Funding opportunities

Using Stem Cells to Make, Study, and Sort Useful Forebrain Cells

Funding Type: 
New Faculty I
Grant Number: 
RN1-00558
Funds requested: 
$2 938 345
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Unbeknownst to many, stem cells have been used to treat patients for over four decades. Although not known at the time, bone marrow transplants – which are used commonly to treat blood disorders and cancers – are nothing more than transplants of hematopoetic stem cells. As a physician-scientist, this means to me that stem cell-based therapies for other human disorders are not questions of if, but when. My goal is to help us turn ‘when’ into ‘now’, by investigating mouse neural stem cell (NSC) findings with important clinical potential, determining the strength of their potential, then turning them into clinical realities by using human embryonic stem cells (hESCs). As a neuropathologist who specializes in the problems of infants and children, mice and their NSCs have helped us greatly to understand how and why brain development sometimes goes wrong. Now, with the advent of hESC culture systems, we have cells that provide better models of human brain disorders and can be used to treat them. In our first Aim, we will try to make choroid plexus epithelial (CPE) cells in a dish. Although not famous, CPE cells are extremely important for brain health and have tremendous clinical potential. The inability to grow CPE cells in culture has been a roadblock, but we have solved at least part of this puzzle. In this Aim, we use mice to figure out the factors needed to make CPE, then apply this knowledge to ESC cultures. We also look for adult CPE cells that can grow. Either avenue, if successful, would open up a number of direct applications for CPE cells in drug testing and clinical therapies. In the second Aim, we try to make neural stem cells that can ‘sort’ themselves. Sorting is the process of purifying desired cells away from unwanted ones, a general issue for regenerative medicine. If given the opportunity, some stem cells may be smart enough to sort themselves, and we have identified a factor that may provide this opportunity. In this Aim, we use mice to understand better what this factor does and use this factor in ESC cultures. If successful, this would provide a simple way to purify one type of stem cell for clinical applications, and the rationale to use self-sorting as a general approach. The third Aim also deals with the problem of sorting. We took an approach shown by others to distinguish highly similar cell types, but not stem cells. Remarkably, this approach not only detects subtle differences between stem cells, but apparently informs us about the kinds of cells that each stem cell can make. To our knowledge, this is a very unique capability. In this Aim, we use this bioengineering tool on a wide range of cells to see how well this principle holds up and to apply this principle to sort cells. If successful, this would be a brand-new way, which could be used alone or along with existing methods, to get the best cells for all sorts of clinical applications.
Statement of Benefit to California: 
Our goal is to use human embryonic stem cells (hESCs) to help us understand human brain development and its disorders better and to make cells to treat these disorders. The project has three Aims: 1) to make choroid plexus epithelial cells, which have significant and largely untapped therapeutic potential, 2) to allow stem cells to sort themselves, which could be a simple general approach for purifying cells, and 3) to enrich for stem cells with specific properties using a bioengineering technology with unique capabilities. If successful in generating and purifying novel cell types with clinical utility, this project should have immediate and direct benefits to the State of California and its citizens, including pharmaceutical companies, basic scientists, clinicians, and patients. In addition, as a culture model for human brain development, the hESC studies should provide insights into developmental brain disorders that could lead to improved diagnostics for patients and their families.
Review Summary: 
SYNOPSIS: The study plan consists of three separate projects. One project deals with optimizing conditions to grow an interesting cell type - the chorioid plexus epithelial (CPE) cell, which is the cellular source of cerebral spinal fluid. A second project addresses the self-assembly of neural stem cells into rosette-like structures. The third project is to determine if a technique called dielectrophoresis (DEP) can be used to segregate neural stem cells by their developmental fate options. The first aim of the project studies the role of Lhx2 in formation of choroids plexus epithelial (CPE) cells in culture. The PI will explore the use of various growth and transcription factors to induce CPE from mouse and human ESC and will also determine if there are adult CPE precursors that could provide an alternative starting point for generation of CPE in culture. The second aim studies how stem cells self aggregate (assemble) under the influence of Lhx2, and to make cortical stem cells that sort themselves. In a completely different line of research, the third aim will test whether Dielectrophoresis (DEP) can be used to separate closely-related stem cells with different fates. DEP sorts cells by inducing a non toxic, frequency-dependent dipole, which is a whole cell characteristic involving multiple cell compartments. Therefore, unlike FACS, DEP sorts cells wholistically and can distinguish different cells in nearly instantaneous unbiased fashion. The technology has been used successfully to separate neurons from astrocytes. The applicant would like to see if DEP can distinguish otherwise similar neural stem cells that are predisposed for alternate fate choices. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This is a very poor study plan from an applicant with "blue chip" clinical and scientific credentials. On a case by case basis, each of the three specific aims is interesting and has some potential. Taken together, however, the three aims undercut each other. Reviewers sensed no vision or unified sense of purpose to the application. Each aim will be discussed separately. The first aim is to grow choroid plexus epithelial cells in culture. The applicant correctly points out that these are interesting, and perhaps underappreciated cell types. Choroid plexus epithelial cells are the source of cerebral spinal fluid. They are also major contributors to the blood brain barrier. The ability to grow and expand the cells would have obvious implications for drug screening and investigation for bypassing this barriers. In addition, CPE represents a potential therapeutic cell type in transplantation therapies, as endogenous CPE secretes growth factors and can provide a supportive environment for repair. Damage to CPE can also contribute to poor outcome in neurodegeneration. It would have been nice to see a fully fleshed out grant proposal on these cells alone. Aim 1 was a laundry list of experiments without enough details to judge their feasibility or likelihood of uncovering important new findings. The second specific aim is to learn how stem cells self assemble into rosette like structures. In preliminary studies, the applicant has identified a gene product (Lhx2) that enables the assembly of these rosettes in mosaic mice. The applicant does not do a good job of selling this specific aim. Why are these rosettes important? What fundamental problem in neuroscience or neurological disease will be addressed by understanding the assembly process? The scientific/clinical rationale for studying Lhx2 and rosette formation is not well articulated. Moreover, specific aim 2 is completely orthogonal to the first specific aim, and also to the third aim. The third specific aim is to segregate closely-related stem cells into separate pools with alternative fates using dieletrophoresis (DEP). DEP technology has been around nearly a decade so the method itself cannot be considered novel or original. However, the application of DEP proposed by this applicant is interesting and it would be nice to see if he could get it to work. This is the most creative of the three specific aims; however, one wonders where the applicant's heart lies. How would he prioritize these three aims? The Research Plan was essentially written in point form without sufficient detail or big picture overview to understand or judge. Each aim is a laundry list of single experiments without details such as the age of animal or tissue sample used for micro-array studies. It is also not carefully edited - throughout he refers to FigX. In preliminary results the Figure legends (example Fig.2) do not correlate with graphs and there is not enough detail (what kind of explant tissue was used in Fig.1, for example) to judge the significance of the results. Also a magnifying glass is needed to read much of the text in the figures. Finally, mouse ES cells form rosettes poorly and only in certain conditions - it is not clear whether he knows this. He will be highly dependent on collaborators for aims 2 and 3. Given 1) the strong scientific background of this applicant and 2) his exemplary track record, reviewers were prepared to see a cohesive yet innovative and sophisticated study plan. The application in hand falls far short of this expectation. The study plan is a loose collection of projects with no cohesive vision, no central scientific question, and no apparent goals. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant received a bachelor of science degree in biology from the Massachusetts Institute of Technology. His M.D. and his Ph.D. (neurosciences) are from UC San Diego. Clinical training at several Harvard teaching hospitals was followed by a period of postdoctoral research with noted neurogeneticist Chris Walsh. The applicant was very productive at both the pre-doctoral and postdoctoral levels. First author publications in top-tier journals such as Neuron and Science resulted from both of these training intervals. There are two gaps in time (1993-2001, 2001-2005) where there were no publications. The applicant joined the faculty of UC Irvine in 2001 where he serves now as an Assistant Professor of Pathology. Several research articles have already appeared from his his own laboratory, and these have been published in well-regarded journals such as Development and the Journal of Neuroscience. The applicant has also generated some research support for his laboratory from private foundations; however, no major research grant support from the NIH is apparent to date. The applicant has five Ph.D. students in his lab, including two in the UCL Medical Scientist Training Program. He has published 3 papers as a PI since 2001 (Dev, J Neurosci Res, J Neuro Sci). He has three grants (Whitehall, NIH/NINDS KO, March of Dimes). He states this CIRM New Faculty Award would be instrumental in achieving his goals and overcoming obstacles. It would not only provide the necessary time and resources to get the mESC and hESC cultures and analytical tools in place, but also to generate meaningful data from them. These studies are not currently funded and he thinks would be premature by NIH RO1 standards. There will be two main avenues by which he will receive formal evaluations and feedback from mentors – one from his home department (Pathology & Laboratory Medicine) which provides a supportive home base for physicians-scientist and its research faculty, including an open door policy and frequent ad hoc mentoring by the chairman (Michael Selsted), and the other through a Faculty Oversight Committee of the SCRC that oversees all of the research and training conducted in the Center, and advised PIs and trainees on any and all aspects of their work. He states that currently his clinical responsibilities (including neuropathologist for the National Human Neural Stem Cell Resource, Children’s Hospital of Orange County) constitute much less than 25% effort, which allows him the necessary time to do research. During the course of this award, his clinical duties (the surgical and autopsy neuroplathology service at the UCL Medical Center) will remain at a low percentage effort to facilitate his research. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant's department chair at UC Irvine has provided a strong letter documenting institutional support. Dr. Monuki was given a generous research start package. His current and future salary are supported by a full line FTE from the state of California. He is protected from excess teaching and clinical duties. Research space is ample and key core facilities are available. His chairman states that Dr. Monuki has had, and will continue to have, the necessary facilities, collaborative resources, and administrative support to ensure his success. In addition to his own resources, these include equipment and resources in his department and the Institution. He will continue to have administrative support from his department. The University of California at Irvine has a commendable track record of promoting the career development of junior faculty. UCI views the support of outstanding young investigators as an integral part of the commitment to stem cell research. It is making a major commitment to stem cell research and the new Sue and Bill Gross Stem Cell Research Center should supply all the core needs along with the equipment given to him in his start-up package. DISCUSSION: This investigator has good training and good mentorship and is a good physician-scientist although he has two significant gaps in publication. The choroid plexus model and Aim 3 are interesting. However, the proposal is diffuse, with disparate and shallow aims that read like a laundry list of experiments. The grant was poorly conceived, poorly written, poorly organized, and contains mistakes.
Conflicts: 

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