Next Generation Sequencing for Nanoscale Functional Analysis of Stem Cells
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.
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.