Cardiovascular disease (CVD) is the leading cause of death in the United States. Over one million Americans will suffer from a new or recurrent heart attacks this year and over 40 percent of those will die suddenly. In addition, about two-thirds of the patients develop congestive heart failure; and in people diagnosed with CHF, sudden cardiac death occurs at 6-9 times the general population rate. Heart transplantation remains the only viable solution for severely injured hearts; however, this treatment is limited by the availability of donor hearts. Therefore, alternative strategies to treat end stage heart failure and blocked blood vessels are needed. The objective of this proposal is to determine whether human embryonic stem (hES) cell can be used for repairing the heart. Our collaborator Advanced Cell Technology (ACT) has recently succeeded in identifying conditions for the reproducible isolation of hES cells which have the characteristics of cells which form blood vessels and heart muscle. This proposal will assess whether the hES cells can form new functional blood vessels and repair injured heart muscle in a rat model of heart attacks. Results from these studies will help develop new therapies for treating patients with heart attacks.
Cardiovascular disease (CVD) is the leading cause of death in California and the United States. Over one million Americans will suffer from a new or recurrent myocardial infarction this year and over 40 percent of those will die suddenly. In addition, about two-thirds of myocardial infarction patients develop congestive heart failure. The 5-year mortality rate for CHF is about 50%, and in people diagnosed with CHF, sudden cardiac death occurs at 6-9 times the general population rate. Heart transplantation remains the only viable solution for severely injured hearts; however, this treatment is limited by the availability of donor hearts. It is estimated that health care costs for CVD is over 18 billion dollars a year. Additionally, the morbidity associated with CVD cost California and the nation billions of dollars a year. Therefore, alternative strategies to treat end stage heart failure and ischemia are needed. (Source: American Heart Association. Heart Disease and Stroke Facts, 2004, Dallas, TX: AHA 2004; American Heart Association. Heart Disease and Stroke Statistics-2006 Update, Dallas, TX: AHA 2006).
The field of regenerative medicine is important to California and the nation. Advances in the technology to find cell based therapies will be revolutionary in their impact on patient care. Human embryonic stem (hES) cells have the potential to become all of the cells in the human body, and their unique properties give researchers the hope that from these primitive cells new therapies can result that may be available in time for the looming health care crisis. This project is focused on a pre-clinical application of a specific hES cell based therapy for myocardial regeneration and an antibody targeting technology to direct stem cells to injured organs. This project will benefit California in several ways including: 1) support for UC trainees, 2) potential of developing important clinical trials in CA based on results from this proposal, and 3) enhancement of the biotechnology industry in CA which would lead to the creation of new jobs in CA and an enhanced tax base.
SYNOPSIS: This proposal takes advantage of a novel resource, human embryonic stem cell (hESC)-derived embryonic progenitor cells and hESC-derived hemangioblasts (isolated and characterized by Advanced Cell Technology i.e. ACT). The Specific Aims are:
1. To test the hypothesis that hESC-derived progenitors that express high levels of angiogenic factors can induce angiogenesis, decrease myocardiac infacrtion (MI) size and improve left ventricular (LV) function in rats.
2. To test the hypothesis that the hESC-derived hemangioblasts (+/- hES-derived progenitors) can induce functional recovery after rodent ischemia-reperfusion injury.
3. To test the hypothesis that hESC-derived progenitors that express high amounts of myocardial specific message can improve LV function in a rat ischemia-reperfusion MI model.
Although the PI states the proposal is risky the preliminary data are fairly compelling.
IMPACT & SIGNIFICANCE: This proposal explores the possibility of treating ischemic cardiomyopathy in an established small animal model with several types of hESC-derived progenitor/differentiated cells populations with hemangioblast/angiogenic/cardiac attributes. If the studies achieve their goals, they could also be applicable to other forms of ischemia such as organ, limb, and cerebral ischemia. The improved treatment of cardiovascular disease in a less invasive way with a cellular therapy would be highly significant.
The impact of the research derives largely from the testing of three important hypotheses:
1. hESC-derived embryonic progenitors expressing high levels of angiogenic factors induce angiogenesis, decrease infarct size and improve LV function in a rodent ischemia-reperfusion model;
2. hESC-derived hemangioblasts alone and/or in combination with hESC-derived embryonic progenitors, expressing high levels of angiogenic factors, induce angiogenesis in a rodent ischemia-reperfusion model;
3. hESC-derived hemangioblasts alone and/or in combination with hESC-derived progenitor cell lines, which express relatively high levels of mRNAs found in myocardial cells, improve LV function in a rodent ischemia-reperfusion model.
These are important hypotheses as they combine the identification of cells of myocardial lineage with that of cells which will optimize blood flow into the ischemic/infarcted zone of the heart. A critically important aspect of the research and one which may be a major contributor to the success of the studies is the use of bispecific antibodies for delivery of hESC-derived cells to regions of cardiac injury.
The significance of the research derives from its potential to identify cell lineages that will optimize both myocyte replacement and blood flow in jeopardized regions of the heart. Additional significance comes from gaining increased experience with bispecific antibodies as a delivery tool.
In summary: Cardiac disease is a major cause of morbidity and mortality and stem cell therapies for failing or infarcted hearts deserve intensive study. The PI incorporates two novel resources into the proposal, the hemangioblast and progenitor cells derived and cloned by ACT and, the bifunctional antibodies which may be useful generally for aiding stem cells in finding the right location where they are needed for repair.
QUALITY OF RESEARCH PLAN: Overall, the research proposal is of high quality. As presented, the work is ambitious but do-able. It is ambitious in terms of man-power: about 3 FTEs (or a bit less) are assigned, the PI seems spread out on a lot of other projects, and the only full-time devoted person is a technician.
The PI has a record of productivity and so the work is likely to proceed as outlined.
The research plan is well-thought-out and presented. Although somewhat repetitive in details, the experimental plan is described with satisfactory clarity to perceive the intended experiments. Three different hESC-derived cell populations (hemangioblasts, putative cardiac progenitors [clone #34], and derivatives expressing some angiogenic molecules) will be studied for their ability alone and in combination to contribute functional and anatomical myocardial repair in a coronary ligation model of rat myocardial ischemia. The investigators has extensive experience with this model and it is appropriately conducted in an immunodeficient rodent model. In aim 1, cells expressing high levels of angiogenic factors will be screened and characterized and then the best line chosen for further study. It is anticipated that promotion of vessel formation in a HUVEC in vitro assay will predict in vivo angiogenesis. Improved function will be ascertained by echocardiography and improved perfusion by microSPECT imaging, techniques that the investigator has used previously and has established collaborations. Aim 2 involves parallel studies with hESC-derived hemangioblasts with and without angiogenic molecule expressing cells. They will compare intramyocardial and intravenous administration and use GFP-labeled cells to identify the survival/migration/position of transplanted cells by immunofluorescence microscopy, which is an important aspect of these studies. Aim 3 will focus on the effects of hESC-derived myocardial cells again with and without hemangioblasts. Although it is not precisely determined at what stage these cells are or whether they represent multiple different cardiac cell types, these studies are important to carry out. After intramyocardial injection, animals will be assessed for function by echocardiography, arrhythmic potential by ventricular fibrillation threshold testing, and electrical communication by high density optical mapping. The provenance of the new myocardial cells will be determined by IHC co-expression studies with HLA and other markers.
The availability of a large number of hESC-derived cell lines from ACT provides a useful and important starting point. Beyond this, the strategy explores the establishment of a vascular bed in its own right, using a clinical trial approach to perform studies in an appropriately powered number of animals and to evaluate them via echocardiography as well as via studies using fluorescent microbeads and fluorescent dyes. The effects of the cell implants to alter infarct size and contractile function as well as angiogenesis all will be determined. Experiments are performed in vitro and in vivo. Both high density mapping and optical mapping of hearts are proposed to evaluate electrical communication. Ventricular fibrillation thresholds will be used to assess proarrhythmic potential.
One reviewer expressed some concern that the design of the experiments does not take important controls into account. The main criticism is that the design of experiments is not optimized to determine whether the in vitro assays actually predict function in vivo. For example, the cells that are most angiogenic in vitro will be used in vivo. Why not compare to cells that are poor at angiogenesis, so that the predictive power of the in vitro assay can actually be determined? Similarly in Aim 3, why not compare cells from clones that express myocardial genes to those that do not? These seem like lost opportunities to really get an integrated picture of the biology.
STRENGTHS: One strength of the proposal is the collaboration with ACT which provides access to cell lines and characterized clones of progenitor populations have a lot of potential for myocardial repair but have not been extensively studied. The preliminary data demonstrate that the cell lines, imaging tools and expertise are available to do the proposed studies, and support the directions taken and methods used by the investigators. Experience with the animal models is also a strength. The Principal Investigator (PI) presents a well-thought-out series of experiments that build on the PI’s strengths and the expertise of his collaborators. The synergistic collaborations with ACT and with Drs. Dae and Gao for radiological and nuclear imaging in small animals add to the feasibility of the studies proposed. One reviewer pointed out that a specific strength is the importance of optimizing both myocardial mass and myocardial perfusion in settings of cardiac ischemia and/or failure. This project is feasible with high pay off if successful.
WEAKNESSES:-One reviewer felt that the main weakness is a lack of cohesiveness between the in vitro assays and the in vivo assays. It would also be of interest to test other factors in vitro that have been associated with bone marrow stem cell mobilization, to see if the cells are responsive to these signals, such as GM-CSF. Finally, given that VEGF can have independent cytoprotective effects beyond promoting new vessel growth, it would enhance the proposal to have VEGF (and receptor) abundance measured in the hearts (and blood), so that they can independently be analyzed (in addition to vessel formation) as mediators of improved heart function.
Another reviewer noted the following specific concerns:
1: The proposal is designed to test hypotheses within a clinical trial framework. Therefore, we may learn whether an approach is/is not effective, but will not necessarily learn why the outcome is positive or negative.
2: While the use of bispecific antibodies is exciting, there is no attempt made to understand whether and/or the extent to which complexing with the antibodies modifies the function of the cells to be delivered, including their ability to further differentiate/proliferate in the infarction setting.
3: since arrhythmogenicity has been identified as one possible outcome of hESC administration, more than a ventricular fibrillation threshold should be studied as a means to understand whether arrhythmias do or do not occur. The mapping experiments to be performed will address whether cell activation is synchronous or dyssynchronous, but will not identify likelihood of arrhythmias. It is probable that telemetry is the most accurate means of doing this.
A third reviewed noted minor weaknesses and made specific recommendations:
·It is possible that nude rats, which retain some B cell function, will demonstrate some immunological activity to hESC-derived grafts. Anti B cell or anti NK cell treatment may be necessary to prevent destruction of cells.
·What is the anticipated nature of the angiogenic compound producing cell populations? What do these cell lines/populations represent? This alone would be quite interesting and may be revealed by these studies. However, it is not clear yet that these cell lines produce or secrete large amounts of protein, as only gene expression profiles have been partially characterized.
·It might have been useful to propose using the BiMab technology with IV hemangioblasts in Aim 2 as another experimental group but this was not considered. The BiMab technology is proposed briefly but these experiments as written are not logical. The epitope on injured myocardium that would be recognized by the antibody is not indicated, nor is preliminary proof of concept data to demonstrate efficacy provided. The majority of the studies can be performed without this technology.
·Also in Aim 2, it would be important to study where GFP labeled hemangioblast cells home to in their studies.
·Angiogenic, hemangioblast and myocardial cell lines are not well characterized, and have not been shown to have a stable phenotype.
DISCUSSION: This is a proposal to test ACT's hESC-derived progenitor lines in cardiac ischemia models. While acknowledging some risk to the proposal given that not much is known about the cells, the reviewers found the preliminary data compelling. A major issue is that the PI does not propose to do the experiment that would prove their hypothesis. The reviewers suggest including a cell line that does not express angiogenic genes as a negative control. The animal model system(s) are nicely worked out and there is a nice synergy with the models and the imaging studies. The radiology and imaging capacity is strong at this institution. These studies are along the lines the field should be going. One reviewer would have liked to see the molecular biology integrated a bit better with the functional studies - that is making the connection between the in vitro assays and the in vivo model.
Another missing experiment is to use telemetry to test the spontaneous incidence of arrhythmia. One reviewer felt that the missing experiments were a problem with this application, and that the PI should have known better since he is an expert in arrhythmia.
One reviewer liked the plans to characterize the ACT cells using immunohistochemistry as opposed to just gene expression. (The ACT hemangioblast and cardio lines seem to have some characterization beyond transcriptional profiling.) This reviewer also liked the examination of five different culture conditions, including plating at low density and low oxygen; the testing of expanded vs. diluted lines, cryopreserved and not the group-selected line 34 for further studies. Another reviewers pointed out that since the angiogenic lines are not well characterized, they could be endothelial progenitors; there is also no comparison to what is currently in the literature.
The microPET work is good, and the PI should try to correlate this with other imaging work. The MiMAP technique is also interesting in allowing the cells to home, but it was noted that this is not developed at all (although another reviewer pointed out that this group has published on the technique before). Other big questions are whether the investigators will be able to image at the single cell level, and whether the PI has underestimated the immunology barriers with the xenograft work (even though the in-vivo model in mice is nice).
The reviewers hope that the PI will integrate the biology better with the functional analyses. These cells (the progenitor lines from ACT) are a novel resource which makes them innovative and interesting. The field still don’t know how cells are making a vascular bed; most people are simply popping in hESCs and “getting heart”. We need to understand the relationship between vascularization and heart function, and with the right techniques, this group will get the answer. For example, VEGF works as a cryoprotective as well as a vasculogenic, and while the PI didn’t acknowledge this complexity, reviewers hope that they look at this.