Basic Biology V
A central goal of CIRM is to develop methods for differentiating embryonic stem cells into defined tissues for regenerative medicine. This is currently an inefficient process because each cell type requires a unique differentiation regimen that its frequently inefficient. It has been difficult to identify a pathway in the cell that would have a general positive effect on all tissues. We believe we have found such a pathway that lies at the core of gene expression. We have designed a series of experiments in human and murine ES cells to test our idea. The simplicity of our idea is that it involves a protein termed HP1γ that substitutes a positively acting factor, Med26, for a negatively acting enzyme termed Cdk8. Our proposal is designed to provide a firm mechanistic understanding of this pathway and to target it using molecular methods. If successful, our work will pave the way for testing Cdk8 chemical inhibitors to promote differentiation in a wide range of systems. This is not an unrealistic goal as pharmaceutical companies are now targeting Cdk8 because it is also involved in cancer.
Statement of Benefit to California:
A major goal of CIRM is to create the framework for generating stem cell therapies that will be developed by California universities, hospitals and biotechnology companies. An understanding of how stem cells differentiate into various tissue types is essential for translating stem cell therapies into the clinic. Our research focuses on a general mechanism that we believe all stem cells use during differentiation. Our study provides both knowledge of how this mechanism influences differentiation and studies to apply it to neurons.
The in vitro differentiation of embryonic stem cells (ESC) into different cell lineages is currently an inefficient process, and each cell type requires a unique differentiation regimen. This Exploratory Concepts proposal is based on the hypothesis that the protein HP1γ can promote differentiation of human ESC toward any lineage by reprogramming the core transcriptional machinery to favor expression of lineage-specific genes, and the corollary idea that by manipulating expression of HP1 γ or associated proteins it may be possible to greatly improve the efficiency of differentiation of ESC toward many different lineages. To test these hypotheses, the applicant will first test whether overexpression of HP1γ promotes differentiation of human and mouse ESC (Aim 1), and then will study the effects of overexpression and knockdown of two HP1γ-associated proteins on differentiation of ESC (Aim 2). Finally, the applicant proposes to examine how the physical interaction of HP1 γ with a second protein might promote ESC differentiation (Aim 3). Novelty and Transformative Potential - If modulation of HP1γ or associated proteins can be demonstrated to promote much more efficient differentiation of human ESC to multiple lineages in vitro, that result would potentially be transformative to the field. Feasibility and Experimental Design - There were insufficient plans to demonstrate that the neural stem cells (NSC) differentiated using the proposed methods are bona fide NSC. It is critical to show that the proposed method of differentiation does not alter the nature of the differentiated cells. - There was concern that differentiation of only one cell type (NSC) would be studied. In order to be transformative, the new methodology should be applicable for differentiation of multiple cell types. - Reviewers expressed some concern that HP1γ has not been reported by others to be associated with the RNA Pol II complex, despite years of studying that complex. - Aims 2 and 3 are not focused on the key question, which is whether overexpression of HP1γ aids in the differentiation of human ESC, and probably should not be done if the results Aim 1 do not support the hypothesis being tested. Principal Investigator (PI) and Research Team - The PI has a long track record of high-quality publications in the study of molecular control of transcription, the main area of this grant proposal. - The collaborating teams are well chosen for their role in the project according to their area of expertise. Responsiveness to the RFA - The identification of a novel technique to increase the efficiency of differentiation of human ESCs into various lineages could transform the ESC field and is therefore responsive to the RFA.
- Alexander Meissner