Five million people in the U.S. suffer with heart failure, at a cost of $30 billion/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. Human embryonic stem cells grow and divide indefinitely while maintaining the potential to develop into many tissues of the body, including heart muscle. They provide an unprecedented opportunity to both study human heart muscle in culture in the laboratory, and advance cell-based therapy for damaged heart muscle. We have developed methods for identifying and isolating specific types of human embryonic stem cells, stimulating them to become human heart muscle cells, and delivering these into the hearts of mice that have had a heart attack. This research will identify those human embryonic stem cells that are best at repairing damaged heart muscle, thereby treating or avoiding heart failure.
More than 90,000 people in California suffer with heart failure, at a cost of ~$540 million/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. This research will identify human embryonic stem cells that are able to repair damaged heart muscle, thereby treating or avoiding heart failure. The medical treatments developed as a result of these studies will not only benefit the health of Californians with heart failure, but also should result in significant savings in health care costs. This research will push the field of cardiovascular regenerative medicine forward despite the paucity of federal funds, and better prepare us to utilize these funds when they become available in the future.
SYNOPSIS: The investigators identify and characterize myocardial cells derived from hESCs and use these to model myocardial development and explore cell therapies for myocardial diseases. Their goals are to determine subpopulations of hESCs that develop into subspecialized cardiomyocyte types, to identify the developmental stage at which hESC-derived myocardial cells most effectively engraft into host tissue, and to demonstrate that hESC-derived cells can be used to improve cardiac function following myocardial injury. The specific aims are to: 1) identify specific subpopulations of proliferating hESCs that preferentially differentiate into cardiomyocytes; 2) determine the developmental stage at which hESC-derived myocardial cells most effectively engraft in vivo and the role of the tissue environment in cardiomyocyte subspecialization; 3) demonstrate the effects of cell therapy with hESC-derived myocardial cells in a mouse model of myocardial infarction.
IMPACT AND SIGNIFICANCE: This is a potentially high impact project as it systematically explores hESC properties from the point of view of identifying potential markers for the cardiac lineage and then attempts to optimize their development into that lineage. They state accurately that they have concentrated on developing methods and reagents that are readily transferable, and ultimately will allow them to evaluate new hESC lines for their cardiogenic properties. In other words, having commenced to work on lines that are federally permitted, they believe the information gained will be transferable to lines not currently available via federal support. The statement that the “observations made will provide new insight into human cardiac myogenesis, and offer novel approaches to myocardial regeneration.” is an accurate one.
The significance of the project is seen in an approach that initially uses standard techniques to identify the markers that characterize those hESCs that go along a cardiac lineage and then to do so in a chamber-specific and tissue-specific fashion. They will then proceed to determine which developmental stage most favors successful engraftment in vivo, as well as the characteristics of the environment that influence engraftment, and finally they will test the robustness of the cells delivered as a therapy for myocardial infarction.
Given the incidence of infarction and heart failure in the US today and the limitations in treatment, this represents a carefully thought out and bold step into obtaining information that can bring us along the road to new therapies through an understanding of mechanism.
Many cardiology groups are working on stem cell repair for MI, and it is also probably one fo the most active translational areas for hES research. In this regard the application is not unique. However, the applicant is fearless in adapting new technologies to this work, and has put together a fairly comprehensive plan to move the field forward.
The proposal is of high scientific and clinical significance, which largely relates to its potential value for the development of a routine and reproducible strategy for the isolation of enriched populations of cardiac progenitors from differentiating human ES cells. This is absolutely essential to eventually allow human ES cells to be used effectively as models of human disease, as well as for any potential clinical applications down the road. The isolation of these committed progenitors might conceptually alleviate the potential problem of the formation of teratomas that is a problematic feature of the parental ES cells themselves, lead to the identification of the pathways that guide the formation of the progenitors, their renewal, and directed differentiation into specific progeny.
QUALITY OF THE RESEARCH PLAN: The high quality of the research plan stems from the application of techniques that are within the investigators’ capabilities to test a series of important hypotheses, the results of which will certainly guide further research and will likely bring us further along the way to understanding how hESCs might be used as therapy for one of the major public health problems in the US today. Each specific aim is carefully thought out, and the measures to be brought to bear will all help the field move forward. To illustrate: for specific aim 1 (identify specific subpopulations of proliferating hESCs that preferentially differentiate into cardiomyocytes) they will differentiate specific hESC subpopulations into cardiomyocytes in vitro, and characterize developmental- and chamber-specific gene expression and action potential propagation. They have already established methods for hESC sorting based on surface markers, will attempt to force them into a cardiogenic lineage by reasonably described methods, and will then further characterize them via gene expression as well as electrophysiologically, via action potential properties. For specific aim 2 (determine the developmental stage at which hESC-derived myocardial cells most effectively engraft in vivo and the role of the tissue environment in cardiomyocyte subspecialization), they will develop reporter hESC lines and "molecular beacon" strategies for isolating myocardial cells at specific developmental time points, and determine their engraftment and differentiation in mouse myocardium in vivo. This approach will be facilitated by using reporter ESC lines (obtained from Bruce Conklin) and so-called molecular beacon FRET analysis that will facilitate isolation of cells at specific developmental time points. Both GFP and surface markers will be used as intentionally redundant guidelines for following engraftment and further differentiation in mouse heart in vivo. For aim 3 (demonstrate the effects of cell therapy with hESC-derived myocardial cells in a mouse model of myocardial infarction) they will evaluate cardiac function and electrical conduction in a mouse model of myocardial infarction following transplant with hESC-derived myocardial cells. Here, standard means for quantifying ventricular function, in vivo as well as optical mapping to study integration of cells will be done.
Overall the quality of the proposed research is high. There are some difficulties in interpreting the feasibility of the timeframe for the work because of the way the research plan is presented:
1- The combinatorics for the sorting in the first aim are not well explained. Will the cells be sorted for expression each marker individually (or in what combinations)?
2- The use of FRET is both a strength and a potential weakness, as it may take time to make the system work. Also in Aim 2 it is not apparent how ‘pure’ the various differentiated populations will have to be to get a clean answer about particular gene expression patterns vs. engraftment ability.
Although there is confidence expressed that the whole myocardium conduction studies will work, there are no preliminary data presented though the PI states that mouse studies have been performed. It would have been helpful to know what changes in the mapping were observed after mouse coronary ligation, for example, to have a sense of the sensitivity of the technique for this proposed research.
The proposal, particularly the first two specific aims, is of high quality. The approach is rigorous, carefully designed and well thought-out. The study has risks that are inherent to the system, as there are no well accepted protocols or markers to purify the cardiac progenitors from human ES cells, and the means to expand them and maintain their potency also has not been established. Thus, there are no guarantees that the experimental approach will ulitmately be successful, but at the same time the plan is extremely well-thought out and is likely to yield information that will be valuable in establishing human ES cell based systems as models of human cardiovascular biology and disease.
STRENGTHS: The strengths of the proposal are that it approaches an important medical issue, myocardial infarction and resultant heart failure, uses an innovative approach of the investigators and skilled collaborators and consultants to evolve a strategy for working with hESCs to solve the problem, and works with protocols that will definitely advance our knowledge and might provide cells to be used for therapy. At this stage in the hESC field, one cannot ask for much more. The experimental designs are innovative and the likelihood of at least partial success is high.
The applicant has some novel methods to offer for this research. The co-culture system of labeled (sorted) and unlabelled supporting hES for experimentation, for example, is a nice system that can yield clean experiments for statistical analysis. Also the relatively non-invasive method of delivering cells into myocardium under high resolution ultrasound guidance is also a very useful tool for these studies
The identification of atrial vs. ventricular markers is also a plus, but it would be nice if cells that generated these characteristic markers were later analyzed (compared) for engraftment ability--It is possible that ventricular types might be better at engrafting?
1) The preliminary data for first two specific aims and the expermiental design is excellent
2) There is a high likelihood that well-characterized, available antibodies that have been used to FACS sort other human cells, in the hands of experienced investigators, may prove quite useful in developing a sorting strategy that significantly enriches the population of cardiac precursors from differentiating human ES cells. A combination of negative and positive markers, even if they are not completely cell type restricted, could be valuable here.
3) The lentiviral vectors that will be employed have been well characterized.
4) The scientific environment at UCSF is superb for these studies, with world class expertise in human ES cells and cardiovascular developmental biology available on site. There is clear evidence that the group of investigators involved is a cohesive, as evident from the preliminary data to date.
WEAKNESSES: An important weakness, as recognized by the authors, is that given the number and heterogeneity of surface markers in undifferentiated hESCs they may not be able to identify which of the markers available is /are indicative of future development into a cardiogenic (much less chamber-specific) cell line. Indeed this step is the Achilles heel of the proposal, as without the proper identification of cells the project cannot move forward. Yet the importance of making this attempt is so critical, not only to their research, but to research in the field, that this is an attempt that should be made.
Another concern is that although the authors have listed a variety of markers and properties that cardiomyocytes derived from hESCs share with mature cardiomyocytes, one cannot make the assumption that this commonality of properties indicates that commonality of function will see in the mature hESC-derived myocyte. To their credit, the investigators recognize the issue here and attempt to bring in environmental factors as an additional important determinant of properties.
Another weakness of the proposal as written (although not as conceived) is that there were major problems with the referencing that made the proposal more difficult to review than should have been the case. The authors are requested to review their references 48-55 with regard to how they are used in the text to convey assurance of their abilities to make and evaluate the murine myocardial infarct. In fact, the mouse infarct model has been used by the investigative team, although the writing of the grant makes it difficult to perceive this.
A concern is the potential overlap with federal grants. If in fact the proposed work is to be done uniquely on non-federally-approved cell lines, this would be reassuring. Nonetheless there should be administrative review of this proposal in light of already funded grants to make any adjustments in funding needed to ensure that overlap is eliminated.
The potential for tumor formation is handled by relatively short-term experiments (60 days) in which cells are incubated under the kidney capsule, then analyzed histologically for teratoma. Then the cells that can form teratomas are not used for the cardiac transplant studies. It seems that this is an opportunity lost to determine whether the chosen teratoma assay actually predicts tumor formation in the heart over the long-term.
Enthusiasm for the proposal was lowered by the overlap of this proposal with others. There are three grants held by the applicant that are cited as having overlap with the CIRM proposal, totaling over $800,000 in support, which is a significant concern. Both the NIH grant and the MDA grant have the same two specific aim summaries as Aims 1 and 2 in the CIRM grant.
1) The proposal is extremely ambitious. If the first two specific aims cannot be accomplished, the third is a moot point. The investigators should be encouraged to forgo the studies on attempting to examine the potential therapeutic benefit of the progentiors for regenerative therapy. It would first be important to make sure that the efficiency of conversion to the cell types of interest was sufficiently robust to justify the in vivo work. Simply injecting the cells into a normal heart to check for differentiation potential would be sufficient, with mulitple controls for fusion, etc.which would be necessary. This would allow focus on the devleopment of the first two specific aims, which is where the impact and novelty of the study lies.
DISCUSSION: Although there are some weaknesses, the high impact of the project, the preliminary data, the careful planning and the experience of the investigators all led to considerable enthusiasm for this proposal.