Buried deep inside the brain are cells known as choroid plexus epithelial (CPe) cells. Although not as famous as other cells in the nervous system, CPe cells perform a large number of important jobs that keep the brain and spinal cord healthy. They produce the fluid (known as cerebrospinal fluid, or CSF) that bathes the brain and spinal cord with many nourishing chemicals, which promote normal nervous system health and function, learning and memory, and neural repair following injury. In addition, CPe cells protect the brain and spinal cord from toxins – such as heavy metals and the amyloid-beta peptide associated with Alzheimer’s disease – by absorbing them or preventing them from entering the nervous system altogether by forming the so-called blood-CSF barrier. Accordingly, as CPe functions diminish during normal aging or in accelerated fashion in certain diseases, memory loss, Alzheimer’s disease, and a number of other neurologic and neuropsychiatric disorders may ensue or become worse. The ability to grow and make CPe cells should therefore enable many clinical applications, such as CPe cell replacements, transplants, and pharmaceutical studies to identify beneficial drugs that can pass through the blood-CSF barrier. However, all of these potential applications are limited by the current inability to make and expand CPe cells in culture. Our published and preliminary studies suggest that it should be feasible to generate CPe cells in culture. Our broad goals are to study how CPe cells form during normal development, then use this information to make human CPe cells for clinical applications. To achieve this goal, our approach will be to use mice to study how the CPe develops normally, then use both mouse and human stem cells to make CPe cells in culture. Our published and preliminary studies have defined one critical factor for this process (known as Bmp4) and identify candidate factors that work with Bmp4 to regulate whether or not CPe cells are formed. In Aim 1, we test whether a molecule known as Fgf8 provides CPe “competency” – i.e. whether Fgf8 allows cells to become CPe cells when exposed to Bmp4. In Aim 2, we test whether a gene known as Lhx2 prevents cortical cells from becoming CPe cells in response to Bmp4. In Aim 3, we manipulate Bmp4, Fgf8, and Lhx2 in hESC cultures to make human CPe cells. If successful, this proposal should greatly improve our understanding of normal CPe development and enable a number of CPe-based clinical applications with significant potential to improve human health.
Our proposal to study choroid plexus epithelial (CPe) cell development and to make CPe cells in culture for clinical applications should benefit the State of California and its citizens in a number of ways. In the short term, this project will provide employment, education and training in stem cell research for a handful of California residents, and will support California-based companies that provide supplies for the stem cell and biomedical research communities. In the longer term, success in making CPe cells in culture should enable many new CPe-based clinical applications, stimulate CPe studies and applications by stem cell companies, and enable screens to identify agents that allow for passage of therapeutics across the blood-CSF barrier, which remains a significant roadblock to the development of pharmaceuticals for neurological and neuropsychiatric disorders. Such outcomes would ultimately stimulate investment in California-based companies and benefit the health of many California citizens, which may reduce the economic burden of health care in the state.
The goal of the proposed research is to understand the development and differentiation of choroid plexus epithelium (CPe), a cell type that has received little attention in stem cell biology. Though primary diseases of the choroid plexus are rare, understanding choroid plexus development is important for therapeutic manipulation of the blood-cerebrospinal fluid barrier (CSF), secreted products from choroid plexus are likely important in general for central nervous system diseases, and the proposed work is potentially important for understanding the normal physiologic maintenance of CSF integrity. In Aim 1 the applicant will examine the interactive role of growth factors for their ability to promote CPe specification. In Aim 2 the role of a candidate negative regulator of CPe specification will be examined. In Aim 3, the applicant will manipulate the growth factors and the negative regulator to optimize differentiation of CPe from human embryonic stem cells (hESCs).
Reviewers were excited about the proposal for its clinical relevance. The applicant’s preliminary data suggesting that the major growth factor chosen for study is indeed a critical instructive factor in choroid plexus development was found to be compelling by the review panel. The preliminary data also support the further investigation of the second growth factor chosen for study in CPe development. The proposal to carry out classical developmental biology studies using mouse embryonic stem cells (mESCs) is a strong component of the plan. The applicant proposes staged work, leaving the hESC work for later in the research timeline, capitalizing on the information obtained from the mouse cell work. Based on this plan of action, the feasibility that the applicant will be able to coax choroid plexus development from hESCs is high. The institutional environment, especially with the chosen mentors for the project, was also considered a major strength of the proposal. Reviewers felt that the learning curve for in the initial studies would be steep, but that the project is novel and worth pursuing. The application presents an elegant approach to understanding development of the parts of the brain.
Although the mouse studies were logically outlined, the reviewers thought that insufficient detail was presented in the experimental plans. A weakness of the application was the superficial presentation of the mouse genetics. A large number of crosses were discussed, and so the clarity of the complex plan was not optimally presented. The proposal would have been strengthened by more rigorous presentation of quantitative endpoints particularly for the in vitro studies and better tabulation of all the conditions and genotypes to be tested. Another concern is that the applicant still has to make the reporter system needed to read out choroid plexus development, and this may prove more difficult than acknowledged. The applicant does not state how the reporter system construction will be validated. Reviewers were concerned that the plan of manipulating only three factors to generate choroid plexus development (Aim 3) was somewhat naïve, and that the differentiation might be more difficult that acknowledged in the application. Finally, despite the strong developmental biology background of the applicant, the evidence for strong hESC training was not presented, but reviewers felt that this inexperience was balanced by the support of experienced investigators in the institution.
The applicant has been at the institution for 7 years, after a post-doctoral position with a leader in cortical development. The applicant has been an assistant professor since 2004. The applicant has strong developmental biology credentials, and so has exactly the background of people who should be drawn into hESC research. The applicant is productive, and recently had a Science publication, as well as some independent funding. The applicant has already obtained good independent funding – An NIH-funded KO2, a March of Dimes grant and an R21 award from NIH.
The institution has assigned adequate space to the PI and this project. The institution has assembled a strong team of stem cell researchers, which form the appropriate intellectual and infrastructure environment for these studies. The commitment to the investigator by the institution is enthusiastic and will help to assure success of the project.