Because of their ability to develop into most of the specialized cells and tissues of the body, stem cells have the potential to replace diseased or dysfunctional cells with healthy functioning ones. It is the hope of the scientific and medical communities that the use of stem cell based therapies to treat diseases such as Parkinson’s disease, diabetes, heart disease and rheumatoid arthritis, etc. will one day be routine. Because this research field is still in its infancy, a number of scientific challenges must be overcome before the promise of stem cells can be harnessed. To this end, efforts aimed at increasing our understanding of the growth conditions, cellular biology and genetic events involved in stem cell survival and differentiation are key. While over 100 distinct stem cell lines have been derived, less than 20 are available in sufficient quantities for research purposes and of these, only a very limited number have been studied with respect to understanding how stem cells grow and develop into target cells. Clearly there is a great need to significantly expand the number of cell lines to allow comparative analysis of growth conditions, signaling and gene expression processes. These studies will help clarify how these cells can be grown to sufficient quantites to be used clinically and will also help determine at what stage these cells have maximum therapeutic potential.
We are interested in understanding how proteins called NF-kappaB factors regulate the expression of genes involved in stem cell survival, growth and differentiation. In adult stem cells, NF-kappaB has been implicated in promoting cell survival. In contrast, almost nothing is known about the action of these factors in embryonic stem cells and whether they play a similar protective role. We propose to generate stem cell lines carrying a potent inhibitor of all NF-kappaB action and use these cells to assess the impact on stem cell survival and progression to cells that make up the central nervous system, namely, neurons and glia. These cells will provide a powerful experimental platform to explore the biology of stem cell survival and neuronal differentiation as it relates to a specific gene regulation program.
Using the limited number of stem cell lines currently available, researchers have demonstrated that despite sharing some key characteristics, these lines also differed markedly. This highlights the importance and necessity of studying how certain genes are turned on or off in order to maintain both the survival and differentiation of stem cells. The studies proposed herein will provide important insights into how NF-kappaB regulates stem cell survival and differentiation. This information will ultimately advance our efforts at generating stem cells with therapeutic potential for use in the clinic.
Statement of Benefit to California:
Experts predict that stem cell research holds the potential to help up to half of all Americans who suffer from diseases, including Parkinson’s and Alzheimer’s diseases, stroke, spinal cord injury, heart disease, arthritis and cancer. Because of their ability to develop into most of the specialized cells and tissues of the body, stem cells have the potential to replace diseased or dysfunctional cells with healthy functioning ones. This regenerative medical technology represents one of the most exciting medical advances to date and may be the only hope for those suffering from what we now refer to as 'incurable diseases'. Despite its infancy, early results from stem cell therapy trials have prompted significant optimism in the scientific community that these therapies will one day be routine. However, there remain several scientific challenges that must be overcome before promise of stem cells can be harnessed. One major challenge involves identifying the desired stem cell type and once identified, determining the optimal culture conditions to form progenitor cells that will ultimately differentiate into the desired therapeutic cell type. A second challenge will be to determine how embryonic stem cell progression through the various differentiation stages is regulated and at what stage these cells posess maximum therapeutic potential. The studies proposed herein are aimed at advancing our understanding of the molecular mechanisms governing viability, pluripotency and differentiation of embryonic stem cells. Using a variety of biochemical, molecular biological and bioinformatics approaches, we will explore the mechanisms by which NF-kappaB regulates specific genes in both undifferentiated human embryonic stem cells and differentiated neuronal progenitor cells. Together, these studies will provide important insights into how NF-kappaB contributes to embryonic stem cell biology and ultimately to neuronal development and repair to treat neurological disorders. Notwithstanding the obvious enormous health and quality-of-life benefits that would accompany the development of effective stem cell therapies, the financial health care savings for the state of California could be sizable. As such, we believe these studies will benefit the citizens of California personally and financially, as well as positively impacting society at large.
SYNOPSIS: This proposal aims to clarify the role of NF-κB in hESC survival and differentiation. Transcripts encoding NF-κB-related signaling intermediates are enriched in hESCs; activation of NF-κB transcriptional regulators are critical for adult SC survival. In Aim I, the PI will assess which NF-κB complexes are expressed in hESCs and determine the effect of NF-κB chemical inhibitors as well as lentivirus transduced I-κB inhibitor. In Aim II, the PI will determine whether NF-κB-regulated expression of miRNAs impacts hESC gene expression. During brain development, miRNA expression changes dynamically and some miRNAs have cell lineage-specific expression. Of interest, Dicer-deficient zebrafish show abnormal development. The PI will assess the levels and identity of miRNAs in hESCs and also determine the effect on these miRNAs on inhibition of NF-κB.In aim III, the PI will evaluate the role of NF-κB on differentiation by transducing the lentivirus--κB inhibitor at different stages.
INNOVATION AND SIGNIFICANCE: The growth and differentiation of hESCs depends on a better understanding of the signaling mechanisms and regulators of gene expression programs. This proposal involves investigations of the effect of NF-κB expression with respect to hESCs. Some of the hypotheses are novel, but overall the proposal lacks innovation.
STRENGTHS: The proposal is well-written, organized, and involves hypotheses that are testable. The outcome of some of the experiments (e.g., related to specific aim II and miRNAs) may be of interest even if NF-κB is not important with respect to survival and differentiation of hESCs. The PI, a Staff Research Investigator at the Gladstone Institute since 2005, is an expert on NF-κB, most recently involved in a study of a role for NF-κB in spatial learning and memory. The PI has also enlisted the help of a number of key collaborators.
WEAKNESSES: The reviewers cited several weaknesses with this proposal. First, the proposal is poorly rationalized with insufficient background that fails to convincingly argue for a major role for the NF-kB system in hESCs. The PI plans to study hESCs from several sources without a clear rationale as to why these particular cells were chosen. Second, the hypotheses are not very compelling. Analysis of NF-kB target genes is unduly focused on micro RNA's and analysis of NF-kB function in neuronal differentiation using hHSC will scratch the surface but unlikely to provide deeper developmental insight. Third, the proposal is risky since the results may be negative. Finally, the proposal seems too ambitious.
DISCUSSION: The proposed experiments are of interest from a descriptive standpoint. One reviewer noted that NF-kB inhibits many genes and therefore it may be difficult to interpret results. Another reviewer thought that the proposal is poorly rationalized with insufficient background and foundation to convince the reviewer of any major role for NF-kB in hESCs. A key weakness of the proposal is that it scratches the surface of neuronal differentiation and has no strong developmental context. The applicant has prior experience studying NF-kB in a different context and is trying to move into the hESC field but had insufficient arguments to support the proposed research.