Heart disease is the number one cause of death and a leading cause of disability in the California. About 40,000 heart attack victims are admitted to California hospitals annually, and over 5,000 of these patients die. When a heart attack occurs, blood flow to the heart muscle is cut off, and eventually portions of the muscle of the heart die and are replaced by non-contracting scar tissue. Heart muscle does not adequately regenerate. In this proposal we hypothesize that the beating, immature cardiac cells derived from human embryonic stem cells can be injected directly into the heart attack scar, and that these cells will survive, grow, develop their own blood supply and eventually improve the contractile function of the entire heart. Improved contraction would help reduce the possibility of heart failure, dilatation of the ventricle and possible death. Before human embryonic stem cells can be injected into a human heart, these cells will need to be tested in standardized animal models. The rat heart attack, which we propose to use in this series of studies, is a standard, well characterized model with many factors in common with heart attacks in humans. Our proposal should help to answer the question of whether an injured heart can be rebuilt using human embryonic stem cells. Our proposal will also test the mechanisms by which treatment with human embryonic stem cell therapy may be beneficial: examining whether the injected cells couple electrically with host cells and/or whether the injected stem cells produce humoral factors that may benefit the function of the host heart.
Statement of Benefit to California:
Heart disease is the number one cause of death and a leading cause of disability in the State of California. Approximately 40,000 heart attack victims are admitted to California hospitals annually, and over 5,000 of these patients die. Heart attacks occur when an atherosclerotic plaque ruptures within a coronary artery. A blood clot forms and cuts off additional flow of blood to the muscle of the heart. Heart cells do not receive enough oxygen and nutrients and these heart cells die. In general, heart cells do not regenerate and following a heart attack, dead heart muscle is replaced by a thin, collagenous, non-contracting scar. The purpose of our study will be to determine whether immature heart cells derived from human embryonic stem cells can replace the scar with viable beating muscle. If the heart can be rebuilt with cells derived stem cells, this has the potential to reduce suffering, disability, and death associated with one of the most common causes of illness and death in the State of California.
SYNOPSIS OF PROPOSAL: The investigators propose to test the hypothesis that cardiomyocytes derived from hESCs can repair hearts post-myocardial infarction. They propose to inject the scar with hESC-derived immature cardiomyocytes and speculate that a functional syncytium will be formed with host myocardium and/or a paracrine effect will occur. Pilot studies have shown the feasibility of implanting hESCs in rat hearts where they can be identified, survive and differentiate. They will test the effects of the implants on global and regional ventricular function and remodeling and coronary flow after permanent and reperfused coronary artery occlusions in athymic nude rats, and assess LV infarct wall thickness, dilatation, and vascularity. Creation of functional syncytium with host myocardium will be assessed via intracellular Ca transients in adjacent cells. They will also test whether late injection (1 month post coronary occlusion) confers a benefit on remodeling. IMPACT AND SIGNIFICANCE: Heart disease remains a major killer in the US and its component states: of major concern is the occurrence of heart failure resulting from scar formation and compromised ventricular function in the post-infarct period. The availability of means to heal the infarct by turning scarred, dysfunctional regions into normally contractile myocardium functionally integrated into the cardiac syncytium has been a major focus of research in the US and Europe. To date, results have involved autologous or allogeneic administration of cardiac precursor cells or mesenchymal stem cells, and have been disappointing. The entire premise of this research is based on the ability to differentiate the hMSCs into a myocardial precursor line that is capable of repairing myocardium. As such it is an up or down project; success will confer great benefit, but there are a number of reasons why it might fail. In other words, there will be either great impact or no impact. The general design follows that of a clinical trial with the trial population being the athymic nude rats. While this obviates concerns regarding rejection of the cells administered, it leaves open the question of whether rejection would complicate the use of hESCs in this setting. The hESCs themselves are a line approved by the US government, and so the research is eligible for support by federal sources, but the investigators believe, perhaps rightly so, that a study so focused on developing what they refer to a “practical, therapy-oriented” project is low. This is understandable, as the research really is focused on a strategy for success rather than an understanding of mechanism regardless of whether there is success or failure. The proposal, if successful, would have clinical significance as it would suggest that human ES cell derived cardiomyocytes can be effectively utilized for cardiac muscle regeneration. From a scientific standpoint, the proposal has less of an impact, and is largely designed as a pre-clinical study, establishing proof of concept for a direct therapeutic endpoint of a population of enriched ES derived cardiomyocytes. QUALITY OF THE RESEARCH PLAN: The research plan is straightforward. The authors state they will use the method of Mummery et al to lead hESCs along a cardiac lineage. The method involves co-culture of hES cells with murine visceral-endoderm–like cells. This initiates differentiation to beating muscle which shows sarcomeric marker proteins, chronotropic responses, and ion channel expression and function consistent with cardiomyocytes. This essential step will be taken in Singapore and the cells shipped to the investigators’ lab. The cells incorporate GFP as a marker. It is not stated whether or how steps to optimize the differentiation of the cells will be taken, nor are the methods clear for cell purification. The research utilizes protocols incorporating 35 rats per group. The first step is to prepare the rat model, then inject cells (or medium, or cell-conditioned medium), follow the animals for 3 months, and then study in vivo hemodynamics, ventriculography, ejection fraction, segmental wall motion and regional myocardial blood flow, all techniques with which the investigators have great familiarity. Remodeling will be studied via gross and histologic assessments, all of which are standardized, and cells will be identified via GFP fluorescence, as well as by study of a number of standard markers. The second aim uses time as a variable to determine when the best time for cell administration is from the points of view of optimal cell survival and cardiac function. Cell-conditioned medium will be used to determine if there is a paracrine function of the administered cells. Also the media will be analyzed for a variety of factors that might mediate the paracrine effect. For all the proposed objectives, 5 animals will be shipped to Indiana University for study of “functional coupling” of the implanted cells with adjacent myocardium. This work, to be done by Loren Field, uses imaging of Ca transients in a two photon laser source to image the simultaneity and sequence of electrical activation across implanted and native cells. The quality of the research plan lies in the use of a clinical trial design to ask a series of yes-no questions regarding the ability of cells to survive and to improve function. Interesting data will be obtained from the paracrine experiments as well as the functional coupling experiments. However, in all experiments, the goal is more to identify effect rather than to ask mechanistic questions about effect. The research plan is appropriate for the type of routine study that has been proposed. The source of the cardiomyocytes that are utilized are human ES cells that have been driven into the cardiac lineage according to protocols that have been developed by Christine Mummery. The cells will originate from Singapore from ES Cell International, and the studies represent a rigorous evaluation as to their potential therapeutic effects, including some experiments to attempt to determine if any realized therapeutic benefit might be cell autonomous or due to a paracrine effect. While thorough in their design, the studies are not highly innovative and do not break new ground in the areas that are really pivotal to realizing the potential of human ES cells, which would be based on improving ways for their isolation, renewal, and directed differentiation. It is unlikely that a sufficient number of cardiac cells could be harvested with the Mummery protocol to allow a meaningful regeneration of cardiac muscle in patients in global dysfunction without the advances noted above, let alone problems related to teratomas. At the same time, it is likely that even in the best hands; the efficiency of grafting of the cells will be low in any event, again underscoring the need to address more fundamental questions. STRENGTHS: The strengths of the proposal lie in the use of a clinical trial design to test the contribution of the cells to regional and global cardiac function and blood flow in an animal model, as well as to determine whether there is a paracrine contribution. From the potential of learning about mechanism, the most exciting experiments will be done at Indiana University, where the electrical propagation of impulses from cell to cell will be tested. The experience of the investigator and his well established leadership position in the field of cardiac injury following myocardial infarction are stengths, as is the clinical importance of the problem. WEAKNESSES: There are several weaknesses of the proposal. First, the answers to be obtained are of a yes-no variety which, while being robust with regard to a trial design do not help in understanding mechanism. The cells to be administered are not adequately characterized. Are the cells pre-cardiac, early differentiated, or more differentiated? How will the purity and quality of cells be assessed? The problems with the cell population trump any positives of this application. The labeling of the cells, while helpful in identifying those which fluoresce, doesn’t allow for the possible loss of fluorescence with proliferation and differentiation, the extent to which autofluorescence of the myocardium will confound the signal from the implanted cells, or the ability to really track what subset of cells persist, multiply or die. Although one can comment on the structural characteristics of those cells which are clearly labeled, this does not clearly reflect the fate of the overall population. Other possibilities for use here include FISH, and the use of iron, ferritin or other particles as labels that can be followed over time with MRI and other imaging techniques such as PET. All of these are far from perfect, but in general have been found to be superior to GFP and other fluorescence techniques over time. The paracrine effect experiments have the potential to give exciting results, but are set up more as a survey of potential factors rather than as an organized exploration of them. In addition, it is difficult to understand how the extent of paracrine versus cell growth effects will be worked out. How does one accurately compare the mobilization of cells and quantify cell numbers with administration of cells on the one hand and administration of paracrine factors on the other? The experiments on functional coupling make use of an excitation-contraction uncoupler to provide the non-contractile environment necessary for the optical mapping of the Ca signals. While this tells about simultaneity and temporal sequence of the signals it doesn’t provide information about homogeneity of contraction. More quantitative information about coupling could be derived from disaggregation of cell pairs, measurement of gap junctional conductance and mapping of connexin isoforms and distribution in the hearts. DISCUSSION: There was concern that the project suffers from a lack of hypothesis and is at risk for returning no useful information in the event that it fails. Reviewers felt that it was primarily a "yes or no"-style of proposal. One reviewer was slightly optimistic at the thought that if the applicant could confirm the presence of the well-characterized desired cell-type, the project might have a chance of success, but went on to speculate that even under optimal conditions, it would be unlikely that cells of adequate quality or number would result. There was general agreement that the most interesting parts of the proposed work would be done outside California in Singapore and in Indiana. To further strengthen the proposal for consideration for funding by CIRM, a reviewer suggested that collaborations with other California researchers such as the USC group or others with direct expertise in this area could be helpful. It was recommended that the applicant could strengthen the proposal by integrating more cutting edge ES cell technology that could couple with the considerable strengths of the current group in animal models systems. Specifically, more attention should be paid to isolation and characterization of the starting cells.