Epigenetic gene regulation during the differentiation of human embryonic stem cells: Impact on neural repair
$2 516 613
Statement of Benefit to California:
SYNOPSIS OF PROPOSAL: The investigators are proposing to study the relationship between DNA methylation and neural differientation of human embryonic stem cells (hESCs). They will analyze 8 lines for their efficiency in neural differentiation, determine genome-wide DNA methylation patterns of human promoters during this process, inhibit expression of DNMTs to modulate this process, and correlate methylation variations with efficacy of neural repair in a mouse stroke model. IMPACT AND SIGNIFICANCE: The role of DNA methylation in differentiation is central but poorly understood, and even less clear in hESCs. The choice of a well defined cell biological model, relevant to potential stroke therapy, suggests a high degree of significance of the results to be obtained by the study. The individual approaches are not novel, but the combination of cell and molecular biological approaches is. The applicant has developed reliable protocols for directed neural differentiation of hESCs. The researchers propose to study DNA methylation, effects on gene expression and whether they can be reversed with the DNMT inhibitor AzaC. A stroke model using immunocompromised mice and transplantation experiments are proposed to study methylation effects on neural repair in vivo. QUALITY OF THE RESEARCH PLAN: This is a well focused proposal that takes on the difficult problem of hESC epigenetics by choosing a particular differentiation system, namely neural differentiation, and combines it with multiple biologic replicates and careful validation. Three Wisconsin/UCSF lines and five lines from UCLA will be studied with careful attention to culture condition and analysis with differentiation markers, backed up by preliminary data. They have already established a differentiation protocol for NPCs. The lines will be characterized with neural and glial markers, and genome-wide DNA methylation analysis will be performed. The researchers plan to use MeDIP, in collaboration with Howard Cedar who has published on this method. It is encouraging that they will use this approach rather than hybridization to CpG islands as in their recent HMG paper. Nevertheless there are significant problems with the antibodies recognizing CpG rich sequences, not simply methylated sequences. Still they plan validation using independent methods and have shown their ability to do this. An experimental manipulation of the system is proposed whereby DNA methylation will be blocked. They thoughtfully propose knockdown of DNMT’s, noting that DNMT3A is expressed in stem cells. They propose two molecular approaches and realize that 5-azacytidine might have independent toxic effects and thus avoid it. The use of an experimental manipulation adds a dimension to the experimental plan that brings it beyond a simple phenomenological study. The relevance of the methylation study to a mouse stroke model will be addressed by introducing cells into SCID mice that have been experimentally given cortical infarcts. Perhaps a bit unfocused, the direct application to a therapeutic model is compelling, especially in the context of CIRM. However, it will be somewhat difficult to determine what specific methylation changes might be related to enhanced migration and differentiation of transplanted cells. This is a very well written application with a careful and detailed experimental plan that appears to be quite feasible. With the proposed use of SCID mice for xenotransplantation, the researchers avoid the issues associated with external immunosuppression. The PI has many excellent collaborators who can help him with many aspects of the experiments. This is an outstanding grant proposal. STRENGTHS: The focus and PI are excellent, the proposal is well written, the experimental plan is feasible, a well defined biological system is proposed, the preliminary data are good, careful methylation analysis and validation are proposed, and an attempt has been made to define causal relationships with experimental manipulation. The translation of these studies to a rodent therapeutic model is also a strength. WEAKNESSES: There are no significant weaknesses noted. DISCUSSION: This proposal was ranked as the best among those read by one of the reviewers. The applicant is taking a central stem cell question, the role of epigenetics, and approaching it "head on". An epigenome profile will be created using immunoprecipitation of methylated DNA and oligonucleotide arrays. The team will look at neural differentiation with a variety of markers and selection in culture to assess effects of manipulation of methylation marks. The applicant is collaborating with reseachers at Hebrew University in Jerusalem, has worked on 3 cell lines, and plans to add 5 additional lines to the studies. The discussants agreed that the objectives of the research plan are exciting and the proposed functional studies with the mouse stroke model are important. The approaches were regarded as nicely complementary. One reviewer believed that useful information was almost certainly going to result from the proposed studies, and others agreed that the proposed work is "state of the art".