in this case, and embeds themselves in an implanted scaffold.

In their first experiments, Lee and his team implanted scaffolds into the leg muscles of rats. After retrieving them several weeks later, it was clear that the muscle stem cell population (muscle satellite cells) not only migrated into the scaffold, but other stem cell populations had also taken up residence in the scaffolds. These scaffolds were also contained an interspersed network of blood vessels only 4 weeks aster transplantation.

In their next experiments, Lee and others laced the scaffolds with different cocktails of proteins to boost the stem cell recruitment properties of the implanted scaffolds. The protein that showed the most robust stem cell recruitment ability was IGF-1. In fact, IGF-1-laced scaffolds had four times the number of cells as plain scaffolds and increased formation of muscle fibers.

“The protein [IGF-1] effectively promoted cell recruitment and accelerated muscle regeneration,” said Lee.

For their next project, Lee would like to test the ability of his scaffolds to promote muscle regeneration in larger laboratory animals.

Culture Medium from Endothelial Progenitor Cells Heals Hearts

Endothelial Progenitor Cells or EPCs have the capacity to make new blood vessels but they also produce a cocktail of healing molecules. EPCs typically come from bone marrow, but they can also be isolated from circulating blood, and a few other sources.

The laboratory of Noel Caplice at the Center for Research in Vascular Biology in Dublin, Ireland, has grown EPCs in culture and shown that they make a variety of molecules useful to organ and tissue repair. For example, in 2008 Caplice published a paper in the journal Stem Cells and Development in workers in his lab showed that injection of EPCs into the hearts of pigs after a heart attack increased the mass of the heat muscle and that this increase in heart muscle was due to a molecule secreted by the EPCs called TGF-beta1 (see Doyle B, et al., Stem Cells Dev. 2008 Oct;17(5):941-51).

In other experiments, Caplice and his colleagues showed that the culture medium of EPCs grown in the laboratory contained a growth factor called “insulin-like growth factor-1” or IGF1. IGF1 is known to play an important role in the healing of the heart after a heart attack. Therefore, Caplice and his colleagues tried to determine if IGF1 was one of the main reasons EPCs heal the heart.

To test the efficacy of IGF1 from cultured EPCs, Caplice’s team grew EPCs in the laboratory and took the culture medium and tested the ability of this culture medium to stave off death in oxygen-starved heart muscle cells in culture. Sure enough, the EPC-conditioned culture medium prevented heart muscle cells from dying as a result of a lack of oxygen.

When they checked to see if IGF1 was present in the medium, it certainly was. IGF1 is known to induce the activity of a protein called “Akt” inside cells once they bind IGF1. The heart muscle cells clearly had activated their Akt proteins, thus strongly indicating the presence of IGF1 in the culture medium. Next they used an antibody that specifically binds to IGF1 and prevents it from binding to the surface of the heart muscle cells. When they added this antibody to the conditioned medium, it completely abrogated any effects of IGF1. This definitively demonstrates that IGF1 in the culture medium is responsible for its effects on heart muscle cells.

Will this conditioned medium work in a laboratory animal? The answer is yes. After inducing a heart attack, injection of the conditioned medium into the heart decreased the amount of cell death in the heart and increased the number of heart muscle cells in the infarct zone, and increased heart function when examined eight weeks after the heart attacks were induced. The density of blood vessels in the area of the infarct also increased as a result of injecting IGF1. All of these effects were abrogated by co-injection of the antibody that specifically binds IGF1.

From this study Caplice summarized that very small amounts of IGF1 (picogram quantities in fact) administered into the heart have potent acute and chronic beneficial effects when introduced into the heart after a heart attack.

These data are good enough grounds for proposing clinical studies. Hopefully we will see some in the near future.

Culture Media from Mesenchymal Stem Cells Heals Injured Lungs

Acute lung injury and acute respiratory distress syndrome remain major causes of death and suffering despite advances in management of these conditions. The incidence of these conditions is expected to double in the next 25 years, and treatment for it is largely supportive.

Fortunately, mesenchymal stem cells (MSCs) from bone marrow have been used in experimental models to treat lung injury in rodents. MSCs can engraft into lung tissue and become lung tissue (or at least turn into cells that sure look a whole lot like lung tissue). MSCs can also suppress the types of immune responses that tend to really chew up lung tissue. Thus, MSC administration seems to improve the condition of lungs that have been attacked by infections or damaging agents.

However, the rates at which MSCs engraft into lung tissue is rather low; too low, in fact, to account for the benefit provided by MSCs. Therefore, MSCs appear to help repair lung tissue by means of “paracrine” mechanisms. This 50-cent word simply means that MSCs repair the lung by secreting molecules that promote lung healing.

To test this hypothesis, researchers in the laboratory of Bernard Thérband from the Ottawa Hospital Research Institute in Ottawa, Canada has grown MSCs in culture, and used the growth medium after the MSCs had been removed from it to treat mice that suffered from lung injuries.

To induce lung injury, mice were treated with isolated bits of bacterial cells that are known to promote acute lung injury. Then a group of these lung-injured mice were treated with conditioned medium from bone marrow MSCs that had been grown in culture dishes.

The MSC-conditioned medium decreased lung inflammation, and disruptions of the blood vessels in the lung normally observed during lung injury. Therefore, the lungs did not fill up with liquid and pus. However, the conditioned medium did not prevent the weight loss associated with lung injury. The overall tissue architecture of the lung tissue was much more normal in the mice treated with the conditioned medium from MSCs than in the untreated mice. Conditioned medium from other cultured cells had no such sanative effect.

MSC conditioned culture media also modified the activity of white blood cells in the lung. Instead of charging forward into lung tissue and damaging it in response to damage, the white blood cells (so-called “alveolar macrophages”) worked with the lung tissue to help heal it.

Finally, when Thébaud and his colleagues examined the molecules secreted into the medium by the MSCs, they discovered that the culture medium was filled with lots of interesting molecules, but one in particular caught their eye:  Insulin-like growth factor-1 (IGF-1). This molecule has all kinds of healing properties, and it seemed to Thébaud and company that IGF-1 could be responsible for a good portion of the healing. Therefore, they infused the lung-injured mice with purified IGF-1, and, wouldn’t you know, the lungs showed rather robust healing after being damaged with bacterial bits.

Thus MSCs provide lung healing properties and they do so by means of the molecules they secrete. Many of these healing properties can be recapitulated by infusing IGF-1.

Such experiments provide hope that future clinical trials for such treatments are not far off.