Stem Cell Factor Delivery into Heart Muscle After Heart Attack May Enhance Cardiac Repair and Reverse Injury


Stem Cell Factor or SCF is a small peptide that circulates throughout the bloodstream and eventually finds its way to the bone marrow where it summons bone marrow-based stem cells to the sight of injury for tissue repair purposes. Unfortunately, it takes injured tissues time to express SCF at high enough levels to recruit bone marrow stem cells to come and accelerate tissue healing. This is particularly the case in the heart after a heart attack. For this reason, scientists are trying to find new and better ways to increase SCF production in the damaged heart.

To that end, cardiologists at the Icahn School of Medicine at Mount Sinai have discovered that delivering SCF directly to damaged heart muscle after a heart attack seems to augment heart muscle repair and regenerate injured tissue.

“Our discoveries offer insight into the power of stem cells to regenerate damaged muscle after a heart attack,” said lead study author Kenneth Fish, Director of the Cardiology Laboratory for Translational Research, Cardiovascular Research Center, Mount Sinai Heart, Icahn School of Medicine at Mount Sinai.

In this study, Fish and his colleagues used gene transfer to administer SCF to the heart shortly after inducing heart attacks in a pig model system in order to test its regenerative repair response. Fish and his coworkers developed a novel SCF gene transfer delivery system that stimulated the recruitment and expansion of adult cardiac stem cells directly to injury sites that reversed heart attack damage. In addition, the gene therapy improved cardiac function, decreased the death of heart muscle cells, increased regeneration of heart tissue blood vessels, and reduced the formation of heart tissue scarring.

“It is clear that the expression of the stem cell factor gene results in the generation of specific signals to neighboring cells in the damaged heart resulting in improved outcomes at the molecular, cellular, and organ level,” says Roger J. Haijar, senior study author and Director of the Cardiovascular Research Center at Mount Sinai. “Thus, while still in the early stages of investigation, there is evidence that recruiting this small group of stem cells to the heart could be the basis of novel therapies for halting the clinical deterioration in patients with advanced heart failure.”

The cell surface receptor for SCF is the c-Kit protein, and cells that possess the c-Kit protein are called c-Kit+ cells. c-Kit+ cells not only respond to SCF, but serve as resident cardiac stem cells that naturally increase in numbers after a heart attack and through cell proliferation are directly involved in cardiac repair.

To date, there is a great need for new interventional strategies for cardiomyopathy to prevent the progression of this disease to heart failure. Heart disease is the number one cause of death in the United States, with cardiomyopathy or an enlarged heart from heart attack or poor blood supply due to clogged arteries being the most common cause of the condition. Cardiomyopathy also causes a loss of heart muscle cells and changes in heart shape, which lead to the heart’s decreased pumping efficiency.

“Our study shows our SCF gene transfer strategy can mobilize a promising adult stem cell type to the damaged region of the heart to improve cardiac pumping function and reduce myocardial infarction sizes resulting in improved cardiac performance and potentially increase long-term survival and improve quality of life in patients at risk of progressing to heart failure,” says Dr. Fish.

“This study adds to the emerging evidence that a small population of adult stem cells can be recruited to the damaged areas of the heart and improve clinical outcomes,” says Dr. Hajjar.

Heart Cells Expressing Stem Cell Factor Show Less Cell Death After a Heart Attack


Stem Cell Factor is a cell surface protein that is expressed by several different cells, including tissue fibroblasts, heart cells, cells in the bone marrow, and blood vessel cells. Stem Cell Factor (SCF) plays important roles in the migration, proliferation, and adhesion of any cell that expresses the receptor for SCF, a molecule called c-kit. Cells that express c-kit include cardiac stem cells, endothelial progenitor cells, and hematopoietic stem cells. When c-kit binds to SCF, the SCF-containing cell activate their Akt /PI3K pathway, and this pathway prevents cells from dying and drives them to divide, differentiate, more, adhere, and even secrete new molecules.

pi3k-resized-600

Fu-Li Xang in the laboratory of Qingping Feng at the University of Western Ontario has done several experiments with SCF in the heart. His goal is to determine if heart cells that have SCF fare better after a heart attack than hearts that do not have quite so much SCF.

To that end, Feng and his team showed that SCF does help heal the heart after a heart attack in 2009 (Xiang et al, Circulation 120: 1065-74). The next step was to determine if SCF could attenuate cell death in the heart that results from a heart attack.

SCF-ckit

 

The strategy behind this experiments involved making genetically engineered mice that expressed lots of SCF in their heart muscle. The particular mouse strain that Feng and his crew made had the SCF gene activated by a heart muscle-specific promoter, but the expression of SCF could be shut off by giving the mice the drug doxycycline. These SCF transgenic mice and normal mice were given heart attacks and then some were treated with a doxycycline while others were given a drug called LY294002, which inhibits the Akt pathway. These animals were then analyzed three hours after the induced heart attack and the amount of cell death, the size of the infact, the number of stem cells that moved into the heart were all measured.

 LY294002
LY294002

The upshot of all this work is this: SCF decreased the amount of cell death by about 40%. Also the size of the infarct was also smaller. These benefits were abrogated by the co-administration of either doxycycline or LY294002. When a search for molecules that are indicative of cell death were examined, the results were completely unsurprising: the markers of cells death like fragmented DNA or caspase-3 were decreased in the SCF mice and this attenuation was abrogated by co-administration with doxycycline or LY294002.

Other experiments examined the activation of the Akt/PI3K pathway in the SCF-expressing animals, and it was quite clear that the SCF-expressing animals showed a robustly active Akt/PI3K pathway compared to the non-SCF-expressing mice.

A different experiment examined the presence of c-kit-expressing cells in the hearts of these mice. Remember that c-kit expressing cells are stem cells that have been recruited to the heart by the SCF. Once again, it was exceedingly clear that the SCF-expressing mice had hearts with a large excess of c-kit-expressing cells and this recruitment of stem cells was abrogated by neutralizing c-kit with an antibody against it. The incoming stem cells also tend to secrete a host of interesting molecules that help heal the heart, and one of these molecules, HGF (hepatic growth factor), which also goes up in concentration in the hearts of the SCF-expressing mice, is blocked by a drug called crizotinib. If SCF-expressing mice were pre-treated with crizotinib, the infarct size tended to be just as large as the non-SCF-expressing cells.

Feng and his group also examined the resident stem cells in the heart, the cardiac stem cells population, which, by the way, also express c-kit. These cells also were induced to express HGF and IGF (insulin-like growth factor) as a result of SCF, and if the c-kit receptor was blocked with an antibody, then this effect was abrogated.

There is a lot of data in this paper, but the news is almost all good. Basically SCF will recruit stem cells to the heart after a heart attack and this recruitment happens quickly (within 3 hours) and does the heart a world of good. Translating this work into human patients will not be easy, but SCF is available. If it could be localized to the heart by some means soon after a heart attack, there is good reason to believe, based on these pre-clinical results that it would do the patient quite a bit of good. The next piece is figuring our how to go about doing just that.

A Home A Stem Cell Could Love


In our bodies, stem cell populations live in specific places that are specially designed to accommodate them known as “stem cell niches.” Stem cell niches host and maintain stem cell populations, but the dependence of particular stem cells on their niche varies. For example, in the fruit fly, Drosophila melanogaster, the germ line stem cell niche can drive stem cells that have already begun to differentiate to revert into undifferentiated stem cells (see Brawley C and Matunis E. Science 2004;304:1331–4 and Kai T and Spradling A. Nature 2004;428:564–9). However, hair follicle stem cells do not revert when they return to their niche even if this niche has been depleted of stem cells (see Hsu Y-C, Pasolli HA, Fuchs E. Cell 2011;144:92–105). Also, blood cell-making stem cells that normally live in bone marrow can leave their niche in the bone marrow without losing their stem cell properties (Cao Y-A, et al., Proc Natl Acad Sci USA 2004;101:221–6). Finally, neural stem cells can exist and even self-renew outside their niche (Conti L, et al., PLoS Biol 2005;3:e283).

In order to properly grow stem cells in culture and manipulate them for therapeutic purposes, scientists have attempted to recapitulate stem cell niches in culture but only with very limited success.

Nevertheless, trying to get stem cells that have been introduced into a patient’s to engraft or make the new body their home has required a better understanding of stem cell niches.

To that end, Professor Claudia Waskow and her colleagues at the Technische Universität Dresden in Germany have utilized a downright ingenious method to make a mouse that can support the transplantation of human blood stem cells. This is despite the species barrier and, these mice do not need to have their own resident stem cell population obliterated with radiation.

How did Waskow and others do this? They used a mutation of a receptor called the “Kit receptor” to facilitate the engraftment of human cells. “What is the Kit receptor,” you ask? The Kit receptor is a protein in the membranes of blood stem cells that binds a soluble protein called stem cell factor (SCF). Stem cell factor drives certain types of blood cells to grow, and also mediates stem cells survival, proliferation and differentiation. Activation of the Kit receptor can also cause blood stem cells to leave the bone marrow and move into the peripheral circulation.

The Kit Receptor - AKA CD117
The Kit Receptor – AKA CD117

In the mouse model system designed by Waskow and others, the human blood stem cells grow and even differentiate into all blood-specific cell types without any additional treatment, and this includes the cells of the innate immune system. This is a milestone discovery because such cells normally do not form properly in “humanized” mice, but in Waskow’s experiment, these immune cells were efficiently generated. Significantly, these transplanted stem cells can be maintained in the mouse over a longer period of time compared to previously existing mouse models.

“Our goal was to develop an optimal model for the transplantation and study of human blood stem cells,” says Claudia Waskow, who recently took office of the professorship for “animal models in hematopoiesis” at the medical faculty of the TU Dresden. Before, coming to TU Dresden, Dr. Waskow was a group leader at the DFG-Center for Regenerative Therapies Dresden where most of the study was conducted.

Waskow’s team used a naturally occurring mutation of the Kit receptor and introduced it into her laboratory mice that lacked a functional immune system. This circumvented the two major obstacles of blood stem cell transplantation: the rejection by the recipient’s immune system and absence of free niche space for the incoming donor stem cells in the recipient’s bone marrow. Typically, the animal or the patient is treated with radiation to deplete the bone marrow of resident stem cells. This step, known as conditioning, creates usable space in the bone marrow for the implanted stem cells to take up residence and set up shop. However, irradiation is toxic a whole host of different cell types, not just bone marrow stem cells, and, unfortunately, has several strong side effects.

This Kit mutation in the mouse modifies the stem cell niche of the recipient mouse so that the resident stem cells are easily displaced by the human donor stem cells that possess a functional Kit receptor. This replacement works so well that irradiation was unnecessary, which allowed the study of human blood development in a physiological setting.

Waskow would like to use this new model system to study diseases of the human blood and immune system or to test new treatment options.

These data show that the Kit receptor (also known as CD117) is important for the function of human blood stem cells in a transplantation setting. Further work will concentrate on applying this new knowledge about the role of the receptor to improve conditioning therapy in bone marrow transplantation patients.