Funding opportunities

Development of High Throughput Functional Genomics Screening in Stem Cells

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01026
Funds requested: 
$1 059 840
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Cell-based functional genomics technologies are greatly accelerating the pace of scientific discovery in multiple fields of biological research. The convergence of high-throughput technologies with the use of RNA interference (RNAi) as a gene silencing tool has facilitated loss of function studies in mammalian systems in a genome-wide basis. In such studies, one can essentially turn off every gene in a cell, one gene at a time, to understand the individual role of each gene in the context of the whole cell. To perform such analysis, human cells need to be cultured in thousands of small wells, with a different gene-specific interfering RNA efficiently introduced (transfected) into each well of cells. This approach is now enhancing the analysis of a variety of cellular functions and the genes that control them, leading to unanticipated new findings. However, the technical hurdles of culturing human embryonic stem cells and their efficient transfection have blocked the application of such approaches to stem cell research. Gene function analysis in human embryonic stem cells is currently limited to studying a small number of candidate genes already suspected to play a role. The overall goal of this proposal is to enable broad functional genomics analysis in the area of stem cell biology and regenerative medicine. Our objective is to develop and validate high-throughput RNAi screening methods in several widely used human embryonic stem cell (hESC) lines. This will enable discovery of both anticipated and unanticipated gene products controlling stem cell fate. Towards this end, we will undertake two parallel approaches to systematically address and overcome the limitations on high throughput genomic screening in hESCs, and to demonstrate the potential of the approaches developed. Utilizing our expertise in functional genomics, stem cells, and high throughput screening, and building on recently reported advances in the study of hESCs, we will establish robust new methods. The specific aims are: 1) the development and optimization of high throughput screening methods with small interfering RNAs in various hESCs; 2) The adaptation of lentiviral-shRNA screening methods to hESCs to enable long term studies of gene function; and 3) Perform several functional genomics screens of hESCs with the methods we have developed, leading to iterative improvement of the screening methods, as well as novel insights into the genes and pathways that regulate stem cell growth and differentiation. Developing such tools will considerably accelerate the pace of discovery in stem cell and regenerative medicine research, enabling for the first time, global insights into key genes and molecular pathways controlling stem cell cellular pluripotency and cell fate determination.
Statement of Benefit to California: 
Human embryonic stem cells and their differentiated products are at the center of many promising approaches being developed for regenerative medicine. Some of the genes and pathways mediating stem cell self-renewal and differentiation have already been characterized, but many remain unknown. Our proposed studies in this application, once completed, are expected to develop new functional genomics research tools that will significantly accelerate the understanding of the mechanisms controlling stem cell growth and differentiation. Functional genomics screening, which allows systematic and rapid assessment of the role of every gene in a cell, has brought great insight to many areas of biology, but this technology has not yet been developed for the rapid analysis of human embryonic stem cells. While we anticipate discovering some important new participants in the biological pathways regulating stem cell differentiation in this proposal, the major value of our proposed studies is to develop functional genomics methods that will be subsequently used by a broad spectrum of stem cell researchers. Because our new methods are predominantly being developed for human embryonic stem cell lines that are widely being studied in California, but are not approved for use in Federally funded research, these tools will be of particular value to California researchers. More broadly, the tools that we propose to develop will accelerate identification of new therapeutic targets or pathways in all areas of regenerative medicine and cell replacement research. This will help to open up new opportunities for future stem cell applications and therapies, continue to attract scientists from other states to California, and stimulate biotechnology development, all this having a positive effect on our State.
Review Summary: 
The goal of the proposed project is to develop methods for high throughput screens of human embryonic stem cells (hESC) using RNA interference (RNAi). Several different methods for RNAi delivery and optimization of cell culture appropriate to large-scale screens will be systematically tested with four different hESC lines. Multiple assay parameters including toxicity and retention of stem cell identity will be evaluated. In a final aim, the applicant proposes to evaluate and refine the identified optimal approach by performing small scale RNAi test screens. The proposed research has a high potential impact. The availability of a high-throughput method for screening gene knockdowns in hESC using RNAi would provide a valuable tool for gaining insight into molecular pathways controlling pluripotency and differentiation. This information would inform the development of procedures to efficiently differentiate hESC to cell types of therapeutic value. Reviewers viewed the feasibility of the project to be a significant problem. Other researchers have failed in the development of efficient transfection protocols for hESC, and the proposal contains no novel insights or approaches to overcome this serious obstacle. It is unclear that the parameters and conditions proposed by the applicant will significantly improve transfection efficiencies. In addition, the experimental design was thought to be too complex, as it includes multiple variables, conditions and measurements without a discussion of its feasibility. Finally, reviewers felt that experimental approaches and goals were inadequately prioritized; for example, the applicant provides no information regarding the criteria used for selecting, as proposed, the 10 most relevant genes that emerge from the small scale screens. The principal investigator (PI) is highly qualified and has had extensive experience in cell biology and with studies of cell signaling. Co-investigators supply needed expertise in RNAi, high throughput screens, and functional genomics. The research team appears well qualified to carry out the proposed research. However, reviewer enthusiasm was diminished by the low percent effort committed to the project by the PI and by two key senior collaborators. There is concern about adequacy of oversight and responsibility in executing the proposed research.
Conflicts: 

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