Human embryonic stem cells (hESC) have the remarkable capacity to replicate indefinitely and differentiate into virtually any cell type in the human body. Maintaining this pluripotent cell state requires the precise control of hundreds, if not thousands of proteins in the cells, a process known as gene regulation. Recently it has been shown that adult human cells can be induced to revert back to earlier stages of development and exhibit properties similar to hESCs. The exact method for "reprogramming" is still being optimized but currently requires inserting multiple genes into adult cells and then exposing them to the appropriate environment suitable for hESC growth, to produce these "induced pluripotent stem (iPS) cells". Generation of patient-specific iPS cells will be of tremendous benefit to disease-related biomedical research and therapy. It is of interest that many of these genes are hESC enriched or specific to pluripotent stem cells, thus understanding the regulation of genes important for pluripotency is of strong benefit to reprogramming as well.
Genes are regulated at many different levels, beginning with the production of RNAs in the nucleus (transcription), and ending with the generation of proteins from processed RNAs in the cytoplasm (translation). While much is known about the transcriptional control of gene expression involved in maintain the pluripotency of stem cells, relatively little is known about what happens to the RNAs after transcription (post-transcriptional control, or PTC), before translation. RNA binding proteins (RBPs) associate with RNAs during this intermediate stage, several of which bind directly to RNAs (targets), while others interact indirectly with RNAs via small non-coding RNAs called microRNAs to change the expression of target RNAs.
The goal of the proposed research is to produce a comprehensive map of RNAs that are targeted by RBPs important for pluripotency in stem cells, as well as uncover how these RBPs regulate their target RNAs. We will use a modification of a high throughput biochemical strategy to identify the precise location on RNAs that are in contact with carefully chosen RBPs. We will isolate and sequence millions short nucleotides representing stretches of these RNAs and map them to the human genome, together representing the complete post-transcriptional controlled regions of pluripotent stem cells. Completion of the proposed research is expected to improve our understanding of the gene regulatory mechanisms in human pluripotent stem cells, which in turn will facilitate the development of new strategies for stem cell based therapeutics and enhance reprogramming of patient-specific adult cells.
Our research is aimed at providing the foundation for understanding the molecular mechanisms that maintain the pluripotent state of human ES cells and enhance reprogramming of adult cells. This in turn helps us to design novel strategies to distinguish differentiated from pluripotent stem cells for mass production of cells for therapy, manipulate stem cells to differentiate into specific cells types and enhance reprogramming of patient-specific adult cells for disease modeling and screening of compounds for new drugs. In particular, the generation of disease-specific and genetically diverse stem cell lines aided by our research will have great potential for California health care patients, pharmaceutical and biotechnology industries in terms of improved human models for drug discovery and toxicological testing. This knowledge base will directly support our efforts as well as other Californian researchers to study stem cell biology and design new therapies, and keep California's position as a strong leader in clinical research developments.
This proposal examines the post-transcriptional networks that regulate pluripotency in human embryonic stem cells (hESC) by identifying targets for stem cell-enriched microRNAs (miRNAs) and RNA binding proteins (RBPs). RBPs can modulate gene expression either directly, by binding messenger RNAs (mRNAs) targets and regulating their subsequent processing, or indirectly via the action of small noncoding miRNAs. The first mode of regulation will be explored in Aim 1, where the applicant proposes to use high-throughput sequencing techniques to identify hESC mRNA targets of an RPB with a known role in pluripotency. In addition, the applicant will explore whether alternative splicing and translational regulation are the means by which this RPB exerts its effects. In Aim 2, the applicant proposes to use similar methodologies to identify mRNA targets of a second RBP, whose effects are exerted indirectly via a miRNA-mediated mechanism. For the final aim, the applicant proposes to synthesize the information gathered in Aims 1 & 2 to generate genome-wide maps of RPB and miRNA regulation and develop new strategies for inducing pluripotency in somatic cells.
Reviewers agreed that this proposal addresses a major unsolved problem and could have a significant impact if successful. They noted that comprehensive data describing post-transcriptional regulatory mechanisms in hESCs would be an important contribution to our global understanding of pluripotency in human cells. Reviewers appreciated the proposal’s innovative approach and its focus on molecular and cellular mechanisms. One reviewer noted that the use of powerful technologies coupled with advanced computational analysis provides a creative, combinatorial approach to an important problem.
Reviewers found the research plan to be well-written and straightforward, following a logical approach in all three specific aims. They praised the high quality of the preliminary data, noting that it provides strong evidence of feasibility and complements the applicant’s recently published work. Reviewers’ major concern was a lack of description as to how mRNA candidates would be pared down to a manageable number for validation and follow-up. They would have liked to see an estimate of the number of targets expected and a strategy for prioritization. One reviewer worried about the feasibility of Aim 3, given the lack of detail provided and its dependence on Aims 1 & 2, but other reviewers were less concerned. In general, reviewers appreciated the sophisticated research plan using state-of-the-art technologies and were confident the proposal would generate a wealth of useful data.
Reviewers described the applicant as a talented junior investigator with a limited but impressive publication record. They had no doubts that the assembled research team will have the necessary expertise and resources to successfully execute the proposed specific aims.
Overall, reviewers were very enthusiastic about this proposal. They appreciated its innovative approach, strong preliminary data and potential to make a significant contribution to our understanding of hESC pluripotency.
- Chad Cowan