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RN1-00529-1: Noncoding RNAs in Cell Fate Determination
Recommendation: Recommended for funding
Scientific Score: 85
First Year Funds Requested: $579,745
Total Funds Requested: $3,028,926
Public Abstract (provided by applicant)
The human body is composed of thousands of cell types, which all came originally from embryonic stem cells. Although all these cell types have the same genetic blueprint, different genes are active in different cells in order to give each its distinctiveness. The process by which the genes remember whether they are in liver, brain, or skin cells is called “epigenetics.” A central problem in regenerative medicine is to understand the epigenetic program so that human embryonic stem cells can be efficiently turned into the cell types required for each specific patient. Conversely, by manipulating the epigenetic program, adult cells may be reprogrammed into primitive cells that can turn into other cell types to repair diseased or damaged tissues.
The goal of the proposed research is to better understand the epigenetic program in human embryonic stem cells and adult cells. We want to tap into the natural mechanisms by which the body normally “remembers” what kinds of cells reside in each tissue and apply them to regenerative therapies. Specifically, the research will study the roles of a newly discovered type of genes, termed “noncoding RNAs”, in stem cell epigenetics.
A better understanding of how cells remember their own fates can improve regenerative medicine in several ways. First, by appreciating the roles of noncoding RNAs in this process, specific noncoding RNAs can be used as markers to track and predict when cells are acquiring or forgetting specific cell fates. For instance, it may be possible to learn from the pattern of noncoding RNAs that an embryonic stem cell is ready to become brain cells, which can be used to treat a patient with stroke. Second, beyond tracking cell fate, noncoding RNAs may be used to directly manipulate stem or adult cell fates. By introducing noncoding RNAs from different cell types, embryonic stem cells or adult cells may be directly reprogrammed into the desired cell type. While these potential application are far in the future, we believe that better knowledge of this new level of gene regulation will one day lead to more facile and efficient manipulation of cell fates for regenerative medicine.
Statement of Benefit to California (provided by applicant)
The proposed research can benefit the state of the California in three ways. First, the research will generate important knowledge on new ways to manipulate cell fate potentials of stem cells and mature adult cells. The focus of this research is to explore the genetic circuitry that locks cells into particular fates, whether it is to become skin, muscle, or brain. Better understanding of these circuitries could allow human embryonic stem cells to be directed to become particular tissues—and remember such instructions permanently. Alternatively, interference with these circuitries could allow adult cells to be reprogrammed into stem cells, where they can be used to generate damaged tissues. This information could speed the development of regenerative medicine in California, benefiting patients with currently untreatable diseases.
Second, the proposed research will generate new tools for stem cell research and regenerative medicine. As a direct result of this work, we will provide a complete genetic and epigenetic characterization of some of the first human embryonic stem cells created in California. This information will allow future investigators, physicians, and potential patients to better utilize them in research and therapy, or conversely appreciate potential limitations or risks associated with these embryonic stem cell lines. Moreover, we are likely to generate derivatives of these embryonic stem cell lines that have altered potential to become specific cell types. Such cell lines with properties of “directed differentiation” may be particularly useful for treatment of diseases where deficits of specific cell types are known.
Finally, the proposed research will train young scientists to become skilled in human stem cell research. Graduate Ph.D. students and postdoctoral fellows in this California-based institution will gain the hands-on experience and expertise of manipulating human stem cells and of reprogramming adult cells. The training and experience of these young scientists will prepare them to develop new regenerative therapies, launch new companies based on stem cells, or teach future students about regenerative medicine. Creating a cadre of well-trained individuals would be a vital step toward making California a central hub for regenerative medicine.
Review
SYNOPSIS: Expression of lineage specific genes in human embryonic stem (ES) cells, and conversely maintenance of pluripotency, is controlled in part by epigenetic modification. Recent data show that non-coding RNAs (ncRNAs) may target certain epigenetic modifications to specific genes. The general goal of this proposal is to explore the roles of ncRNAs in determining human ES cell pluripotency and cell fate determination. The Principal Investigator (PI), an Assistant Professor, proposes to study the role of ncRNAs in epigenetic regulation. The PI proposes to identify ncRNAs complexed with specific epigenetic modification proteins in hES cells and more differentiated cells, and then examine the genomic targets. In Aim 1 the PI will use a battery of antibodies to isolate the proteins and then compare the ncRNAs that are obtained. S/he will do this with hES cells and ES cells differentiated to endoderm/ectoderm. In Aim 2 the functional significance of these ncRNAs will be studied by mapping the genome wide sites to which they bind, and by strategies designed to manipulate the expression of both the ncRNAs and the chromatin modifying factors with which they interact.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The control of epigenetic regulation in ES cells is important and relevant to pluripotency and reprogramming, and in controlling lineage specific gene expression. This proposal contains very well-designed studies and is innovative in terms of its technology and focus on ncRNAs. The PI has an outstanding track record and is a pioneer in this area. The PI is highly experienced in the proposed genome mapping technologies and has excellent collaborators; if these experiments are going to be successful s/he is the one to get them done. In the first aim, different subunits of certain complexes involved in epigenetic modification will be examined for ncRNA binding in embryonic and somatic cell lines. This important aim addresses a significant question as it may identify novel ncRNAs and reveal their potential role in cellular differentiation. Aim 2, which proposes to characterize the genomic binding sites of newly identified ncRNA(s), will be critical for understanding their role in directing cell fate and controlling gene expression. Most of the tools and expertise are already in hand to perform the experiments proposed, and other technology is provided by collaborators.
This is certainly an emerging area of great interest, and one minor criticism might be that the study of ncRNAs is in its infancy with respect to their function in epigenetic modification. Even for the ncRNA(s) proposed here, the mechanism of action is still uncertain, and it is somewhat unclear how far the PI will be able to take these studies. There is a diversity of mechanisms for recruiting chromatin modifiers to gene promoters, such as via their interactions with other transcriptional regulatory proteins, and it may be too soon to tell how important ncRNAs will be relative to these other pathways. It could be argued that a more cautious approach would be to conduct pilot studies of the type described here, but on a smaller scale, in order to first demonstrate the possible validity of this model to gene regulation in ES cells. Going “all in” with a large scale, comprehensive attack is bold, but possibly premature given how little of the underlying biology is understood. Will these ncRNAs prove to be critical? Is the number of ncRNAs to be studied sufficient to get major insights? Are hESCs the best test system for study of ncRNAs? Would the mouse be more tractable at this time?
It might have been good to propose some experiments on the existing ncRNAs in case no other ncRNAs are identified, in which case both aims would not be very informative. However, if the PI’s prior experience is any guide it is almost certain that many hundreds of ncRNAs will be identified by the approaches outlined in this proposal. In fact, little is said about how s/he would sort through this potentially vast array of ncRNAs for those that will be subsequently studied at a functional level. Two other minor concerns were noted. First, as noted by the applicant, Aim 1 depends on having highly specific antibodies for isolating components of the epigenetic modification complexes. There are no specific details about how these proposed antibodies will be quality-controlled. These reagents are essential for these experiments, and it is difficult to evaluate their feasibility without additional information about their utility and specificity. Second, little detail is given on the methodology for manipulating ncRNAs expression in human cells and on the feasibility of the genomic mapping approach. Preliminary data on these techniques would have strengthened the proposal.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI is a physician-scientist who obtained his/her MD and PhD degrees in 2000, having done graduate work with a Nobel laureate. Postdoctoral studies were performed with a well-known scientist. At the conclusion of a postdoctoral fellowship, the applicant was appointed an Assistant Professor, and in 2005 s/he became a member of the applicant institution’s cancer center. The applicant’s CV is excellent — s/he is very productive, has numerous first and now senior author papers in the top journals, and s/he has received two prestigious scholar awards. The PI’s current support is very extensive, and the lab is tackling diverse problems in gene regulation, and is very well funded to do that. In addition, the PI is an active physician. There is some question as to whether another large-scale project would be appropriate at this stage, and the comments above regarding a pilot scale approach might also be considered in this context. The PI has outlined an excellent career plan – the environment is excellent, and the PI has excellent collaborators and mentors. This investigator may well become a leader in the field.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The institutional commitment is superb. They are providing excellent support to the PI, and have a superb track record with trainees. While no specific comments are made in the letter are made about ongoing support, the PI’s lab space is very adequate, his/her interactions with colleagues outstanding, and the shared resources are outstanding.
DISCUSSION: The reviewers considered this an excellent, innovative, and nicely written proposal from a PI with excellent training and an excellent team of collaborators. The only real issue is how many ncRNAs will be found? Is human the right system, or should s/he look in the mouse first? These are only minor concerns as this PI, a physician-scientist who studied with excellent PhD and post-doctoral mentors, is the type of investigator that CIRM wants to fund.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- Odorico, Jon
- Wagers, Professor Amy