Basic Biology II
There are now viable experimental approaches to elucidate the genetic and molecular mechanisms that underlie severe brain disorders through the generation of stem cells, called iPS cells, from the skin of patients. Scientists are now challenged to develop methods to program iPS cells to become the specific types of brain cells that are most relevant to each specific brain disease. For instance, there is evidence that defects in cortical interneurons contribute to epilepsy, autism and schizophrenia. The experiments proposed in this grant application aim to understand basic mechanisms that underlie the development of cortical interneurons. We are discovering regulatory elements (called enhancers) in the human genome that control gene expression in developing interneurons. We have three experimental Aims. In Aim 1, we will study when and where these enhancers are expressed during mouse brain development. We will concentrate on identifying enhancers that control gene expression during development of specific types of cortical interneurons, although we hope to use this approach for additional cell types. Once we identify and characterize where and when these enhancers are active, in Aim 2 we will use the enhancers as tools in human stem cells to produce specific types of cortical interneurons in the test tube. The enhancers will be used to express proteins in the stem cells that will enable us purify only those cells that have specific properties (e.g. properties of cortical interneurons). In Aim 3 we will explore whether the human brain produces cortical interneurons in the same way as the mouse brain; this information is essential to identify molecular markers on the developing interneurons that could be used for further characterization and purification of the interneurons that we care generating in Aim 2. We want to emphasize that while the experiments focus on cortical interneuron subtypes, our work has general implications for the other types of brain cells our labs study, such as cortical and striatal neurons. In sum, the basic science mechanisms that we will discover will provide novel insights into how to generate specific types of neurons that can be used to study and treat brain diseases.
Statement of Benefit to California:
Large numbers of California residents are stricken with severe medical disorders affecting the function of their brain. These include epilepsy, Parkinson’s Disease, Alzheimer’s Disease, Huntington’s Disease, Autism and Schizophrenia. For instance, a recent report from the Center for Disease Control and Prevention [www.cdc.gov/epilepsy/] estimates that 1 out of 100 adults have epilepsy. In California, epilepsy is one of the most common disabling neurological conditions, with approximately 140,000 affected individuals. The annual cost estimates to treat epilepsy range from $12 to $16 billion in the U.S. Currenlty up to one-third of these patients are not receiving adequate treatment, and may benefit from a cell-based transplantation therapy that we are currently exploring with our work in mice. There are now viable experimental approaches to elucidate the genetic and molecular mechanisms that underlie these neuropsychiatric disorders through the generation a stem cells, called iPS cells, from the skin of patients. Scientists are now challenged to develop methods to program iPS cells to become the specific types of brain cells that are most relevant to each specific brain disease. For instance, there is evidence that defects in cortical interneurons contribute to epilepsy, autism and schizophrenia. The experiments proposed in this grant application aim to understand basic mechanisms that underlie the development of cortical interneurons. We are discovering regulatory elements (called enhancers) in the human genome that control gene expression in developing interneurons. Our experiments will help us understand fundamental mechanisms that govern development of these cells. Furthermore, we have designed experiments that harness these enhancers to drive the production of specific subtypes of these cells from human stem cells. This will open the door to making these types of neurons from iPS cells to study human disease, and potentially to the production of these neurons for transplantation into patients whose interneurons are deficient in regulating their brain function. Furthermore, the approach we describe is general and readily applicable to the generation of other brain cells. Thus, the results from these studies will provide essential and novel basic information for understanding and potentially treating severe brain disorders.
EXECUTIVE SUMMARY This proposal focuses on understanding the development of cortical interneurons, a cell type implicated in a range of neurological diseases. The applicant hypothesizes that genetic regulatory elements, called enhancers, drive the expression of cell type specific markers that can be used to produce and isolate cortical interneurons from human embryonic stem cells (hESCs). The applicant proposes three Specific Aims. Aim 1 will focus in the identification and characterization of human enhancers that drive gene expression at different stages of interneuron development in transgenic mouse models. In Aim 2, s/he proposes to develop enhancer driven reporters to allow selection of hESCs in the process of differentiation toward an interneuron fate. Finally, in Aim 3, the applicant proposes to compare the generation of cortical interneurons from hESC, the human brain and the mouse brain. This will allow the applicant to identify molecular markers on the developing interneurons that could be used for further cell sorting and purification of interneurons. Reviewers agreed that this proposal addresses a significant obstacle in regenerative medicine: the inability to efficiently produce specific cell types from pluripotent precursors. The generation of enriched populations of human interneurons from hESCs would permit studies into their therapeutic relevance for disorders such as epilepsy, schizophrenia and autism. Reviewers also appreciated that the proposed approach for purifying precursor cells from hESCs could have implications for differentiation strategies for a variety of other cell types. The reviewers found the research plan to be logical, well-organized and feasible. They found its approach to be supported by high-quality preliminary data. Reviewers noted that the profiling of enhancers and markers in primary human tissue is a key strength of the proposal. They also appreciated that differentiated interneurons would be assayed for function in vivo. Reviewers did make some minor criticisms of the research plan. They noted that the applicant does not demonstrate proof-of-principle for using enhancers to purify interneurons from mouse ESCs or embryonic brain. In addition, the approach relies on there being a good correlation between the function of enhancers in vivo and in vitro. The possibility that these may not correlate is not discussed. Finally, reviewers noted that the two cell-surface markers mentioned in Aim 3 are not restricted to the interneurons of interest; they are also expressed on other neuronal populations as well as glial and endothelial cells in the brain. Thus these markers may not be good choices for purifying cells from a heterogeneous population. Reviewers described the Principal Investigator (PI) as a talented investigator with a strong publication record and many important contributions to his/her field. Furthermore, the PI is collaborating with two co-investigators who are world-class neurobiologists with track records of extraordinary achievement. The reviewers had no doubts that the research team is qualified to carry out the proposed studies. Overall, reviewers were impressed with this proposal, in particular its strong preliminary data and outstanding research team. They felt that the proposed studies, if successful, would have a major impact in the fields of stem cell biology and regenerative medicine.