Understanding differentiation of human embryonic stem cells (hESCs) provides insight into early human development and will help directing hESC differentiation for future cell-based therapies of Parkinson’s disease, stroke and other neurodegenerative conditions. The PI’s laboratory was the first to clone and characterize the transcription factor MEF2C, a protein that can direct the orchestra of genes to produce a particular type of cell, in this case a nerve cell (or neuron). We have demonstrated that MEF2C directs the differentiation of mouse ES cells into neurons and suppresses glial fate. MEF2C also helps keep new nerve cells alive, which is very helpful for their successful transplantation. However, little is known about the role of MEF2C in human neurogenesis, that is, its ability to direct hESC differentiation into neuronal lineages such as dopaminergic neurons to treat Parkinson’s disease and its therapeutic potential to promote the generation of nerve cells in stem cell transplantation experiments. The goal of this application is to fill these gaps. The co-PI’s laboratory has recently developed a unique procedure for the efficient differentiation of hESCs into a uniform population of neural precursor cells (NPCs), which are progenitor cells that develop from embryonic stem cells and can form different kinds of mature cells in the nervous system. Here, we will investigate if MEF2C can instruct hESC-derived NPCs to differentiate into nerve cells, including dopaminergic nerve cells for Parkinson’s disease or other types of neurons that are lost after a stroke. Moreover, we will transplant hESC-NPCs engineered with MEF2C to try to treat animal models of stroke and Parkinson’s disease. We will characterize known and novel MEF2C target genes to identify critical components in the MEF2C transcriptional network in the clinically relevant cell population of hESC-derived neural precursor cells (hESC-NPCs). Specifically we will: 1) determine the function of MEF2C during in vitro neurogenesis (generation of new nerve cells) from hESC-NPCs; 2) investigate the therapeutic potential of MEF2C engineered hESC-NPCs in Parkinson’s and stroke models; 3) determine the MEF2C DNA (gene) binding sites and perform a “network” analysis of MEF2C target genes in order to understand how MEF2C works in driving the formation of new nerve cells from hESCs.
Statement of Benefit to California:
Efficient and controlled neuronal differentiation from human embryonic stem cells (hESCs) is mandatory for developing future clinical cell-based therapies. Strategies to direct differentiation towards neuronal vs. glial fate are critical for the development of a uniform population of desired neuronal specificities (e.g., dopaminergic neurons for Parkinson’s disease (PD)). Our laboratory was the first to clone and characterize the transcription factor MEF2C, the major isoform of MEF2 found in the developing brain. Based on our encouraging preliminary results that were obtained with mouse (m)ESC-derived and human fetal brain-derived neural precursors, we propose to investigate if MEF2C enhances neurogenesis from hESCs. In addition to neurogenic activity, we have shown that MEF2C exhibits an anti-apoptotic (that is, anti-death) effect and therefore increases cell survival. This dual function of MEF2C is extremely valuable for the purpose of transplantation of MEF2C-engineererd neural precursors. Additionally, we found MEF2 binding sites in the Nurr1 promoter region, which in the proper cell context, should enhance dopaminergic (DA) neuronal differentiation. We hypothesize that hESC-derived neural precursors engineered with MEF2C will selectively differentiate into neurons, which will be resistant to apoptotic death and not form tumors such as teratomas. We believe that our proposed research will lead us to a better understanding of the role of MEF2C in hESC differentiation to neurons. These results will lead to novel and effective means to direct hESCs to become neurons and to resist cell death. This information will ultimately lead to novel, stem cell-based therapies to treat stroke and neurodegenerative diseases such as Parkinson’s. We also believe that an effective, straightforward, and broadly understandable way to describe the benefits to the citizens of the State of California that will flow from the stem cell research we propose to conduct is to couch the work in the familiar, everyday business concept of “Return on Investment.” The novel therapies and reconstructions that will be developed and accomplished as a result of our research program and the many related programs that will follow will provide direct benefits to the health of California citizens. In addition, this program and its many complementary programs will generate potentially very large, tangible monetary benefits to the citizens of California. These financial benefits will derive directly from two sources. The first source will be the sale and licensing of the intellectual property rights that will accrue to the state and its citizens from this and the many other stem cell research programs that will be financed by CIRM. The second source will be the many different kinds of tax revenues that will be generated from the increased bio-science and bio-manufacturing businesses that will be attracted to California by the success of CIRM.
SYNOPSIS: The proposal seeks to determine if Myocyte Enhancer Factor-2 C (MEF2C) can instruct human embryonic stem cell (hESC)-derived neural progenitor cells (NPCs) to differentiate into neurons, including dopaminergic neurons, and to promote recovery in stroke and Parkinson’s disease models. The aims are to: 1) Determine the function of MEF2C during in vitro neurogenesis from hESC-NPCs using wild type, constitutively active and dominant negative mutants of MEF2C in order to determine cell fate after modulation of MEF2C levels. 2) Investigate the therapeutic potential of MEF2C in Parkinson’s and ischemia models by transplanting MEF2C-engineered hESC-NPCs into Parkinson’s or ischemia mouse models in an effort to improve functional recovery. 3) Determine genome-wide DNA targets of MEF2C and perform network analysis of MEF2C targets using serial analysis of chromatin occupancy (SACO) and weighted gene coexpression network analysis (WGCNA) IMPACT AND SIGNIFICANCE: This proposal seeks to use, essentially, a one gene approach (i.e., MEF2C), to determine if expression in hESCs can direct progenitors towards neuronal phenotype. The significance of this proposal lies in the fact that it would be a major step forward if, certainly, a single gene approach could be used to selectively differentiate hESCs into neurons. Of course, these must be resistant to cell death and be non-tumorigenic as well. Using such a directed approach to isolate and expand large numbers of neuronal cells would be quite useful in transplantation studies and for therapeutic based cell approaches. The importance of this application is in developing improved means for highly directed differentiation of hESC-NPCs to generate neurons of importance in tissue replacement. This group has shown MEF2 proteins play roles in neuronal survival and differentiation and can promote differentiation of P19 cells into neurons and that MEF2C binds within 1.3 kB of the Nurr1 gene 5’ region. This finding may be important for cell-based therapies for Parkinson's disease, as the function of transcription factor Nurr1 is critical for the development of neurons with dopaminergic phenotype, and overexpression of Nurr1 in neural precursors directs them towards the dopaminergic (DA) lineage in vivo if placed into the proper context. QUALITY OF THE RESEARCH PLAN: Overall, this research proposal is well-designed and feasible. The quality of the proposal really hinges on the experience of the investigator and the importance of the problem. As we are reminded in this proposal, the PI was the first to clone MEF2C and has now shown, at least in mouse cells, that it has the capacity to direct neurogenesis. The preliminary data supports this and shows that at least one promoter that is a direct target of MEF2C is activated and that it seems to exhibit an anti-apoptotic function and therefore increases cell survival. The research plan is, pretty much, very standard and benign. It proposes over, under expression in hESCs, use of dominant negatives and knockdowns and finally characterization of direct targets using SACO technology. The major detraction of this proposal is that it is simply transference of using MEF2C in the mouse to the human system where it is unclear whether we will learn anything new. The experimental approach is extremely straightforward and in addition, is essentially a one-gene approach. The preliminary data are reasonable but not completely convincing. It is significant that the proposal includes biologic characterization of MEF2C directed neuronal cells via transplantation and this would be the first step of pre-clinical developments of this. The PI has also established important collaborations. One reviewer is unsure that the proposal conforms to the RFA designed to support mature ongoing studies of hESCs, as this scientist does not have a record in this field. However, it is a reasonably promising new direction if MEF2C could be used to expand and stably maintain these hESCs for transplantation. STRENGTHS: The strengths of this proposal are several. First, the importance of the research topic is high and the PI has experience in the analysis of MEF2C. In addition, the PI has assembled a team of collaborators that is outstanding as well as institutional support. There is an over-riding hypothesis being tested and multiple techniques are brought to bear on the project including the use of in vivo systems for analysis of MEF2C defined neuronal phenotypes. Substantial preliminary data suggest that the factor of interest plays a role and the research plan is feasible. WEAKNESSES: The weaknesses cited for this proposal include the fact that this is a single gene approach, there are marginal and ill-defined differences between expanding well established principals on MEF2C from the mouse to human hESCs, and it is unclear whether this is a promising new direction as a specified criterion in the RFA. A fundamental weakness is that no preliminary data is presented on hESCs for any aim of this application. Thus, while there is no doubt about the ability to carry out the proposed research, the lack of any data on hESCs does weaken enthusiasm. DISCUSSION: The PI is an expert with MEF2C (i.e., cloned MEF2C) and from a neurology perspective (electrophysiology, etc.) these are extremely elegant studies. The directed differentiation of dopaminergic neurons with this factor addresses a really important problem. The PI has the neural precursor cells, which is the most interesting aspect of this grant. The PI has done all the work in the mouse, and making human cells using this transcription factor is very important to test. Unfortunately, this is an interesting but benign proposal in that the strategy is extremely basic (over and underexpression studies, examining where MEF2C sits on chromatin), and the PI doesn’t know what he wants to do with these cells. A wide range of techniques is brought to bear with electrophysiology and multiple therapeutic models. The MEF2C genetic analysis to find MEF2C targets has all the problems of a fishing expedition, but it is being done by investigators who are considered to be "good fisherpeople". The PI could have done some of these experiments using the human population that is proposed. One needs to believe in the continuity between the mouse and human cells, otherwise there is no project if the human cells can’t be made. One reviewer felt that MEF2C needs to be tested in human cells because of the work in mouse. This maybe one of the factors that plays a role in neurogenesis and thus should be tested, and this is the best lab to do the work. The molecular biology is not stellar or imaginative, but it is good. Steven Goldman just published on creating domaninergic neurons from hESCs but not with directed differentiation. These cells became tumorigenic, probably because they lacked an essential purification step. This work will be more careful.