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RS1-00164-1: Induction of cardiogenesis in human stem cells via chromatin remodeling

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

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 (provided by applicant)

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.

Review

SYNOPSIS: The long-term goal of this proposed research is to devise a strategy to reprogram human embryonic stem cells (hESC) 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 genes with a specific component of a chromatin modifying complex can activate several cardiac genes in cultured cells. The first Aim will determine whether hESC lines can be induced toward a cardiac fate by the forced expression of these genes. Aim 2 will use technology that modifies gene expression to test whether the specific chromatin remodeling factor is required for the normal or amplified development of cardiac cells. Aim 3 will examine the role that certain molecular changes in the chromatin remodeling factor play in cardiac differentiation of hESC.

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 a specific chromatin remodeling factor can enhance cardiac differentiation in ES cells by co-expression with known cardiogenic genes or by modifying induced cardiac differentiation. The investigator further proposes to ask whether molecular changes in the chromatin remodeling factor are 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 hESC. Another innovative aspect of this proposal is the insight into the requirement for a combination of genes 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 genes and chromatin factors required for effective directed differentiation of ES cells toward the cardiac myogenic fate.

STRENGTHS: There is a gap in knowledge 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 applicant’s institution, including a nascent hESC facility, and the demonstration that a particular cocktail of genes is sufficient to activate several cardiac genes in a certain cell type and may also expand the field of cardiomyocytes in certain mouse tissue systems. The layering of multiple factors is novel and interesting, and could substantially move the field forward if successful. Also, the potential that a similar cocktail might direct hESC to cardiomyogenic differentiation is important to test.

WEAKNESSES: A main concern is the assumption that the cocktail of genes effective to transform cultured cells to a cardiac myogenic phenotype will also be effective with ES cells. The ES cell transcription program and chromatin structure appears to be substantially different than those of differentiated cell types; in particular, the apparent general repression of differentiation genes by Polycomb complexes. 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 the experiments with the cocktail of genes and the specific chromatin remodeling factor. Because the project depends on this initial demonstration, data from a proof-of-principle experiment with mouse ES cells would be valuable, although not required for SEED proposals.

The experimental plan for Aim 3 does not indicate which molecular changes in the chromatin remodeling factor or how many different changes 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 molecular changes be designed to test them? Moreover, the molecular changes proposed do not always create an effective tool. While not a requirement for SEED grants, no preliminary data is provided to justify Aim 3. In particular, the activity of the molecular change in another assay dependent upon this factor 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 modifications of gene expression of hESC, 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 a lot of interest and attention. The PI is very well trained and has access to good reagents. The approach of looking at a gene activated chromatin remodeling complex is a valuable approach, but there is limited detail or rationale presented. Reviewers noted that there were problems with the structure of the experiments. Two types of information were stated without detail: 1) the nature of the molecular changes and 2) the induction of cardiac phenotype in cells. For example, what is the nature of induction in these specific cells? How many genes are up-regulated, which ones, and what is the nature of the phenotypic change? One suggestion would be to differentiate human embryonic stem cells (hESC) into mesoderm first, then ask whether these genes can move the cells further towards a cardiac state. Also, Aim 3 was very brief. Which molecular changes in the chromatin remodeling factor will be made, and will multiple changes be required?

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None