Coronary heart disease is one of the leading causes of death in humans. Coronary arteries are blood vessels that supply the heart muscle with blood and oxygen. Thus, blockage of a coronary artery deprives the heart muscle of blood and oxygen, causing injury to the heart muscle. Sudden blockage of the coronary artery causes heart attack (also known as a myocardial infarction) will result in the death of heart muscle. In adults, heart muscle cannot regenerate itself effectively. The dead heart muscle is replaced by scar tissue which jeopardizes the function of the heart. Regeneration of lost heart muscle by stem cells is one strategy to restore heart function after a myocardial infarction. Embryonic stem cells (ES cell) can differentiate into heart muscle cells spontaneously or by induction with chemicals and growth factors. Thus, ES cells could be a very useful source for repairing injured heart. However, none of the current methods available can efficiently induce 100% of ES cells to differentiate into heart muscle. While, injection of a mixture of differentiated heart muscle cells and undifferentiated ES cells into the heart may cause tumor formation. Thus, we propose to develop a method to select differentiated heart muscle cells. We plan to label the ES cell derived-heart cells with a surface marker so that the differentiated heart muscle cells can be isolated from undifferentiated ES cells and other cell types. We will use adeno-associated virus (AAV) to deliver the cardiac-specific surface marker gene, because this virus is not associated with any human diseases, will not stimulate a strong immune reaction in humans and can infect undifferentiated and differentiated human ES cells. The labeled heart muscle cells will be isolated from a pool of undifferentiated ES cells and other cell types based on the specific surface marker. We will then analyze the purity and function of the isolated cells, and the ability of these cells to regenerate heart muscle. Our ultimate goal is to find an effective way to isolate pure ES cell-derived heart cells to repair damaged myocardium. This study will lead to the development of a more efficient cell therapy for coronary heart disease.
Each year, approximately 1 million Americans suffer a heart attack and 40% of them are fatal. Heart disease is America’s number one killer, claiming nearly 1400 lives daily. In California, the death rate associated with heart disease is 504/10,000 for people older than 35 (data collected in 1969-2000). A heart attack results in the death of heart muscle caused by the sudden blockage of a coronary artery by a blood clot. In adults, heart muscle cannot regenerate itself efficiently. The dead heart muscle is replaced by scar tissue, which leads to the gradual loss of heart function. Medical and surgical procedures are often needed to restore the blood supply to the remaining heart muscle and to prevent future heart attacks. However, patients usually need to take medication long-term to slow down the progressive process of the disease. Although many advanced treatments have been developed, none can cure the disease. The cost of medicine and surgery are quite high. The health care system may simply not be able to meet the needs of patients or control these spiraling costs, unless the therapeutic focus switches away from maintenance and toward its prevention and cure. The treatment and cure of heart disease can potentially be accomplished through the use of new regenerative medical therapies including a special type of human cells, called stem cells. Our research will overcome some of the obstacles and will lead to the development of an effective and safe therapeutic strategy for coronary heart disease. The results obtained from this study will eventually improve the quality of life, prolong life and reduce the burden of long-term health care for Californians.
SYNOPSIS: The PI focuses this proposal on a novel method for enhancing the purity of differentiated hESC populations so that they will be mostly cardiomyocytes. The specific aims are: (1) to test the relative ability of AAV serotypes to infect hESC-derived cardiomyocytes; (2) To use a transfected GATA-4 promoter to isolate cardiomyocyte progenitor cells from hESCs (3) To use truncated flt driven by the AAV-transfected GATA-4 to sort out cardiomyocytes and (4) to characterize cardiomyocytes in vitro (and in vivo) derived using the techniques in Aims 1-3.
Understood, but not stated directly in the proposal, is that the GATA-4 driven flt expressing cells will be compared to MLC-driven flt expressing cells for the relative ability to generate more cardiomyocytes that work in the functional assays.
SIGNIFICANCE AND INNOVATION: First Reviewer: The most valuable part of the proposal and most innovative is the evaluation of various AAV serotypes for their ability to infect hESCs. However, the grant only proposes a look at differentiated hESCs vis-a-vis AAV, and the work would be significantly more important for the field if undifferentiated cells were examined.
Second Reviewer: The proposed research rates highly on both innovation and significance. A major problem facing the therapeutic application of hESC-derived products is the potential for contaminating hESCs in the differentiated cell product. Contamination with undifferentiated cells raises the tumorigenic potential of this class of products -- potentially altering the risk/benefit ratio of using such products clinically. Therefore, the approach proposed by the investigator, to uniquely tag the differentiated cell in order to provide a mechanism to both positively and negatively select for the desired cell phenotype has the potential to enhance the safety margin of these types of products.
STRENGTHS: First Reviewer: The strength of the proposal is the PI’s experience in cardiology research and in the use of AAV vectors. Aim 1 probably carries the most importance for hESC researchers, in that AAV vectors may be useful for many applications in the field.
Second Reviewer: The investigator has extensive experience and expertise in both the use of AAV vectors generally, and in the use of these vectors to deliver to specific cell types using tissue-specific promoters. The investigator has pilot data using the proposed promoter to induce tissue-specific regulated gene expression in cardiac myocytes -- showing that there is proof of concept data to support his strategy. He also has experience in working with murine embryonic stem cell cultures as well as in vivo models of cardiac disease.
The overall strategy that the investigator has proposed is well-designed and has a high degree of feasibility. Potential problems have already been considered by the PI and addressed in the proposal. The markers chosen for the positive and negative selection strategies provide a number of advantages, as outlined by the investigator on p.4, all of which I agree with. Finally, the investigator proposes to take the project step-wise through a series of steps that are well-considered, from choosing the most appropriate AAV serotype, to choosing the most appropriate tissue-specific promoter, and then testing the cell surface marker strategy, to finally assessing whether the marking strategy has any impact on the gene expression and/or in vivo function of the transduced, selected cells.
WEAKNESSES: Pushing hESCs to cardiomyocytes is a fairly hot issue in research now, and there are many approaches that could be taken to increase the efficiency and yield of this differentiation process. The approach chosen by the investigator is novel. However, its success may be limited because GATA-4 is involved in differentiation of other lineages besides cardiomyocytes, and the surface marker chosen may have unintended downsides. Also the stage of differentiation at which the AAV vectors are to be used is not defined: Are undifferentiated HES cells infected and then followed for the reporter constructs, or is the investigator going to induce cardiomyocyte differentiation (how) and then infect with AAV at some point along the differentiation protocol? (Only ‘differentiated HES cells are mentioned in the Aim. How differentiated?) Aim 2 uses AAV GATA4-GFP and it is really not clear why this is done, since it delays the analysis of the GATA-4-flt cells. Aim 3 is to ‘select’ a suitable surface marker but only one marker is proposed. There are a lot of places where this approach could go wrong, but they are acknowledged.
When the final constructs are made with flt expression, and the sorting is done, it would also be a good idea to sort for cardiomyoctyes that are not flt expressing, to monitor cardiomyocyte differentiation in the whole culture.
It is unclear why only non-beating rat cardiomyocytes will be used for the integration studies.
DISCUSSION: The evaluation of AAV serotypes to infect cardiomyocytes is valuable in this work because the PI is an expert on AAV, and the attempt to develop methods for purifying differentiated from uindifferentiated cells is this proposals greatest strengths. The strategy for tagging cells using positive and negative selection seems feasible, and the preliminary data are also good. The PI seems to be reaching to optimize cardiomyocyte differentiation which makes the proposal complicated. In the end, there may be too many ways for this experiment to fail, however. For example, even though truncated flt may not signal, the binding of ligand may still have some effect. Discussion also centered around there not being any real description of what the promoter construct is, and how validated the promoters are. GATA-4 seems to be too early a promoter to use for selection, thus it may decrease the feasibility of the experiment. Finally, the PI did not consider/discuss the fact that AAV rarely integrates, and in a proliferating cell population the viral genome will become diluted out.