Over 5 million Americans suffer with congestive heart failure, and 500,000 new cases are diagnosed each year. Unfortunately, these numbers are likely to worsen over the next two decades, as the total number of heart failure patients is expected to rise by over 50%. Ischemic heart disease is the most common cause of heart failure, and stem cell transplantation may represent the first realistic clinical strategy for actually reversing the deleterious effects of what has been considered irreversible damage to the heart resulting from myocardial infarction. However, significant hurdles must still be overcome before successful human translation of this strategy can be realized; in particular, a means is needed both for preventing the rapid death of most of the transplanted cells and for encouraging their functional contribution to the heart. We propose a strategy, termed Matrix-Assisted Cell Transplantation (MACT), that has been developed by a collaboration of bioengineers and cardiac surgeons/cell biologists to improve the efficacy of stem cell transplantation into a heart injured by MI. This strategy is based upon novel biomimetic polymers that will: 1) provide a foundation for increased stem cell survival, and 2) enhance the functional capacity of transplanted stem cells in an animal model of MI. The goal of our research proposal is to establish artificial extracelluar matrices that enhance the survival and performance of cells transplanted into ischemically damaged heart muscle and that, within a 4 year period, can begin pre-clinical testing for preservation of cardiac function and prevention of post-MI heart failure.
Statement of Benefit to California:
The proposed research will benefit California by preserving and strengthening California’s position as a leader in the emerging field of human stem cell therapeutics. Through the recent passage of Proposition 71, the voters of California have identified stem cell research as a key area of focus for the state, with anticipated positive impacts including: creation of biotechnology jobs, attraction of leading researchers to Californian universities, creation of valuable intellectual property, and advancement of therapeutics beneficial to California’s residents. This proposal will bring California’s diverse technical resources to bear towards this goal by combining the expertise of leading academics in the fields of biomaterials and bioengineering with world-leading clinicians to the field of human embryonic stem cell therapeutics. The use of the synthetic matrices developed in this collaboration for the manufacture of hESC-derived cell therapeutics will increase the potential availability of such treatments to patients, while at the same time reducing the risk of zoonoses from such therapies. More than 2 million Californians stand to benefit from development of hESC-based therapies in our program of heart failure treatment, many more could be affected by the translation of our technology to similar therapies for additional degenerative disorders such as Parkinson’s disease, Alzheimer’s disease, diabetes, rheumatoid arthritis and osteoporosis.
SYNOPSIS: The investigators propose to deliver an injectable artificial extracellular matrix (ECM) to act as a stem cell carrier and protective microenvironment; a mechanical support to the myocardial wall; and to suppress post-myocardial infarction (MI) congestive heart failure (CHF). They call this Matrix-Assisted Cellular Transplantation (MACT). Specific aims are to: 1) create a synthetic protocol to differentiate human embryonic stem cells (hESCs) into cardiomyocytes and characterize the cells via immunohistochemistry, flow cytometry, protein/gene expression, and electrophysiology; 2) identify the integrins presented by hESC-derived cardiomyocytes and develop peptide ligands that promote adhesion of cells to materials via the integrins; 3) develop biomimetic artificial ECMs to optimize survival and proliferation of hypoxic cardiomyocytes in vitro; 4) assess survival and growth patterns of cardiomyocytes, after MACT into infarcted mouse hearts. This will allow them to assess whether enhanced cell survival correlates with improvement in post-MI cardiac function. IMPACT & SIGNIFICANCE: This proposal focuses on a huge, important medical problem, i.e. improving current medical treatment of CHF which carries a 50% mortality rate by 5 years into its clinical course. The use of an artificial ECM as part of the strategy may be generalizable to other areas of stem cell research. CHF is a serious public health problem. Better treatment of MI and the resulting tissue damage that cannot be repaired naturally would significantly reduce CHF. Cell therapies have generated significant excitement for treating/repairing infarcted cardiac tissue and therefore embryonic cells are of interest. Most approaches to stem cell therapy for heart disease to date have depended on injection of cells into the peripheral vasculature, into the coronary vessels or into the myocardium. While numbers of cells administered has been controlled, there has been variable success in seeing them localize to sites where their impact would be greatest, and still less success in demonstrating impact that is physiologically significant, as opposed to merely statistically significant. Greater success has been seen in animal models in which the pathologic environment has been more controllable than in human subjects. Several groups have been experimenting with biomaterials to aid in delivering cells, localizing cells and maintaining their presence at sites. Experimentation has also focused on the therapeutic potential of the biomaterials themselves. The impact and significance of the work done to date by this team is that they have developed a synthetic ECM system the structure of which is completely known, and which is a polymer hydrogel. Working with this ECM they can independently vary matrix stiffness and the quality of cell adhesion. They have shown that over the short term this matrix can be used to maintain hESCs in culture. The fact that the matrix is biodegradable by matrix metalloproteinases is useful as the persistence long-term of the matrix in cardiac tissues after cells have had a chance to integrate and remodel might be a drawback. An advantage of their matrix design over that of other matrices is that its simplicity permits addition of signaling components and systematic exploration of the conditions that will optimize cell survival and differentiation. Given the complexity of existing matrices, it is far more difficult to interpret which of their components is important to any outcome achieved via their manipulation. The part of the project that deals with biomaterials development and application has a highly significant potential in that the knowledge about biomaterials will be important in advancing this field. However, the hESC project is of exceptionally high risk in the sense that it depends entirely on the ability of the investigators to force hESCs along a cardiac lineage as a result of their growth on the matrix. If this does not occur with the matrix “as is”, then the numbers and combinations of factors that might be needed to bring about such an outcome is daunting. Also, whether the function of the matrix and its potential for being biodegraded will occur in the infarct and/or heart failure environment is not certain. QUALITY OF THE RESEARCH PLAN: The application justifiably pays a great deal of attention to the qualities of the matrix itself. In aim 1 a clear plan is presented for differentiating hESCs into cardiomyocytes and characterizing them via immunohistochemistry, flow cytometry, protein/gene expression, and electrophysiology. Regrettably, the plan as proposed depends on the work of others using Matrigel as a substitute for preliminary data and on an intention to replace the Matrigel with their own matrix and then to vary ligand type and density to optimize conversion to cardiomyocytes. There are simply no preliminary data to demonstrate any success or potential for success here. Aim 2 identifies the integrins presented by hESC-derived cardiomyocytes and proposes to develop peptide ligands that promote adhesion of cells to materials via the integrins. The experimental plan here is a logical one, although it again depends upon one of the subset of peptides they have identified as potentially providing the right outcome. Aim 3 develops biomimetic artificial ECMs to optimize survival and proliferation of hypoxic cardiomyocytes in vitro. This is an intriguing approach wherein so-called “tunable” hydrogels are used to establish design rules for engineering and improving survival and proliferation for transplanted hESC-derived cardiomyocytes. The problem is that the aim depends entirely on the success of the first aim, regarding cardiomyocyte differentiation from hESCs. Aim 4 assesses the effects of peri-infarct implantation of hESCs on survival and growth patterns of cardiomyocytes in infarcted SCED mouse hearts. The injection will be 1 h after infarction. Again, there is the need for Aim 1 to succeed here (not to mention aims 2 and 3) and the identification of a peri-infarct zone is problematic because the infarct can be unstable in size for a longer period than this. As a result, they may be too far from the infarct to be successful, or alternatively, the site injected may be within the infarct zone. In summary, this proposal presents the application of injectable hydrogel synthetic-biological composite materials for transplanting embryonic derived cardiomyocyte progenitors to treat MI. Cell death is a serious problem/barrier to cell transplantation in cardiac applications. The plan is logical and described in a straightforward manner. It incorporates the establishment of an ECM platform (semi-interpenetrating polymer networks or sIPNs) to facilitate hESC self-renewal, and this possibility is supported by published information from the PI using hESCs. The strategy of using female recipients for transplantation of male-derived cells in order to examine SRY gene copy number by RT-PCR is attractive. The main strength of the application is the materials expertise and analysis. The weaknesses are associated with biological analysis and stem cell biology. The collaboration and cardiac application is new and therefore introduces an element of risk. STRENGTHS: The strengths of the proposal lie in the investigators’ knowledge of the biomaterials to be used and in their innovative development of them to date. The research plan is very direct and straightforward. It incorporates interesting material science and engineering work as well as molecular technology to examine outcomes of matrix-supported hESC product. The team of invesatigators the PI has recruited is strong. The main strength of the proposal is related to the biomaterials design and the ability of the group to rationally design and test a wide array of materials. There are very few materials/engineering groups that also have the capability of to culture and evaluate embryonic stem cells. The testing and optimization protocol is another strength of the proposal. The composite synthetic and peptide gels are innovative and are likely to have a number of potential clinical applications. These materials have not yet been applied to transplanting stem cells and are likely to have a positive impact on cell survival which is a serious barrier to success in the cardiac application of stem cells. WEAKNESSES: One of the reviewers identified a number of weaknesses which together cast serious doubt on the prospects for even partial success of this ambitious study. These are partially enumerated below, and are tied to the individual aims: Aim 1: The investigators state on p5 that the method they will use routinely generates cardiomyocytes of up to 80% purity without further purification and produces yields of cardiomyocytes sufficient for their in vitro and in vivo experiments. Whether this refers to their own work (and their own figure 5e and 5f does not demonstrate cells that can be interpreted as potentially functional mature cardiomyocytes) or that in reference 24 is uncertain. But reference 24 presents results in which a particular environment is used to maintain and expand hESCs and differentiate them into the three germ layers. So it is difficult to see the support for the investigators’ contention. And this is critical to the success of their entire study. As for measuring contraction using electrograms to record peak-to-peak amplitude “and depolarization and repolarization processes,” about all that will be valid from such measurements will be the frequency of contraction, and this can more easily be done via edge detection. Finally, stating there should be no significant problems in differentiating hESCs into cardiomyocytes that fulfill the needs of this study is a particularly disingenuous statement, given the lack of information provided by the reviewers to consider. Aim 2 suffers from the same concern about failure to consider alternatives should the specific peptides selected fail to work out. Aim 3 has some of the most innovative strategies proposed in the application. Yet in describing the experiments, there is insufficient attention to detail. What is the amount of O2 needed by the cells? If they are not contractile and not actively involved in energy expenditure, what do they really need? How will the 1% be maintained, and will it be maintained throughout the study period? As for the H2O2 experiment, how will the mechanisms of effects that occur be tested? For example, will there be any consideration of the p38 system of MAP kinases which figure importantly in the pro-apoptotic effects of free radical generation? If mechanism is not to be understood, then how can one go beyond what may turn out to be an untoward result. Aim 4: Not only is there concern about the method and site for implantation of cells but also about the method used to isolate and implant the cells. Are the cells implanted on the matrix? Are they removed? Does removing them and resuspending them for administration have untoward effects? How will the arrhythmogenic effect of the implant referred to by the investigators be assessed and differentiated from that of the infarct? How will the arrhythmogenic effect of cells versus matrix be understood? And what is meant by the “conductivity” of the matrix-cell complex and how will this be assessed? Another reviewer also found shortcomings in the experimental plan. (1) Although measures of proliferation, apoptosis, and cell death will be collected, there is no attempt to document actual increases in cell mass or number to evaluate success. (2) Major emphasis is placed on survival and proliferation of cardiomyocytes with the sIPNs, but the functional impact of enhanced donor cell survival will be assessed in vitro only by serial electrocardiographic analysis. The experiments in an animal model of myocardial infarcton followed by in vitro analysis of the transplanted cells are laudable, but an in vivo counterpart, to evaluate the functional success of the cells will be very important to the eventual success of this work. It is not clear whether any of the data in Fig 6 of the Preliminary Results are collected from in vivo experiments. A third reviewer noted that this is a new area of application for the material and a new collaboration. The initial peptide-biomaterial scaffold that is proposed is the basic RGD peptide used to culture the cells in an undifferentiated state. Some statements in the proposal refer to improving cell survival and also differentiation/purity. It is suggested that some growth factors (Shh and VEGF) may be incorporated into the system but this is vague and a challenging proposition. It is feasible that incorporation of peptides will improve cell survival but choosing peptides to promote (or select) a specific cell lineage will be more difficult and is not thoroughly defined. Understanding the integrin profile of adult cardiomyocytes may also help to improve the rationale. Many of the weaknesses in this proposal are related to the fact that this is a very new project for the group and preliminary data on the topic is not yet there. However, the team is excellent and with the incorporation of a stem cell biologist and/or someone with expertise in cardiac biology many of the weaknesses may be addressed. DISCUSSION: This proposal is based on the belief that ESC-derived cardiomyocyte therapy hinges on cell survival in the host, and providing an injectable artificial ECM creates a protective microenvironment for the cells in the myocardium. The primary reviewer really liked the idea, but the part (s)he liked best was the part (s)he knew the least about. This reviewer believes that success of the proposal hinges on integration into myocardium. Rather than using their own biomaterials, the PI used Matrigel as the matrix in generating the preliminary data. This is a problem because we know very little about what Matrigel really is. Other questions included what is "conductivity" of a matrix-cell complex. The PI’s claim is that the methods used here produce 80% purity of differentiated cardiomyocytes, but it is unclear whose work is this claim based on. There is little experimental detail, for example there is no examination of whether low O2 would help, or what the effects of tension would be. While this was the strongest of the biomaterials proposals from the biomaterials perspective, there was no data relevant to the application of the material to developing cardiomyocytes. All the preliminary data involved cells in an undifferentiated state. The reviewers agreed that this was a nice biomaterials approach. There are not too many strong biomaterial groups that can also work with ESCs. One nice aspect of this proposal was the idea of trying to figure out what integrins are involved in the growth of these cells, and somehow incorporating that knowledge into improving the biomaterial system.