Stem cells generate mature, functional cells after proteins on the cell surface interact with cues from the environment encountered during development or after transplantation. Thus, these cell surface proteins are critical for directing transplanted stem cells to form appropriate cells to treat injury or disease. A key modification regulating cell surface proteins is glycosylation, which is the addition of sugars onto proteins and has not been well studied in neural stem cells. We focus on a major unsolved problem in the neural stem cell field: do different proteins coated with sugars on the surfaces of cells in this lineage (neuron precursors, NPs and astrocyte precursors, APs) determine what types of mature cells will form? We hypothesize key players directing cellular decisions are glycosylated proteins controlling how precursors respond to extracellular cues. We will address this hypothesis with aims investigating whether (1) glycosylation pathways predicted to affect cell surface proteins differ between NPs and APs, (2) glycosylated proteins on the surface of NPs and APs serve as instructive cues governing fate or merely mark their fate potential, and (3) glycosylation pathways regulate cell surface proteins likely to affect fate choice. By answering these questions we will better understand the formation of NPs and APs, which will improve the use of these cells to treat brain and spinal cord diseases and injuries.
The goal of this project is to determine how cell surface proteins differ between cells in the neural lineage that form two types of final, mature cells (neurons and astrocytes) in the brain and spinal cord. In the course of these studies, we will uncover specific properties of human stem cells that are used to treat neurological diseases and injuries. We expect this knowledge will improve the use of these cells in transplants by enabling more control over what type of mature cell will be formed from transplanted cells. Also, cells that specifically generate either neurons or astrocytes can be used for drug testing, which will help to predict the effects of compounds on cells in the human brain. We hope our research will greatly improve identification, isolation, and utility of specific types of human neural stem cells for treatment of human conditions. Furthermore, this project will generate new jobs for high-skilled workers and, hopefully, intellectual property that will contribute to the economic growth of California.
This Track 1 (Fundamental Mechanisms) proposal aims to take advantage of an observation that neuron and astrocyte precursors (NPs and APs) can be distinguished by their membrane capacitance. The group hypothesizes that differential membrane glycosylation patterns may underlie this property and seek to further understand the differences between NPs and APs and how glycosylation may influence their function and unique characteristics. The experiments proposed will define the glycosylation patterns on surface proteins that differ between the lineages, identify the enzymes involved, and determine if the observed differences influence cell fate determination.
Significance and Innovation
- This proposal uses a novel approach to distinguish between two precursor fates (neuron and astrocyte). This project utilizes a measurable phenotypic difference between cell fates to uncover the cause for the phenotypic difference and whether it is instructive in fate choice. If the hypothesis is correct, these studies could have a major impact on the use of stem cells for regenerative medicine therapies and for disease modeling.
- This project is highly innovative. The novel approach utilizes an interesting and understudied characteristic of cells and develops a unique way to enrich for cells of certain fates that does not require killing or substantially altering them.
- Glycosylation in stem cells is an understudied area and knowledge gained has the potential to significantly advance the field.
Feasibility and Experimental Design
- The project employs innovative approaches in its experimental design. The rationale is logical and scientifically sound and is supported by compelling preliminary data. With the extensive experience of the research team and availability of special agents, the project is highly feasible.
- The proposed research is carefully designed to generate meaningful results. The experiments are straightforward and should answer the questions without difficulty. The research team has appropriate facilities available to conduct the proposed research.
- Potential difficulties are acknowledged and alternative plans are provided and discussed. However, it is not clear whether and when alternatives might be employed. The preliminary data supplied for Aim 2 falls a bit short in terms of demonstrating full feasibility. This is of some concern because Aim 3 depends on the success of Aim 2.
- Concerns were raised with respect to whether the proposed analyses of lineage differences would provide useful insights into what these differences mean.
Principal Investigator (PI) and Research Team
- The PI has an independent track record which combines experience in neural stem cell biology with engineering-based analysis platforms.
- The PI’s recent publications in high-profile journals, the preliminary data for this application, and the collaborative interactions of the team involved in this proposed project all support the application.
- The PI has assembled an excellent multidisciplinary team capable of carrying out all aspects of the project.
- The PI's and research team’s level of commitment and the project budget are appropriate for success of the project.
Responsiveness to the RFA
- This proposal is responsive to the RFA. Specifically, the proposed studies utilize human stem cells and human precursor cell lines that give rise to neuronal and astrocyte precursors. The proposed studies target key cellular and molecular mechanisms with relevance to stem cell differentiation to neural fates.