Developing novel markers or improving the current ones for tracking and isolating stem and progenitor cells is still a major challenge. Although cell surface markers and genetic approaches have been developed for isolating and tracking stem and progenitor cells, these approaches have serious limitations. Multiple antibodies against stem cell-specific cell surface molecules are required for stem cell enrichment, thus limiting the use of these markers for tracking stem cells in animals. Genetic markers controlled by cell-specific promoters have limited specificity since few genes have truly stem cell-specific expression patterns. In addition to limited specificity, it is tedious, time consuming, and costly to generate transgenic mice with reporter genes controlled by different cell or tissue-specific promoters. The discovery of RNA interference (RNAi) has revealed a fundamental layer of gene regulation controlled by small non-coding RNAs. Among these small non-coding RNAs, miRNAs are a family of endogenous RNAs (~22-nt long) that mediate post-transcriptional gene repression through imperfect base-pairing to their cognate target mRNAs (1). MiRNA-mediated gene regulation represents a comprehensive layer of genetic programs at the post-transcriptional levels, and has been shown to play diverse functional roles in animal development and in the pathogenesis of cancers. Several lines of evidence have implicated miRNAs in the developmental regulatory decisions of stem-cell maintenance and differentiation in flies and mice. miRNA expression signatures may be used as tissue and lineage markers for stem and progenitor cells from normal and diseased tissues. Since miRNAs can selectively silence gene expression in cultured cells and animals by targeting cognate target mRNAs for either degradation or translational repression. We will develop miRNA sensors, which are reporter genes fused with UTRs containing one or more miRNA complementary sites, to indicate the expression of stem and progenitor cell-specific miRNAs. Up-regulation of stem-cell miRNAs will result in the suppression of the reporter genes, whereas down-regulation of stem cell miRNAs will result in the increase of reporter gene expression. The proposed research will results in novel markers for stem and progenitor cells that are controlled by the miRNA-mediated post-transcritpional genetic programs, which will complement the existing markers for stem and progenitor cells. Thus, the proposed study will provide new markers and further improve the existing markers for stem and progenitor cells. Particularly, stem cell miRNA sensors can be used to monitor and isolate live stem and progenitor cells from animals or differentiated embryonic stem cells.
Statement of Benefit to California:
Specific markers are required for tracking stem cells in vivo and for isolating stem cells from animals and differentiated embryonic stem cells. Such markers are essential for advancing our understanding about stem cell biology and for developing therapeutic applications using embryonic and adult stem cells. However, available marker are inefficient, difficult to use, and costly to develop, and it remains a challenge to develop specific markers for tracking and isolating stem and progenitor cells. So far, only gene regulatory information at the transcriptional levels were utilized to develop stem cell markers. However, recent studies revealed that stem cell development is also controlled by miRNA-mediated gene regulation –– a comprehensive layer of genetic programs at the post-transcriptional levels. The proposed study will develop specific markers for stem and progenitor cells based on their unique miRNA expression signatures. Specifically, we will develop fluorescent sensors that convert stem-cell miRNA expression signatures into distinct fluorescent signals, which can be monitored in real-time using fluorescent microscope and flow cytometry. These miRNA sensors, which are controlled by the miRNA genes at the post-transcritpional levels, will improve the existing markers for stem and progenitor cells and facilitate the identification of stem cells in vitro and in vivo. This technology will have broad applications in stem cell biology and therapeutics, and can be easily adapted for human stem cells. We will be making available the miRNA sensors created by our work for stem cell researchers at California, which will further benefit California and its citizens.