Advancing our understanding of stem cell biology often relies on answers to the following types of questions:
What are the differences in gene expression between a stem cell and the “mature” cell (for example, a neuron or heart cell) made by the stem cell? Answers to such questions can lead to methods for directing stem cells to make specific types of progeny.
How similar are the patterns of gene expression between a “normal” cell and a stem cell-derived cell (for example, a healthy neuron in the brain versus a neuron made from an embryonic stem cell)? Answers to these types of questions can determine exactly how closely a stem cell-derived cell matches the cell it is meant to replace. This information is essential for developing safe and effective therapies.
To best answer these questions, it is necessary to study gene expression in specific cells within their normal setting. This presents a technical hurdle, since the normal setting of a cell is typically within a complex tissue, surrounded by other cell types. To perform these types of experiments using currently available tools, it is necessary to first physically remove cells of interest from all other cells. This type of manipulation can cause unwanted changes in gene expression (giving false results) and is often not technically possible.
I have developed a technique that overcomes this technical hurdle and allows the identification of genes expressed in specific cell types within a mixed population of cells. I propose to develop this technique for the study of stem cells in tissue culture and for the study of stem cells in mice. The tools that are developed as a result of this work will allow previously impossible experiments to be performed and will benefit many areas of stem cell research.
Benefits to the development of regenerative medicine therapies in California: The development of the tools described in this proposal will accelerate the progress of regenerative medicine research in California by making previously impossible experiments available to stem cell researchers. The tools described in this proposal are especially well suited for the discovery of novel biomarkers for clinically-relevant cell types, new genetic methods for directing stem cells to generate specific cell types, and new ways to functionally characterize stem cell-derived cell types; three areas of research that CIRM has identified as needing novel technologies.
Benefits to the pharmaceutical and biotechnology industry in California: The types of research that will be made possible using the proposed tools (e.g. the ability to identify cell type specific changes in gene expression in response to drug therapies or in disease states, using whole animal models) will open doors to novel areas of drug discovery, drug development, and diagnostics. Biotechnology and pharmaceutical companies will likely use these tools to launch new research efforts and ultimately new product development. Given the large presence of such industry in California, this will provide benefits to the state’s economy in the form of new jobs, new investment, and increased tax revenue.
The applicant aims to apply a new method known as RNA Analysis by Biosynthetic Tagging or RABT to stem cells. This technique uses cell- or tissue-specific promoters to drive expression of an RNA-modification enzyme that does not occur naturally in multicellular organisms. Cells expressing the enzyme will label their mRNA, which can then be selectively purified, obviating the need for cell purification. The principal investigator (PI) proposes to apply RABT to tissue culture cells, mouse embryonic stem cells, and enzyme-expressing transgenic mice with the goal of developing a tool for analysis of gene expression profiles in vitro and in vivo. The technique would overcome the current difficulty of identifying and isolating specific cell types among mixed cell populations and tissues. The PI intends to establish collaborations with other stem cell researchers interested in using the new RABT tools.
Reviewers commented that the proposed RABT method might have a broad impact on many aspects of stem cell biology. It may well set a new standard for analysis of the stem cell transcriptome. Although several different applications are discussed, one reviewer noted that potentially the most useful will be the development of a transgenic mouse line that would allow conditional expression of the mRNA modifying enzyme in selected cell types by crossing with mouse lines expressing the Cre recombinase in specific tissues. This tool would provide a novel method for cell type-specific purification of mRNA for gene expression profiling of stem cells and their differentiated derivatives.
Reviewers also felt that the feasibility of the proposed work is high based on the PI’s previous success using the method in mammalian cell lines and specific tissues within the complete Drosophila organism. The proposed project will extend the technology to the mouse model. One reviewer viewed specific aim one largely as a proof-of-principle exercise, and felt that the results may not be worth the effort required to work out conditions for introducing lenti-viral vectors. However, the reviewer felt that the second aim is more important and more likely to succeed. Overall, the proposal was judged to be well designed and feasible.
The PI and co-investigator are relatively new assistant professors and they bring complimentary expertise to the project. A senior research associate and postdoctoral fellow will be recruited to the team. The PI is uniquely qualified for development of new methods based on RABT since he/she was co-inventor of this technology and has used the method in several systems since its origin.
The overall strengths of the application are the potentially high impact of the tool in stem cell research, the likelihood of succeeding in the development of a useful tool, and the PI’s creativity and experience as the inventor of the new methodology. The proposal’s weakness is largely attributed to the PI’s inexperience with lenti-viral vectors and use of mouse models but reviewers viewed these as minor. Development of this tool should prove useful to stem cell scientists.