Human embryonic stem cells (hESCs) have the potential to become all sorts of cells in human body including nerve cells. Moreover, hESCs can be expanded in culture plates into a large quantity, thus serving as an ideal source for cell transplantation in clinical use. However, the existing hESC lines are not fully characterized in terms of their potential to become specific cell types such as nerve cells. It is also unclear if the nerve cells that are derived from hESCs are totally normal when tested in cell transplantation experiments. One of the goals for our proposal is to compare the quality and the potential of eight lines of hESCs in their capacity to become nerve cells. To measure if the nerve cells that are derived from hESCs are normal when compared to the nerve cells in normal human beings, we will examine the levels of gene expression and the mechanisms that control gene expression in hESC-derived nerve cells. Specifically, we will examine the pattern of DNA modification, namely DNA methylation, in the DNA of nerve cells. This DNA modification is involved in the inhibition of gene expression. It is known that if DNA methylation pattern is abnormal, it can lead to human diseases including cancer and mental retardation disorders. We will use a DNA microarray technology to identify DNA methylation pattern in the critical regions where gene expression is controlled. Our recent results suggest that increased DNA methylation is observed in hESC-derived nerve cells. In this proposal, we will also test if we can balance the level of DNA methylation through pharmacological treatment of enzymes that are responsible for DNA methylation. Finally, we will test if hESC-derived nerve cells can repair the brain after injury . A mouse stroke model will be used for testing the mechanisms stem cell-mediated repair and recovery in the injured brain and for selecting the best nerve cells for cell transplantation. Our study will pave the way for the future use of hESC-derived nerve cells in clinical treatment of nerve injury and neurodegenerative diseases such as stroke and Parkinson’s disease.
Neurodegenerative diseases such as stroke are the leading cause of adult disability. Stroke produces an area of damage in the brain which frequently causes the loss of crucial brain functions such as sensory and movement control, language skills, and cognition capability. Stem cell transplantation has emerged as a method that may improve recovery in these brain areas. Studies of stem cell transplantation after stroke have been limited because many of the transplanted cells do not survive, the appropriate regions for transplantation have not been identified, and the mechanisms by which transplanted stem cells improve recovery have not been determined. Also, there have been no studies of human embryonic stem cell transplantation after stroke. For the use of stem cell therapy in stroke patients, human embryonic stem cell lines have to be grown and tested for their efficacy in repairing the brain after stroke. We have recently found that the process of growing human embryonic stem cells in culture introduces genetic modifications in some of these cell lines that may decrease survival of the cells in the brain and impair their ability to repair the injured brain. The experiments in this grant will determine which human embryonic stem cell lines do not undergo this negative genetic modification. The optimum human embryonic stem cell lines will then be systematically tested for the location in the stroke brain that produces survival and integration, and the mechanisms of repair that these cells mediate in the brain after stroke. These studies will specifically test the role of human embryonic stem cells in improving sensory and movement functions after stroke. In summary, these studies will establish protocols for the proper growth of human embryonic stem cell lines, the lines that are most effective for repairing the brain after stroke, and the principles behind how human embryonic stem cells repair the brain. These results are applicable to other kinds of neurodegenerative conditions, such as Parkinsons, Alzheimer’s and Huntington’s diseases, and to the growth and culture of human embryonic stem cells in general for repair of disease of other human tissues.
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".