Cardiovascular disease (CVD) affects more than 71 million Americans and 1.7 million Californians. Recently, engineered cardiovascular tissue grafts, or “patches”, including one made from mouse embryonic stem cells (ESC), have shown promising results as a future therapy for CVD. Our overall goal is to extend these recent results to human ESC as follows. Aim 1: Apply mechanical stretch and electrical pacemaker-like stimulation to hESC-derived heart cells in order to make them stronger and beat at the same time. Current methods to turn hESC into heart cells do not result in the organization required to generate enough strength to support a weak heart and to avoid irregular heart beats. We will use specially engineered devices to apply mechanical stretch and electrical pacemaker-like stimulation to hESC-derived heart cells in order to strengthen them and make them beat in unison. Aim 2: Engineer a cardiovascular patch from hESC-derived heart cells in order to make a potential new therapy for heart disease. Recently, heart cells from mouse ESC, supporting structures called scaffolds, and mechanical stretch have successfully been combined to engineer a cardiovascular patch. We will combine the hESC-derived heart cells from Aim 1, scaffolds, and the same stretch and pacemaker-like stimulation as in Aim 1 to engineer a cardiovascular patch. In addition, we will add a specialized substance called VEGF to our patch so that, potentially, a blood supply will form around it after it is implanted on a diseased heart. We believe a blood supply will be necessary to keep our patch healthy, and in turn, this will allow our patch to help a damaged heart pump better. Aim 3: Assess whether our patch can remain healthy and also strengthen the heart of a rat after it has undergone a heart attack. We will first implant our cardiovascular patch in the rat aorta, the main blood vessel that supplies blood to the body, to test whether the patch remains healthy and whether it can contract and beat on its own. We will first use the aortic position because we feel it will allow us to assess the inherent function of the patch, thus facilitating our efforts to improve its design. After testing in the aortic position, we will implant the patch over damaged heart tissue in a rat that has undergone an experimentally created heart attack. Over a period of several weeks, we will use novel imaging techniques, ultrasonography, echocardiography, and electrocardiography to non-invasively test whether the patch remains healthy and whether the patch helps the damaged heart pump better. We believe the above aims will address questions relevant to hESC-based cardiovascular therapies and will provide vital information needed for safe and effective future clinical translation. As we will evaluate both federally and non-federally approved cell lines, and thus unlikely to receive federal funding, we will need to rely on the support provided by CIRM to carry out our objectives.
Statement of Benefit to California:
Cardiovascular disease (CVD) affects more than 1.7 million Californians and 71 million Americans. The societal and financial impacts are tremendous, with CVD accounting annually for an estimated $8 billion in CA and nearly $400 billion in US health care costs. In the case of chronic illness such as CVD, the state and national health care systems may not be able to meet the needs of patients or control spiraling costs, unless the focus of therapy switches away from maintenance and toward cures. Fortunately, the passage of Proposition 71 in 2004, and the subsequent creation of the California Institute for Regenerative Medicine (CIRM), has created the funding needed to advance human embryonic stem cell (hESC) research that could lead to curative therapies that would benefit both millions of Californians and Americans. Recently, engineered cardiovascular tissue grafts, made from rat neonatal cardiomyocytes (CM) and cardiomyocytes derived from mouse ESC, have shown promising results as a future therapy for CVD. The overall goal of our proposed research is to extend these recent studies to hESC and engineer a hESC-CM based cardiovascular tissue graft as a regenerative therapy for CVD. We believe the objectives of our research will benefit the people and the state of California by addressing questions relevant to hESC-based cardiovascular regenerative therapies and will provide vital information needed for safe and efficacious future clinical translation. Development of cures for diseases such as CVD could potentially improve the California health care system by reducing the long-term health care cost burden on California. In addition, the results of our research may provide an opportunity for California to benefit from royalties, patents, and licensing fees and benefit the California economy by creating projects, jobs, and therapies that will generate millions of dollars in new tax revenues in our state. Finally, stem cell research such as ours could further advance the biotech industry in California, serving as an engine for California’s economic future. We have assembled a multidisciplinary team of experienced investigators to attack the objectives of our proposed research. At the same time, we will train and mentor a new generation of bright students and junior scientists in the areas of hESC biology, regenerative medicine, and technology development. This ensures that an essential knowledge base will be preserved and passed on to both investigators and patients within and beyond California.
SYNOPSIS: The Principal Investigator (PI) is a senior vascular surgeon and researcher, whose work has focused on issues of shear stress and atherosclerosis, and he has made important contributions to vascular surgery. His biosketch suggests that he has only more recently begun work on cardiomyocytes. The first aim of this proposal is to electromechanically condition hESC-derived cardiomyocytes in order to modify their spatial and temporal organization. The second aim is to engineer a graft made of mechanically stimulated hESC-derived cardiomyocytes embedded in matrix, and incorporating VEGF. The third, and final, aim is to assess the function of this graft transplanted into rat aorta and then rat heart. IMPACT & SIGNIFICANCE: Cardiovascular disease, including congestive heart failure, is a major cause of morbidity and mortality in the U.S. Better treatment of myocardial infarction and the resulting tissue damage that cannot repair naturally would significantly reduce congestive heart failure. Cell therapies have generated significant excitement for treating/repairing infarcted cardiac tissue and therefore embryonic cells are of interest and are an important research agenda. Many groups are working on developing grafts or patches to augment heart function. The proposal does not bring in unique techniques in that regard. However, there are many new technologies applied in this proposal and its breadth is novel. This project interrogates the role of the environment (seen as a contractile matrix) in organizing hESC maturation into conducting and contracting cardiomyocytes. Its impact is potentially high. It bases its hypotheses on the role of contraction in remodeling and influencing development of embryonic and fetal cardiomyocytes in the developing heart. Given the important role of stretch and rhythmic contraction on the evolution of the developing heart, the application of this approach to hESCs is timely and important. Moreover, given the pioneering work of the Eschenhagen group in this area, the potential for success in at least developing functioning myotubes is high as well. The significance of this work is that the use of stem cell technologies to repair/replace infarcted/failed tissues rests in part on the development of myocytes but also in part on the ability of the developed myocytes to contract in orderly fashion and to exist in a matrix which is not “blown out” by the high pressures developed in the ventricle. Of concern is not only rupture, but also dys-synchronous contraction or aneurismal dilatation. If the proposed work is brought to fruition, it will address these concerns with a matrix that has been tested for its functionality in this setting. QUALITY OF THE RESEARCH PLAN: The quality of the research plan is high, but the quality of the presentation of the preliminary data tempers enthusiasm for the research plan. This proposal has the least amount of preliminary data of any that one reviewer saw in this round of reviews, and s/he believes the proposal might have been better received as a new SEED proposal. The proposal is considered generally well-organized and logical. The investigators have significant experience in the proposed project and the collaboration is interdisciplinary and will likely produce interesting results. The research plan is designed in such a way that both Aims 1 and 2 can be carried out semi-independently of one another. Aim 3 is dependent on success of Aims 1 and 2. In Aim 1, they will condition hESC-derived cardiomyocytes using a microelectromechanical system (MEMS) device integrated with a bioreactor to stretch and electrically stimulate hESC-cardiomyocytes. They believe this will provide the spatial and temporal organization required to create cells that generate enough force to support a failing heart and avoid arrhythmogenesis. The hypothesis to be tested is that in vivo electromechanical conditions that exist in cardiac development are required for the in vitro spatial-temporal organization of hESC-cardiomyocytes. This is a novel and testable hypothesis, and testing this hypothesis will be of use regardless of whether the outcome is positive or negative. The second aim is to engineer a hESC-CM based cardiovascular tissue graft. Their rationale is that mouse ESC-cardiomyocytes, Matrigel, collagen I, and mechanical stimulation have already been combined to engineer a contractile tissue graft. Hence it is not unreasonable to combine hESC-derived cardiomyocytes from Aim 1, Matrigel, collagen I and IV (to enhance cell adhesion), VEGF (to induce vascularization), and electromechanical stimulation. Their hypothesis is straightforward, suggesting that techniques already effective to this end in murine ESC-cardiomyocytes can be extended to hESC-cardiomyocytes and improved upon by the addition of collagen IV, VEGF, and electromechanical stimulation. In Aim 3, they will assess tissue graft viability and function in rat aorta. This is a useful approach as the extremes of contraction and relaxation seen in ventricles will not come into play here. Moreover, they will either patch the aorta directly with the grafts or – if the forces generated in this high-pressure tube are too great and cause rupture - they will wrap the grafts around the aorta. Once function is tested and established here, the next step will be to patch over acutely infarcted rat myocardium. They will then test function via novel molecular imaging techniques, ultrasonography, echocardiography, and electrocardiography to non-invasively assess in vivo graft viability and function. They hypothesize the tissue grafts will remain viable and will exhibit sustained rhythmic contractions leading to improved cardiac function. The approach is thoughtful and maximizes the possibility of obtaining useful answers to the questions posed. Furthermore, there are a number of potential applications for this proposal including the creation of both a cell population for treating myocardial infarction and a tissue graft that can be implanted directly. STRENGTHS: Several major strengths are identified for this proposal. One strength is the team brought together from engineering, biology and medical disciplines. The experience in animal models is also an important historical track record for the lab and the PI has strong preliminary data with mouse cells. The concept of applying “signals” normally found in the tissue of interest is a strength of the proposal and the rationale and design for each of the aims is clearly delineated. The electrical conditioning and bioreactor is novel and the application to hESC will likely lead to interesting results. Hopefully the conditioned cells will not lose the benefits after removal from the conditioning MEMS device and bioreactor. If cell transplantation causes loss of the conditioning benefits it may be possible to incorporate a method to transfer cells on a material that also allows electrical conditioning. Another strength is in the bringing together in a well-organized study design a variety of up-to-date techniques for stimulation and stretching of the cells, as well as for imaging. A particular advantage of the study design is that negative answers will carry significant value and will help focus future efforts. WEAKNESSES: The main point of the proposal is Aim 1, the electromechanical conditioning of the cells. What if there is no advantage found to this procedure? There are many reasons why the conditioning can fail, including the many and subtle physical and chemical parameters, the precise stage of cells used, the absence of other circulating factors or other influences normally present in vivo. Thus while clearly the systems to test this have been developed, as demonstrated in preliminary data, no preliminary outcomes have yet been noted. Since the coordinated electrical function of the cells is the focus of Aim 1, the analyses would be best focused on this issue. All of the imaging modalities are not needed for these analyses, rather the only ones needed focus on the electrical coordination problem, which is the thorny issue. Do the cells used have VEGF receptors? Which cells will contribute to new vessel formation under influence of VEGF (presumably recruited cells and not the graft itself)?. There appear to be no analyses of the effects of VEGF(VEGF has other functions in stem and progenitor cells in addition to being a growth factor for blood vessels). The major weakness of the proposal, according to one reviewer, is related to the biomaterials portion. The preliminary studies show little benefit for cell survival with the materials tested and there is a question of mechanical integrity and survival in the aortic implants. Addition of a materials collaborator would greatly benefit the proposal and could provide a number of methods to strengthen the material physical properties without changing the bulk biological properties. The incorporation of VEGF did not show any improvement in the referenced paper in preliminary data from the PI’s group. Incorporation of VEGF in the constructs should have some influence and the lack of improvement is likely due to release characteristics that are not optimized. Again, a materials or drug delivery expert may help in addressing this problem and aid in the design of appropriate in vitro experiments to optimize this variable. Another weakness was identified with respect to understanding mechanism, in the use of Matrigel (yet this is also a strength of the proposal with regard to the potential for success). The weakness is that in the event that the approach is successful, it will be difficult to ascertain whether (and which) of the components of the matrix contribute to the success of the effort. A second weakness is the use of the matrix to provide a patch over an infarct. The remodeling that occurs in this setting is likely to be complex and may actually be hampered by the extent to which the tissues within the infarct do or do not remodel. An alternative would be to cut out the infarct and do a full-thickness patch of the left ventricle. Of course, this might result in a “blowout” of the patch if it cannot withstand left ventricular pressure. In other words, no approach is optimal, but after testing the system in the way they have planned, it is important that other forms of patching (including full thickness) be tried. The extent to which the exceptionally complex 3-D geometry of the left ventricle, as well as coordinated contraction, is met by a full thickness patch would be of interest as well. One reviewer commented that when reading the proposal it was difficult for him/her to ascertain whether data were generated in the PIs own laboratory, or data were the product of work done by others. For example the bioreactor paragraph contains references to other labs’ work at the end of sentences that could be taken to imply that all the work was done in the PI’s lab “Recently the Zarins lab has shown that…” It would have been better, in this reviewers opinion, to have a clear delineation of work from the PIs lab and work from other labs. One reviewer suggests that the investigators might make use of the following information in their study design. The investigators propose, among other markers, to study connexin43. The fact that Cx43 is present is of great interest as in immature myocardial cells it often is not in as great a quantity as it is with maturity. Moreover, the work of Kleber and others has shown that Cx43 tends to align along the long axis of myocytes as they are subjected to stretch, thereby facilitating conduction of impulses from cell-to-cell. DISCUSSION: There was some disagreement between reviewers about the amount of the preliminary data relative to other applications, as well as questions about the integrity of the bioscaffold, leading to the comment that this group will need help from a biomaterials expert. Questions from the panel were posed concerning the utility of the imaging experiments and the value of the bioreactor in comparison to others. The imaging experiments were described by reviewers as valuable for providing another line of evidence that the implanted cells are retained. There is little basis for comparing the value of various bioreactors because everyone builds their own. Finally, it was noted as a benefit that the PI is in a large group with murine ESC and hESC experience.