Basic Biology V
Virtually all cells in our body possess an identical genetic code. However, the expression of this code is regulated by a multitude of factors that coordinate which portions of the genetic code can be read and interpreted. We further know that these so called epigenetic factors change drastically through development, and patterns of epigenetic marks control changes to cellular identity. The proposed research aims to site-specifically change key epigenetic marks, altering which DNA instructions are available to pluripotent cells and changing their behavior during cell fate commitment. We describe a technique to bind custom DNA binding proteins (TALES) to critical regulatory regions of known cell fate determining genes. TALE binding changes local DNA chromatin structures and implants instructions for becoming desirable cell types when given an appropriate environmental cue. This novel and potentially transformative approach, termed "epigenetic engineering" (EE), is proposed here to improve stem cell differentiation towards more pure and functionally mature cardiomyocytes. This will not only be the first example of guiding cell fate through gene-specific epigenetic manipulation, but will improve the generation of necessary cardiomyocyte types for cell replacement following heart attack. Lastly, EE also is likely to be adaptable to numerous differentiation strategies for routine production of many regenerative medicine cell types.
Statement of Benefit to California:
Heart disease and myocardial infarction represent the primary cause of death in industrialized nations. In California, greater than 1 out of 4 deaths are due to heart disease. The health and financial burdens are enormous. Currently, the most promising new strategies for heart regeneration involve replacement of dead or damaged tissue with cardiomyocytes (CMs) derived from pluripotent stem cells; however, the control of pluripotent cell fate is problematic. Pluripotent-derived-CMs are typically impure and functionally immature, so they may not contribute to heart contractility effectively or safely. We propose the development of a novel technology, termed “epigenetic engineering (EE),” to control cell fate and improve yields of mature CMs. EE involves the stable manipulation of stem cell chromatin structures, without DNA breaks or mutations, so as to “set the stage” for correct differentiation. When given an appropriate environmental cue, the engineered stem cells can then interpret these instructions to guide their cell fate commitment. This technology will improve the routine production of pure and functional CMs for cell replacement strategies that could significantly augment the current standard of care. Also, and highly important, EE is likely to be adaptable to alternative differentiation strategies (e.g. insulin producing beta-cells) and therefore potentially transformative for regenerative medicine.
The goal of this Track 2 (Exploratory Concepts) proposal is to develop a technology to induce transient epigenetic changes in human embryonic stem cells (hESCs) that induce their differentiation towards specific lineages and silence the differentiation to alternative cell fates. In so doing, the applicants hope to achieve higher purity and yields in the differentiation process. This approach will be tested through two specific aims that will create and test DNA sequence-specific transcription activator-like effectors (TALES) designed to guide hESCs towards a cardiomyocyte phenotype, and then examine whether the TALES impart an epigenetic memory that persists and regulates differentiation. Novelty and Transformative Potential - The proposal to modulate the pluripotent epigenome to direct stem cell differentiation was felt to be novel and somewhat innovative. - While the concept was novel, fundamental flaws in logic and design led to the conclusion that the transformative potential of the proposal would be low. Feasibility and Experimental Design -Technically, the studies outlined to induce the epigenetic engineering in specific loci are sound and feasible. - Reviewers felt that the induced epigenetic memory would be too transient to have a meaningful and sustained effect on differentiation as this “memory” erases with passage of the cells. - Reviewers felt that there are already many ways to direct differentiation of hESC towards specific lineages and a convincing rationale for developing the proposed approach was not found. - The preliminary data provided is not impressive and suggests that the approach does not regulate target gene expression to an extent that would be desirable to affect reprogramming and differentiation. Principal Investigator (PI) and Research Team -The PI and assembled team are skilled and capable of performing the outlined research although the relative contribution by some personnel was minimal. - Productivity of the PI in recent years has been minimal. Responsiveness to the RFA -The goals outlined in the proposal are responsive to the CIRM RFA.