Heart disease is one of the biggest killers in the civilized world, and as populations age, this trend will increase dramatically. Currently the only way to treat failing hearts is with expensive and relatively ineffective drugs, or by heart transplantation. Ideally, we would like to be able to regenerate sick or dead heart tissue. The best strategy would be to make new heart cells that match the patients' cells (to avoid rejection), and inject them into diseased heart so that they could regenerate the sick heart.Unfortunately, current strategies that are planned to do so are ineffectual. We wish to attempt to generate heart cells from human embryonic stem cells by "reprogramming" the stem cells into heart cells. This would be accomplished by turning on heart genes that normally are off in stem cells and seeing if this turns stem cells into heart cells. If this approach is successful, these newly generated stem cells could be used for regenerative therapies in the future.
Statement of Benefit to California:
The proposed research willl likely be of great benefit to the State of California and its citizens, as it will provide to possible means to generate therapeutically relevant heart cells from stem cells. As heart disease is the number one killer in California and throughout the US, our findings will help eradicate this disease.
SYNOPSIS: The long-term goal of this proposed research is to devise a strategy to reprogram human ES cells toward a cardiac lineage by directing chromatin-remodeling complexes to cardiac-specific genes. The key insight behind this approach is the demonstration that co-expression of the cardiac transcription factors Nkx2-5, Gata4 and Tbx5 with the cardiac-restricted BAF60c subunit of the Bmi1 chromatin modifying complex can activate several cardiac genes in cultured fibroblasts. The first Aim will determine whether human ES cell lines can be induced toward a cardiac fate by the forced expression of a similar cocktail of transcriptional activators. Aim 2 will use short hairpin RNA technology to test whether BAF60c is required for the normal or amplified development of cardiac myocytes in differentiating human embryoid bodies. Aim 3 will examine the role that BAF60c activation by phosphorylation plays in cardiac differentiation of human ES cells. SIGNIFICANCE AND INNOVATION: The damaged heart, with its prevalence in western society and its accessibility to manipulation, is a prime target for repair by cell therapy, yet little progress has been made. Stem cell technology that would provide a replenishable, patient-specific source of cardiac myocytes that could repair damage by incorporation into heart tissue would be an important advance. This proposal asks whether the chromatin remodeling factor BAF60c can enhance cardiac differentiation in ES cells by co-expression with known cardiogenic transcription factors or by modifying phamacogically-induced cardiac differentiation. The investigator further proposes to ask whether a phosphomimetic of BAF60c is active in driving cardiac differentiation. This is a novel and interesting hypothesis that could significantly enhance our understanding of cardiac regeneration, and define a novel mechanism for improving cardiac differentiation protocols from hES cells. Another innovative aspect of this proposal is the insight into the requirement for a combination of transcriptional regulators augmented by a particular form of a chromatin modifying complex to induce cardiac-specific myogenesis. The research plan will use state-of-the-art conventional approaches to define the transcription and chromatin factors required for effective, directed differentiation of ES cells towards the cardiac myogenic fate. STRENGTHS: There is a gap in our understanding of directed differentiation into cardiac myocytes. The proposed approach to systematically examine the known genes required for cardiac development is a sound and practical approach. The PI shows a good working knowledge of the strengths of the approaches and limitations in working with these cell lines. The key strengths of this proposal are the accomplishments and inventiveness of the principal investigator who has an outstanding track record in the field of cardiac development, the resources at the Gladstone Institutes including a nascent human ES cell facility, and the demonstration that a particular cocktail of transcriptional activators (Gata4, Nkx2-5, Tbx5 and Baf60c) is sufficient to activate several cardiac genes in fibroblast cell lines and may also expand the field of cardiomyocytes in mouse embryoid bodies. The addition of layering an epigenetic activator with these transcription factors is novel and interesting, and could substantially move the field forward if successful. Also, the potential that a similar cocktail might direct human ES cells to cardiomyogenic differentiation is important to test. WEAKNESSES: A main concern is the assumption that the cocktail of transcription factors effective to transform cultured fibroblasts to a cardiac myogenic phenotype will also be effective with ES cells. The ES cell transcription program and chromatin structure - in particular, the apparent general repression of differentiation genes by Polycomb complexes - appears to be substantially different than those of differentiated cell types, including fibroblasts. Consequently, it may not be feasible to jump from the ES cell state directly to the cardiomyogenic fate. An alternative plan should be in hand, such as using in-vitro protocols to direct ES cells first toward early mesoderm differentiation to release chromatin restrictions before transfection experiments with transcription factors and BAF60c. For example, because BMP4 and activin are effective enhancers of early cardiomyocyte differentiation of human ES cells in a chemically defined culture medium, these cells may be a more optimal starting point. Because the project depends on this initial demonstration, data from a proof of principle experiment with mouse ES cells would be valuable. The experimental plan for Aim 2 does not indicate which of the 35 serines in BAF60c are phosphorylated in response to p38 activation and how many different phosphomimetics or combinations would need to be analyzed to provide a convincing test of the hypothesis. If other signaling pathways are involved, as proposed, how would additional phosphomimetics be designed to test them? Moreover, aspartate substitution for a phosphoserine does not always create an effective phosphomimetic. While not a requirement for SEED grants, no preliminary data is provided to justify Aim 3. In particular, the activity of the phosphomimetic in another assay dependent upon BAF60c function should be provided. While it is an interesting idea, it is too preliminary to support without better experimental validation. From the publication record, it is not clear that the assembled team has established the specific expertise needed for effective culture, differentiation and multiplex transfections of human ES cells, which generally are more difficult than mouse ES cells to manipulate. DISCUSSION: The area of inducing cardiac differentiation from ES cells holds great promise and has captured much interest and attention. The PI is very well trained and has access to good reagents. The approach of looking at a transcription factor-activated chromatin remodeling complex is a valuable approach, but possibly due to the lack of a requirement for preliminary data there is limited detail or rationale. Reviewers noted that there were problems with the structure of the experiments. Two types of information were stated without detail: 1) the role of phosphorylation and 2) the induction of cardiac phenotype in fibroblasts. For example, what is the nature of induction in fibroblasts? How many genes are up-regulated, which ones, and what is the nature of the phenotypic change? There is a lot of information without the particulars. One suggestion would be to differentiate hESC into mesoderm first, then ask whether these transcription factors can move the cells further towards a cardiac state. Also, Aim 3 was very brief. Which Serine residues (phosphorylation sites) on BAF60c will be modified, and will multiple mutations be required?