Funding opportunities

Developmental Regulation of Human Embryonic Stem Cells by microRNAs

Funding Type: 
SEED Grant
Grant Number: 
Funds requested: 
$637 999
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Stem cells are remarkable cells which have two unique properties: self-renewal (self replication) and pluripotency (the ability to regenate a wide range of tissue-specific cells). These properties and the associated potential to use these cells to cure a wide range of degenerative diseases such as Alzheimer’s Disease, Parkinson’s Disease, and heart disease, have made stem cells a subject of intense scientific and medical interest. The recent discovery of cancer stem cells implies that stem cell research will also have important implications for cancer therapy, since cancer stem cells must be targeted by cancer drugs to prevent relapse of tumors. The mechanisms by which stem cells self-renew and differentiate are poorly understood. Several genes have been isolated and are thought to be essential to mammalian stem cell renewal and differentiation. Recent evidence has identified a new and potentially important molecular mechanism for regulating these genes in ESCs. These molecules are small bits of RNA, of approximately 22 nucleotides (nt) in length, and often termed microRNAs (miRNAs). miRNAs appear to play an important part in regulating gene activity. These small RNAs “turn off” genes by directly binding to them and preventing the production of proteins based on the gene’s genetic code. Several recent studies indicate that the types of miRNAs present in stem cells (miRNA “expression profiles”) are different from other cells and tissues. We propose to identify candidate miRNAs that are playing key roles in hESCs and to characterize the effects of their actions on self-renewal and differentiation. Our preliminary data also indicates that miRNA “expression profiles” differ depending on whether a stem cell is differentiating, self-renewing, or quiescent. This project will employ a wide range of techniques in molecular biology, including microarrays, bioinformatics, and bioluminescent reporter gene assays, to determine which specific miRNAs show differential expression between stem cell states and we will identify the genes they target. Having identified candidate miRNAs that play an important role in determining stem cell fate, we will manipulate their expression levels using biotechnology techniques known as RNA oligos and plasmid or viral expression vectors. We will then determine if these manipulations change the cell’s decision-making process in regard to differentiation or self-renewal. This will be done using molecular biology and biochemical assays on undifferentiated and differentiated cells. Ultimately we will investigate the underlying regulatory mechanisms in hESCs that control miRNA expression. We anticipate that our results will contribute to understanding the transitions between stem cells and differentiated cells, as well as normal and cancer cells. This type of information can be invaluable in designing new therapeutic approaches for stem cell replacement or cancer treatment.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) hold the potential to revolutionize human medicine by making cell replacement therapies, drug delivery, or in vivo modifications of cell populations a reality. In degenerative conditions we may be able to replace dead cells with functional new ones derived from hESCs. This approach could be used to treat Parkinson’s Disease, Alzheimer’s Disease, heart disease, diabetes, or paralytic spinal cord injuries. There is also potential for new cancer treatments, as cancer stem cells share many properties of hESCs. This type of medical technology would benefit the citizens of California in several general ways. It could offer hope to Californians suffering from these diseases. It could help relieve the pain and suffering for affected individuals and their families and loved ones. Finally, the economic impact of hESC-based therapies is likely to be significant. Chronic diseases will no longer incapacitate patients and they can return to productive work lives. State government and private expenditures on health care will actually decrease. Companies will organize to commercialize these hESC-based therapies and this will stimulate the California economy by providing new jobs and tax revenue. Most medical economists believe that significant revenues from patents, royalties, and licenses will flow from scientific discoveries pioneered in California based stem cell research centers. This project will also enhance California’s competitive position in biomedical research and push the State to the forefront of research not only in America, but also the world. It will be a reflection of the enormous ongoing investment in science on the part of the state and private institutions, fueled in recent decades by the high-tech and biotechnology industries. All of the research will be done in California. The project focuses on the role of microRNA (miRNA) molecules in controlling the fate of hESCs. miRNAs are recently discovered molecules that play an important part in gene regulation. The expression profiles of miRNAs in stem cells are different from other tissues, and miRNAs may play an essential role in stem cell self-renewal and differentiation. Many potential target genes for miRNAs are essential players in stem cell renewal and differentiation. Some of the most exciting and innovative work on miRNAs has taken place in California and this project will help confirm the State’s leading role in miRNA research and California’s role as a place where miRNA researchers’ specific application to stem cell biology is being studied.
Review Summary: 
SYNOPSIS: This proposal addresses the role of microRNA (miRNA) in the control of stem cell growth, differentiation, apoptosis, and cell cycle control in hESC. Preliminary data reveal differential expression of two miRNA clusters and in individual miRNAs associated with differentiation. The goal will be to analyze miRNAs in a systematic fashion and test roles and targets with RNA oligonucleotide interference. Finally, the expression of RNAi/miRNA machinery components in hESC will be identified. INNOVATION AND SIGNIFICANCE: This is a straightforward, largely descriptive project to survey and study miRNAs in hESC. MiRNAs may play a role in both keeping hESC in an undifferentiated state and a different set of miRNAs may be responsible for producing differentiated hESC. The methods and logic used are largely conventional in the field. The proposed experiments, as such, are feasible. It would be an exciting discovery to uncover a miRNA(s) or target(s) that were responsible for hESC differentiation or “stemness”. STRENGTHS: The PI has experience in RNA dependent gene regulatory phenomena and has enlisted collaborators for work with hESC. The experiments proposed are feasible and it is likely that miRNA effects on self-renewal and differentiation will be identified. The goal of aim 1 is to identify new miRNAs and target genes. The standard way to determine if a given UTR is a miRNA target is to create a miRNA “sensor” construct. This construct is then transfected in vitro into cells and reporter activity is measured. The goal of aim 1 is to identify miRNA targets but it is difficult and expensive to grow hESC. To avoid having to test each potential miRNA target in hESC the investigator has proposed to use NT-2 cells which are similar to hESC but much easier to maintain. WEAKNESSES: There is little conceptually new in this proposal. The PI is encouraged to develop more specific and penetrating hypotheses regarding the roles of miRNAs in hESC pluripotency or differentiation. This proposal is much too ambitious and very unfocused. It will be very difficult to complete the proposed work in the two-year time frame of the CIRM funding mechanism. The first aim of this proposal, identification of new miRNAs in hESC and the validation of miRNA targets, by itself has a good chance of requiring more than 2 years to complete. The amount of time the three proposed Aims may take to accomplish is probably even greater then the 5 year time frame of an R01 application. In the first Aim, the investigator will identify targets of newly identified miRNAs using the miRanda database. There are hundreds of miRNA targets predicted for each miRNA. In addition, it is rare that a single mRNA is targeted by a single miRNA. It would have been very useful if the investigator had provided a strategy outlining how the huge number of target genes that may be uncovered would be sorted. A list of genes, Oct4, Sox2, Nonog etc., are noted as genes that the investigator will pay “special attention” to. If these are the genes that are the most interesting, wouldn’t it have been better to propose experiments to closely examine the UTRs of these genes for miRNA binding sites? The expression of miRNAs that may bind these sites could be identified using a bioinformatics approach and then be confirmed in hESC by performing Northern Blots. By starting with interesting genes and working backwards to identify miRNAs, the investigator may be able to avoid performing an array screen and will not have to deal with 100s (or even 1000s) of miRNA targets. This approach would also help Aim 2 immensely since the investigator would be confirming the expression of known genes upon the removal of various mRNAs. DISCUSSION: There was no further discussion following reviewers' comments.

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