Coronary artery disease is a leading cause of mortality in the USA, and cannot be cured. Although Stem Cell therapy has been initiated in several cases of cardiovascular dysfunction, all current efforts (including a major clinical trial) are aimed at repairing the myocardial component of the heart. To date, little attention has been dedicated at the potential repair of the damaged coronary vasculature. The goal of this proposal is to lay groundwork for coronary vascular therapy based on embryonic stem cell technology. We hypothesize that human embryonic stem cells have the potential to generate coronary progenitor cells that can be clonally expanded. We also hypothesize that this clonally expanded population will retain the potential to differentiate into the diverse components of the coronary vasculature.
Statement of Benefit to California:
This proposal investigates new avenues for the cure of coronary artery disease, which is an increasing cause of mortality in the population of California.
SYNOPSIS: The goal of this project is to generate epicardial progenitor cells (EPCs) from hES-derived EBs, and subsequently coax them to differentiate in vitro into vascular structures. The idea is to translate what is known about EPCs in the mouse to the human system and ultimately to provide a means to regenerate coronary artery tissue. Aim 1 is to isolate lines of EPC. The idea is to use the chicken Gata5 promoter to drive expression of GFP and Puro, in order to select for EPC lineage. The Gata5 promoter has been useful in mice for targeting expression of Cre to the epicardium. A battery of EPC lineage regulatory factors will be used in culture with the hope that these will direct expression of the promoter effectively: FGF, RA, Wnt3a, BMPs, PDGFBB, HGF, etc. A battery of markers will also be evaluated to confirm if lines are EPC-like. If the approach fails, the PI proposes to embark on a high-throughput screen to define the factors, in collaboration with Dr. Mercola. The subsequent Aims determine the potential of EPCs to undergo epithelial-to-mesenchymal transformation (EMT) and to assess the ability of EMT-transformed epicardial cells to form vascular structures - to undergo vasculogenesis and angiogenesis, both in vitro and in vivo. Based on the success of this work, the PI will use hESC-derived epicardial cells in animal models to lay the groundwork for cellular therapy of coronary disease. SIGNIFICANCE AND INNOVATION: The significance for developing a method of isolating and expanding EPCs is very high and could impact a variety of major coronary and heart diseases. The innovation is to translate the known pathways in mouse to the human ES system. This proposal focuses on the utility of hESCs as a tool for the generation of coronary artery precursors. Clearly, cardiovascular disease is the #1 cause of death and disability in our society, and therefore represents an obvious target for new therapeutics. The significance of the proposal, however, is limited by the investigator’s understanding of cardiovascular disease. The PI does not make clear distinctions between coronary artery disease and congestive heart failure, which are two different clinical processes. More importantly, the PI does not adequately address how coronary arterial precursors will help in the setting of an occluded coronary vessel. The PI states in the abstract that “all current efforts. . . are aimed at repairing the myocardial component of the heart”, a statement that is simply not true (see the work of Doris Taylor, among others). The PI fails to distinguish how the proposed approach would be different or better than other ongoing approaches. This is a major limitation of the proposal. STRENGTHS: The PI has experience studying the development of the epicardium in mouse. The investigative team is skilled, the environment excellent. The in vivo data are compelling in that the PI can mark the proepicardium and its derivatives. Assuming the team is able to capture precursors appropriately, the plans for differentiation and in vitro functional assays are straightforward WEAKNESSES: It is not at all clear that the EB system can recapitulate normal epicardial development. Even in the mouse system, the Gata5 promoter is not fully restricted to EPCs. A major potential issue is whether the “selected” cells, once so induced, will maintain any progenitor cell state rather than going on to differentiate. The protocols to generate the EPCs do not differ significantly from those used for subsequent differentiation, so why should they remain stem-like? The combinations of factors proposed for analysis reads very much like a “laundry list” but one that can grow indefinitely, considering different concentrations and timing of addition. When is the selection drug added? What are the benchmarks and quantifiable assays that will determine success or failure? Consideration of experiments in the 2nd and 3rd Aims is difficult pending demonstrated feasibility for Aim1. It seems likely that this project could be more productive at this time in the mouse ES system. There is no evidence that coronary precursors can be derived from ES cells of any species. If aim 1 is unsuccessful, the rest of the aims cannot be implemented. There is no evidence that pure populations of precursors would be captured; the marker also tags derivatives as well as hepatic cells. A clear pathway to identifying the precursors is neither considered nor explained. DISCUSSION: There was no discussion following the reviewers' comments.