Cardiovascular disease is the leading cause of morbidity and mortality in the United States with >50% of mortality attributable to coronary artery disease that leads to a heart attack. A heart attack results from death of heart tissue and leads to loss of heart function. Because the heart is composed of muscle cells that are not able to regenerate, two options exist to protect people from reduced heart function after a heart attack begins: 1) prevent the extent of injury or 2) regenerate the dead tissue and create a more functional heart muscle. The first option is the current approach used but it has only limited effectiveness, in part because it is difficult to predict when someone is going to have a heart attack. The second option, the main goal of this project, is more challenging but offers potential for a wide variety of individuals with injured hearts. Studies with experimental animals have had limited success in terms of generating functional heart tissue using various types of stem cell types. Heart cells exist as a cohesive network that meets the energy demands of the body by pumping blood in a rhythmic manner. The futility of previous studies has been to introduce stem cells into this network in the hope that the cells will be able to graft onto the damaged tissue and create functional networks with existing cells to generate a better functioning heart. Unfortunately, this has not occurred. The cells that are introduced into the damaged heart are eliminated without becoming permanent members of the “cardiac network”. In this study, we propose a new approach that exploits a critical feature of the structure of the heart: cardiac muscle cells are surrounded by another cell type, cardiac fibroblasts, which produce a number of as-yet inadequately defined factors that likely contribute to the health and function of the muscle cells. The goal of our proposed studies is to understand how cardiac fibroblasts are able to maintain and, in the case of human embryonic stem cells (hESC), to create an environment that favors differentiation of cells to be ones able to contract and improve overall cardiac function, in particular perhaps to regenerate dead heart tissue. Fibroblasts are a vital part of the network of cardiac cells. We thus will first determine if hESCs grown together with cardiac fibroblasts are able to coax the stem cells to turn into heart cells. Fibroblasts are known to make and release factors that then act on nearby cells but the full complement of these factors is not known. Our second aim is to determine which factors are released by fibroblasts that are capable of transforming hESC to heart cells. In the final aim, we will test if hESC cells grown with cardiac fibroblasts or factors made by fibroblasts will create an environment, when tested in mice, for long term health and benefit in terms of heart function.
Statement of Benefit to California:
Cardiovascular disease is the leading cause of morbidity and mortality in the United States as well as in California with >50% of mortality attributable to coronary artery disease primarily leading to a heart attack. The current proposal aims to determine if human embryonic stem cells can be coaxed to become heart cells by interaction with cardiac fibroblasts, which in a functioning heart are essential neighbors of heart muscle cells. The information gained from these studies will help in the development of therapies that will restore cardiac function following injury, in particular heart attacks, that destroys cells in the heart that are necessary for normal function. Thus, Californians would potentially benefit from improved health by virtue of success in the proposed studies. Given the widespread occurrence of coronary artery disease and heart failure, success in the proposed studies that would identify key factors produced by cardiac fibroblasts that facilitate health and function of cardiac myocytes would likely lead to the development of novel drugs that exploit such findings and that would thus potentially contribute to the benefit of the California economy.
SYNOPSIS: The PI proposes research to study the role of cardiac-derived fibroblasts in differentiation of hES cells into cardiomyocytes. The aims are (i) to see if co-culture of hES cells with commercially available cardiac fibroblasts induces differentiation of the hES cells into cardiomyocytes (ii) to use proteomics to determine the secreted products of cardiac fibroblasts and (iii) to transplant cardiomyocytes derived from hES co-cultured with cardiac fibroblasts, and determine their role in repair in an acute MI model in mice. SIGNIFICANCE AND INNOVATION: The damaged heart, with its prevalence in western society and its accessibility to manipulation, is a prime target for repair by cell therapy, yet little progress has been made. The PI is probably correct that fibroblasts in the heart have not been characterized sufficiently to determine their role in regeneration and repair of the heart. In that respect the choice of cell focus is innovative. Approaches to enhance the production of effective transplantable cardiomyocytes would be a valuable advance to treatment of cardiac disease. STRENGHTS: The idea that cardiac fibroblasts may be important in heart repair is a good one. In addition, the PI and his group have a strong track record for productive basic research, including differentiation of cardiac myocytes, though not in this area. WEAKNESSES: The main problem is that there is no data available to suggest that cardiac fibroblasts will induce hESC to differentiate into cardiomyocytes. The three aims of the project depend on this ability of cardiac fibroblast cells to induce differentiation of human ES cells toward the cardiomyocyte fate, and there is simply no experimental support for this. Moreover, the fibroblasts are coming from a commercial source, so it is likely that they represent pools of adult fibroblasts. The properties of these cells are not described in terms of known differentiated phenotypes, which are expected to provide the specificity and strength of this proposed effect. There also may be problems with using fetal vs. adult sources for these cells, but there is just very little information available. Another major issue in the design of the co-cultures is that the PI does not state what the matrix is on which the hESCs will be grown. They will be separated from the human fibroblasts in transwells, but presumably something will have to be done so that they are not on feeder layers. This is simply not accounted for in the presentation. In order to keep the hESCs expanding off a feeder layer, will large bFGF doses be used? What is the anticipated effect on the human fibroblasts? Since all the aims depend on the first one, the success of the entire proposal is a bit iffy. In vivo, several groups have shown that ES cells can regenerate cardiomyocytes, whereas bone marrow-derived stem cells generally cannot. So there are no controls for Aim 3. If ES cells already can generate cardiomyocytes in the hands of other groups, then how will the PI show that it is the co-culture of his hES cells that is promoting the regeneration after transplantation? There would have to be a comparison with non-co-cultured hESCs for the conclusion to be made that co-culturing does the trick. While this seed grant RFA was designed to bring in people with expertise outside stem cell biology, to bring their tools and perspectives to stem cell issues. The PI studies caveolae in fibroblasts and other cells, so he is really not an expert in fibroblasts (and their differentiation-inducing potential) which is the expertise necessary for the grant. It is also unclear what exactly the role of Dr. Roth is. Are daily discussions really the reason for 10% salary support? A lot of informative work could happen in vitro before the transplant experiments. For example, in vitro co-culture experiments with cells that can and cannot differentiate into cardiomyocytes in vivo might be informative in determining which genes in the hESCs (vs. marrow) drive cardiomyocyte differentiation. Also, UCSD has a renowned engineering department. Isn’t it likely that mechanical forces regulate gene expression in cardiac fibroblasts, given that they are under constant mechanical strain? This could also be tested. DISCUSSION: There was no discussion following the reviewers' comments.