Basic Biology II
Neural stem cells are self-renewing cells that generate progenitors capable of further differentiation into functional cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. During brain formation, the stem cells of the embryonic cortex generate neurons early during the developmental process but form astrocytes at later stages, suggesting that progenitor fate potential shifts over time. Despite evidence for neuron-restricted and astrocyte-restricted progenitors, little is known about the cellular characteristics that critically differentiate these two cell types from each other. In part, this lack of understanding is due to the shortage of specific markers that will distinguish these cell types. Specific progenitors can be used for transplantation to form a particular type of final, functional cell. Analysis of neuron- and astrocyte-forming neural stem/progenitor cells (NSPCs) by a technique termed dielectrophoresis (DEP) demonstrates that cell behavior in DEP correlates with potential to form neurons. DEP is a label-free, non-toxic and unbiased method for analyzing cells that detects formation of frequency-induced dipoles in cells. The dielectric properties of neuron- and astrocyte-forming mouse and human NSPCs significantly differ from each other and reflect their fate biases such that neuron-forming progenitors become more similar to neurons and astrocyte-forming progenitors to astrocytes. Furthermore, the specific dielectric properties of human NSPCs isolated from brain represent differences in the membrane compartments of those cells. The goal of this proposal is to utilize DEP to determine the cellular properties that discriminate neuron-forming progenitors from other cells. We hypothesize that stem cell fate potential revealed by dielectric signature is due to the cell membrane and that modifications altering the effective membrane thickness or surface area, such as modification of certain membrane components, are the main contributors to the cell fate-specific dielectric signatures. We will test this hypothesis with neural lineage cells derived from human ES cells, iPS cells, and brain. Experiments to test this hypothesis will identify the contribution of membrane and cytoplasmic cellular compartments to cell lineage-specific dielectric properties and test the involvement of membrane modifications in progenitor cell dielectric properties. We expect that NSPC dielectric properties will be a biophysical measure of their fate potential, providing a novel approach for investigating lineage-committed NSPCs and a way to use these cells to obtain specific cell types, such as neurons, after transplantation.
Statement of Benefit to California:
The goal of this project is to determine whether a novel strategy using DEP can serve to identify specific characteristics of stem cell subpopulations. In the course of these studies, we expect to learn more about human progenitor cells that specifically generate neurons, a cell population of interest for basic biological studies, therapeutic approaches, and as a source of human neurons for drug testing. Our hope is that this label-free method for investigating stem cell subpopulations will greatly increase the speed of stem cell research in California and improve our understanding of how to control the composition of cells used in therapeutics.
EXECUTIVE SUMMARY The goal of this proposal is to determine the cellular properties that allow the separation of neural/stem progenitor cells (NSPCs) by dielectrophoresis (DEP). DEP is a technique that can be used to sort cells based on their dielectric properties. The applicant presents preliminary data suggesting that DEP is able to discriminate neurogenic NSPCs from gliogenic NSPCs, and proposes to study the biophysical and biochemical bases of this phenomenon. In Aim 1, the applicant proposes to test the hypothesis that the glycosylated components (protein and lipid) of the plasma membrane of cells are responsible for cell-fate specific dielectric signatures. In addition, the applicant will use DEP on human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) differentiated along the neural lineage to determine when specific dielectric properties arise. In Aim 2, the applicant proposes to profile membrane modifications in lineage-committed NSPCs and use inhibitors to disrupt these modifications in order to understand the biochemical basis for differences in dielectric properties. Reviewers found this proposal innovative but were not convinced it would have a significant impact on stem cell research and regenerative medicine. The application would have benefited from a stronger discussion of the importance of DEP technology to hESC research. Although reviewers agreed that it would be a significant technical advance to make DEP-based cell sorting more reliable, adaptable and robust, they had serious reservations as to whether the resolving power of DEP will ever equal or surpass conventional cell sorting techniques using cell-type specific marker proteins. The reviewers praised the promising preliminary data presented in the application but raised serious concerns about the research plan. A particular concern was that the applicant doesn’t describe how dielectric properties will be measured or what model will be used to extract and interpret dielectric data. Although the applicant includes a letter of support from a collaborator that suggests his system and model may be used, no detail was provided. Given the importance of accurate measurement of dielectric properties to the proposal, this omission substantially weakens the application. Another concern was that the application did not address enough of the cell biology behind the DEP signal differences, and that only very general hypotheses are addressed by the study aims. In Aim 1, the applicant proposes to generate a number of neuronal subtypes but does not provide much detail about how these will be used in further experiments. Additionally, appropriate controls and the interpretation of DEP data in these experiments were not described. In experiments related to Aim 2, reviewers worried that the use of inhibitors may have non-specific effects or induce widespread phenotypic changes that might indirectly alter dielectric properties. Overall, reviewers appreciated the applicant’s focus on mechanism and noted that this is an aspect sorely missing from most previous research on DEP. However, the panel felt that deficiencies in experimental design and data analysis would severely limit the impact of the proposed studies. Reviewers found the PI and assembled research team to be generally qualified to conduct the DEP aspects of the study, and they noted the team’s strong group of collaborators. However, the application suffered from a critical lack of expertise in developmental neurobiology. Additionally, research team’s limited expertise with hESC or iPSC cell culture and differentiation protocols further diminished the reviewers’ enthusiasm. Overall, although reviewers appreciated the novelty of this proposal and its focus on basic mechanisms, they had serious concerns about the project’s feasibility and were not convinced that it would have a significant impact on the field of human stem cell biology.