Funding opportunities
Epigenetic mechanisms of human cell reprogramming
Funding Type:
Basic Biology II
Grant Number:
RB2-01542
Funds Committed:
$1,470,300
Funding Recommendations:
Not recommended
Public Abstract:
Recent advances in our ability to generate cell models of human disease come from the area of regenerative medicine. Patient cells have been reprogrammed to a stem cell like state by the introduction of genes encoding proteins involved in the regulation of gene expression in stem cells, also called transcription factors. A cocktail of four such genes, when introduced into human skin cells, turns these cells into induced pluripotent stem (iPS) cells. By manipulation of the environment in which these iPS cells are grown, using a combination of proteins and small molecules, researchers have been able to generate many different types of cells found in the human body. These cell types can be those that are affected in human diseases, thus allowing researchers to probe the basis for these diseases, and to test possible therapeutics. One limitation of this approach, however, is the poor efficiency of reprogramming and the length of time necessary to induce pluripotency. To circumvent these obstacles, several labs have looked for small molecules that will either replace components of the four-transcription factor cocktail used for reprogramming or enhance the efficiency of reprogramming. Recent studies have shown that a compound called valproic acid improves the efficiency of generation of iPS cells from fibroblasts, and can allow reprogramming with only two transcription factors. This molecule acts by changing the chromosome environment of genes, thereby activating silent genes. However, valproic acid is a relatively weak and non-selective compound. We propose to identify more selective and efficient small molecules that improve the efficiency of reprogramming human cells, with the long-term goal of identifying small molecules that can reprogram cells by activating endogenous cellular genes involved in reprogramming differentiated cells to the pluripotent state. Our laboratory has extensive experience with a class of molecules called histone deacetylase (or HDAC) inhibitors, and we have on hand a library of such molecules, and small interfering RNAs that also reduce the levels of the HDAC enzymes in cells. We will screen these libraries in order to identify the HDAC enzymes that regulate the master genes for pluripotency and to identify small molecule HDAC inhibitors to improve on our ability to generate iPS cells. We will also collaborate with JST researchers in Japan. Since only a single transcription factor (POU5F1/OCT4) is sufficient for generation of iPS cells from neural stem cells, they will focus on the reactivation of POU5F1/OCT4 in neural stem cells by using similar approaches. Once we have identified these enzymes and small molecule inhibitors, this information will advance our understanding of the cellular control mechanism that govern stem cells, and will also make the generation of stem cells from patient samples to be a more efficient process. It will also link to a new pathway to regenerate the human brain.
Statement of Benefit to California:
A major obstacle in the development of new drugs for human diseases is our lack of cell models that represent the tissues or organs that are affected in these diseases. While cancer cells can be grown in the laboratory, and are available for drug testing, the cell types that are affected in many diseases are not readily available. Two familiar examples are brain cells from Parkinson’s and Alzheimer’s disease patients, or cells of the pancreas from diabetic patients, neither of which are available for experimentation and drug screening. However, recent advances in stem cell biology now make it possible to generate such cells, starting from patient skin cells. Skin cells can be turned into stem cell-like cells (induced pluripotent stem cells), which can then give rise to just about any cell type in the human body. However, the process to generate these cell models is time consuming and inefficient. The studies proposed in this application will yield information on how to improve of generation of patient-specific induced pluripotent stem cells, and will likely speed up the process of generating these cell models and screening for new drugs. Development of therapeutics for neurological diseases such as Parkinson’s and Alzheimer’s disease or diabetes would be a major benefit to the people of the State of California, and the nation as a whole.
Review Summary:
EXECUTIVE SUMMARY
The main goal of this proposal is to develop improved methods for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). The project is based on the earlier finding that valproic acid, a general inhibitor of histone deacetlyases (HDACs), can increase the efficiency of reprogramming and substitute for two of the four canonical reprogramming factors (KLF4 and c-MYC) in the production of iPSCs. In the first three Aims, the applicant proposes to identify specific HDACs involved in the regulation of pluripotency genes, perform a chemical library screen to identify small molecules that selectively inhibit these HDACs, and determine whether these small molecule inhibitors enhance iPSC reprogramming. Work in a Partner Principal Investigator’s (PI) laboratory will concentrate on the final two Aims focused on the reprogramming of neural stem cells (NSCs) to pluripotency. In Aim 4, the Partner PI proposes to investigate the molecular mechanisms underlying OCT4 suppression in NSCs, and in Aim 5, HDAC inhibitors identified in Aim 2 will be tested for effects on OCT4 expression and reprogramming in NSCs.
Reviewers agreed that understanding the mechanism of reprogramming is an important area of study. However, they noted that the focus of this proposal, improving reprogramming efficiency, is not as significant an endeavor as overcoming barriers to using iPSCs for clinical applications or for understanding the basic biology of reprogramming. Reviewers also questioned the focus of Aims 4 & 5 on optimizing reprogramming of NSCs, as the value of such studies for clinical applications is obscure. Furthermore, it is not clear that the NSC reprogramming system will yield mechanistic insights beyond those obtained with somatic cells. Reviewers commented that the novelty of the proposal is modest, as there have already been several reports of small molecule screens for enhancers of iPSC generation.
The reviewers found the research plan to be logical but overly ambitious and disjointed. They commented that Aims 4 and 5, which focus on reprogramming of human NSCs, have little relevance to the first three aims, which concern reprogramming of human somatic cells. In terms of feasibility, reviewers noted that the proposal relies heavily on the hypothesis that de-repression of OCT4 or SOX2 can be achieved by HDAC inhibition alone. They were concerned that knockdown of individual HDACs, or even combinations, may have little effect on endogenous OCT4 or SOX2 expression. Alternative approaches were not well developed.
Reviewers praised the assembled research team, noting that the PI and Partner PI have excellent track records and complementary expertise. The PI has a strong publication record in the HDAC field. Reviewers felt that the team is stronger in chemistry than biology but had no doubts that the team is well qualified to carry out the proposed research.
Overall, while reviewers appreciated the strong research team and the proposal’s focus on mechanism, they found the research plan lacking coherence and raised concerns about its feasibility and significance.
Conflicts:
