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RN1-00558-1: Using Stem Cells to Make, Study, and Sort Useful Forebrain Cells
Recommendation: Not recommended for funding
Public Abstract (provided by applicant)
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 (provided by applicant)
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
SYNOPSIS: This 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 a sorting strategy for neural stem cells based an intrinsic mechanism. The third project is to determine if a specific technology can be used to segregate neural stem cells by their developmental fate options.
The first aim of the project studies the role of an intrinsic neural stem cell regulator 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 a potential sorting approach for neural stem cells based on the cited intrinsic neural stem cell regulator, with a goal to make cortical stem cells that sort themselves.
The third aim will test whether a specific technology can be used to separate closely-related stem cells with different fates. This technique sorts cells by inducing a non-toxic whole cell characteristic. Therefore, unlike FACS, this technology sorts cells holistically and can distinguish different cells in a nearly instantaneous, unbiased fashion. The technology has been used successfully to separate neurons from astrocytes. The applicant would like to see if the technology 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 below.
The first aim is to grow choroid plexus epithelial (CPE) 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 barrier. 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. Instead, 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 explore a potential sorting approach based on an intrinsic cell process. The applicant does not do a good job of selling this specific aim. Why is the intrinsic cell process important? What fundamental problem in neuroscience or neurological disease will be addressed by understanding this process? The scientific/clinical rationale for studying the cited intrinsic neural stem cell regulator and the process mediated by it 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 a specific technology. This technology has been around nearly a decade so the method itself cannot be considered novel or original. However, the application of the technology proposed by this applicant is interesting and it would be nice to see if s/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 s/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. It is also not carefully edited – throughout the applicant refers to FigX. In the preliminary results section, 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 perform the intrinsic process proposed for the sorting approach poorly and only in certain conditions – it is not clear whether the applicant knows this. The applicant will be highly dependent on collaborators for aims 2 and 3.
Given the strong scientific background of this applicant and the applicant’s 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 a top-rated undergraduate institution, and an M.D. and a Ph.D. (neurosciences) are from an excellent institution within California. Clinical training at several top teaching hospitals was followed by a period of postdoctoral research with a noted neurogeneticist. 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, although there are two gaps in time (1993-2001, 2001-2005) where there were no publications. The applicant joined the faculty of his/her current institution in 2001, where s/he serves now as an Assistant Professor of Pathology. Several research articles have already appeared from the applicant’s 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 from private foundations; however, no major research grant support from the NIH is apparent to date.
The applicant has Ph.D. students in his/her lab, including students from the institution’s medical scientist training program. S/he has published 3 papers as a PI since 2001 in respected journals and has three grants from the NIH and foundations. The applicant states this CIRM New Faculty Award would be instrumental in achieving his/her goals and overcoming obstacles. It would not only provide the necessary time and resources to get the ESC cultures and analytical tools in place, but also to generate meaningful data from them. These studies are not currently funded and the applicant thinks would be premature by NIH RO1 standards. There will be two main avenues by which the applicant will receive formal evaluations and feedback from mentors — one from his/her home department 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, and the other through a Faculty Oversight Committee of the stem cell research program that oversees all of the research and training conducted in the program, and advised PIs and trainees on any and all aspects of their work.
The applicant states that current clinical responsibilities constitute much less than 25% effort, which allows him/her the necessary time to do research. During the course of this award, the applicant’s clinical duties will remain at a low percentage effort to facilitate the stated research.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant’s department chair has provided a strong letter documenting institutional support. The PI was given a generous research start package. S/he is protected from excess teaching and clinical duties. Research space is ample and key core facilities are available. The chairman states that the applicant has had, and will continue to have, the necessary facilities, collaborative resources, and administrative support to ensure his/her success. In addition to the laboratory’s resources, these include equipment and resources in the applicant’s department and the Institution. The applicant will continue to have administrative support from the department.
The Institution has a commendable track record of promoting the career development of junior faculty. It 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 a new stem cell center should supply all the core needs, along with the equipment given to the applicant in his/her start-up package.
DISCUSSION: This investigator has good training and good mentorship and is a good physician-scientist, although s/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.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- None