Tools and Technologies I
Small RNAs (smRNAs) have been shown to have a key role in the regulation of gene expression. They also show great promise as prognostic and diagnostic biomarkers for cancer and other diseases. Existing microarray technologies for detecting and measuring levels of smRNAs are limited in sensitivity and accuracy, and are not able to discover new smRNAs. Next-generation sequencing can overcome these limitations of microarray technologies, but currently require large amounts of starting material (10 ug, equivalent to 100,000-1 million cells). We propose to develop a nanoscale small RNA sequencing method to enable analysis of smRNAs from small numbers of cells, with an interest in studying extremely small samples sizes (10-100 cells). This would allow analysis of cancer stem cells, biopsies of engrafted cells from stem cell transplant studies, and rare types of stem-cell derived differentiated cells. We will use our method to sequence smRNAs from human fibroblasts and human embryonic stem cells, and compare the results with existing data to verify the assay. We will then demonstrate our method on individual mouse oocytes and early embryos to generate smRNA expression profiles for these two very rare cell types. Since the human embryonic stem cell line to be used is not NIH-approved, this study is not eligible for NIH funding.
Statement of Benefit to California:
Healthcare in California is becoming increasingly expensive, while many common diseases remain intractable to classical drug therapy. The healthcare industry has a great need for improved therapies that can be more effective without significantly increasing costs. The CIRM’s strategic plan has mechanisms in place to ensure that Californian taxpayers benefit from their investment in stem cell research. A key aspect of the plan is to use the power of the focused stem cell research program to develop tools and technologies that can fuel the economy by, jobs, and tax revenues. The technology that we propose to develop is at the cutting edge of diagnostic tools. While we will develop the tools specifically for stem cell research and clinical development, they will also contribute to other areas of medicine, including cancer diagnosis. We propose to develop a nanoscale method for analyzing small RNA molecules in rare populations of cells, including cancer stem cells. Small RNAs, including a class of molecules called microRNAs, have a critical role in determining what genes are active in cells. MicroRNAs have been proposed as potential diagnostic and prognostic biomarkers for cancer and other diseases. Our diagnostic tool will use the powerful “next-generation” sequencing methods that have been recently developed for use in genetic analysis projects, such as the Human Genome Project. Our technology will allow detailed analysis of cancer stem cells, biopsies of engrafted cells from stem cell transplant studies, and rare types of stem-cell derived differentiated cells, and identify potential biomarkers that can be applied to further technology development. This technology has broad applications and can be a key diagnostic tool for both research and medical use, benefiting California by improving healthcare and attracting investment.
This application proposes the improvement and scale-down of methods for preparing and sequencing small RNA molecules (smRNA) from samples containing as few as 10-100 cells. An improvement in existing methods will permit the sequencing and analysis of smRNA from rare stem cell populations such as cancer stem cells or cells at specific points in the differentiation process. The reviewers acknowledged the utility of the proposed technology but were uncertain of its potential to advance stem cell science. Although improved RNA profiling methodologies would be useful, the Principal Investigator (PI) failed to provide compelling justification for the use of stem cells in this effort. As the proposed methods are designed to improve an existing technological platform, some reviewers felt that it might be more appropriate to conduct this investigation in an established, well-characterized cell line. Initial experiments related to the generation of smRNA libraries and scale-down of the technology were regarded as straightforward, but it was unclear why the PI proposed to validate the tool using mouse oocytes and embryos. Specific experiments to analyze rare stem cell populations with the tool were otherwise not proposed despite their stated goal of using the technology with such cells. Furthermore, it was unclear how the RNA isolation from rare populations would actually be achieved. Of additional concern is an odd inconsistency in the proposal of which cells will be used to prepare smRNA libraries. Initially, the PI justifies the proposal to CIRM by claiming that the studies will make use of a federally unapproved hESC line for which s/he cannot seek federal funds. However, the experiments described refer only to the use of an approved line. While the PI demonstrated a clear grasp of the experimental challenges, there appears to be no preliminary data that demonstrates feasibility of the approach and there is no discussion of how data is to be analyzed. Finally, the reviewers questioned the rationale for using oocytes and/or embryos for method validation rather than cancer stem cells or other populations that might add relevance to this proposal. The PI has extensive experience in genomic assays and assay development that are appropriate for the proposed studies. The PI just completed a postdoctoral fellowship prior to joining the applicant organization and was viewed as fairly inexperienced as an independent investigator. However, the strength of the collaborators and the excellent research environment provided some confidence that the PI would have adequate support. Nonetheless, the level of commitment from the PI as well as several key collaborators appeared insufficient in the minds of reviewers. One commented that the sum total of combined efforts might be appropriate, but the descriptions of the individual contributions were vague. The budget was judged to be appropriate, but the applicants failed to provide adequate justification of consultancy and other staff costs. Overall, while the proposed technology could be very useful, the applicants did not make a strong case for its potential to advance the field of stem cell biology.