Stem cells are able to develop into most of the specialized cells and tissues of the body and therefore have the potential to replace diseased 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 Alzheimer’s disease, diabetes, heart disease and other degenerative conditions will one day be routine. Because this research field is still in its infancy, a number of scientific challenges must be overcome before promise of stem cells can be harnessed. In particular, we need to increase our understanding of the growth conditions, cellular biology and genetic events involved in stem cell survival and differentiation are key. While more than 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 studied with respect to understanding how stem cells grow and develop into target cells. Clearly there is a great need to study more 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 quantities to be used clinically and will also help determine at stage these cells have maximum therapeutic potential.
We are interested in understanding in molecular details two key properties of stem cells. First, self renewal is defined as the ability of stem cells to divide indefinitely, in contrast to non-stem cells which are limited in their ability to divide. Second, pluripotentiality refers to the ability of stem cells to differentiate in all cell types that are present in an adult organism. There is growing evidence that these two properties of stem cells are controlled at the central level via the interplay of cellular factors that control the transfer of DNA into RNA. Several key transcription factors have been identified that are unique to stem cells. Our laboratory has specialized during the last 20 years in the study of a family of proteins called histone deacetylases that control the activity of many transcription factors. However, no data exist on the possible role of these proteins in the self renewal and pluripotentiality of stem cells. We propose a series of experiments that will explore the role of histone deacetylases in these critical properties of stem cells.
This information will ultimately advance our efforts at generating stem cells with therapeutic potential for use in the clinic.
Human stem cells have the potential to replace diseased or dysfunctional cells with healthy functioning ones. This new technology represents one of the most exciting medical advances in history. Early results from stem cell therapy trials have prompted significant optimism in the scientific community that these therapies will one day be used routinely to cure a series of diseases ranging from Alzheimer’s disease to diabetes. Before the potential for this new technology can be realized, there remains much to be learned about the biology of stem cells including how stem cells differentiate to different cell types, what are the factors that trigger differentiation and contribute to their viability, etc. Our studies plan to explore the role of human histone deacetylases in human embryonic stem cell biology. Human histone deacetylases are a family of protein that regulate the expression of specific genes in human embryonic stem cells and in more differentiated adult cells. These studies will provide important insights into how histone deacetylases contributes to embryonic stem cell biology and their differentiation. Understanding these basic mechanisms will lay the foundation to therapies for numerous diseased that effect the citizens in this state and the world.
SYNOPSIS: The focus of this proposal is to explore the possible biological role of different HDACs and associated cofactors in pluripotency and self-renewal of hESCs. HDACs are histone deacetylases that are believed to play a role in chromatin and transcriptional regulation. The PI’s laboratory has been involved with the isolation and characterization of human HDACs and has collected 18 of them. This proposal has three specific aims. The first is to evaluate the contribution of HDACs to the differentiation of hESCs into various progenitors. The second is to identify gene targets of individual HDACs in hESCs using microarrays. The third specific aim will identify the cellular partners of individual HDACs in hESCs using immunoprecipitation and proteomics.
SIGNIFICANCE AND INNOVATION: One of the main challenges to moving from the discovery of human embryonic stem cells into the development of therapeutic products is to understand how to control and modify the pathways that control both the pluripotent state and the various states of differentiation. Epigenetic modification via chromatin remodeling has been shown to be a very important way to modulate the state of differentiation. This investigator, who is an expert in the field of chromatin remodelling via histone deacetylases (HDAC), proposes a series of studies that will provide much needed information regarding the role of HDACs in maintaining the undifferentiated pluripotent state and their role in driving differentiation. He also proposes to go beyond the analysis of HDAC expression alone, to include studies of the additional transcription factors (positive and negative) that are recruited due to the chromatin changes, and finally, to correlate expression of specific HDACs (either in a positive way or via knockdown strategy using shRNA) with in vivo differentiation into teratocarcinomas. These studies are not innovative per se, in that they are using reagents and methods that have been applied to many other cell types, but they are innovative in the application of these analyses to hESCs. The results of the studies could provide important information about the pathways and key switches in these pathways that would drive differentiation or maintain pluripotency. This type of information will aid in the ultimate development of specific differentiated cell products derived from hESCs.
STRENGTHS: The PI is an expert in the study of HDACs and therefore, has the reagents and assays readily available to perform the proposed studies: quantitative RT-PCR, specific antibodies for Western blot analysis, and shRNAs that work to specifically knockdown expression of certain HDACs. Further, their preliminary studies in murine ESC have shown that driving differentiation down the neuronal pathway has resulted in selected loss of expression of certain HDACs, but not all, underscoring the basis of their hypothesis.
The use of feeder-free culture conditions allow for a more precise measurement of the influence of the HDACs in the hESCs without the risk of contamination from the feeder cells.
The Gladstone Institute has available a core program that supports the use of embronic stem cells by providing methods and reagents to the other investigators at the Institute. In addition, the Gladstone has a core Genomics facility that has experience using the ChIP on CHIP assay that is proposed by the investigators to map the gene targets of HDAC expression.
The proposal has linked the mechanistic studies of looking at HDAC expression and the interaction of HDACs with transcription factors and genomic targets with functional assays to understand the phenotypic impact on differentiation when certain HDACs are either down-regulated by shRNAs or constitutively expressed. These types of functional outputs add an important dimension to the proposed studies.
In summary, the proposal is well thought out and very feasible, based on the combination of the experience of the PI with the great support core services available through the Gladstone Institute.
WEAKNESSES: In addition to the use of the in vivo differentiation assay (essentially, a gross assay for differentiation, by looking at teratoma formation in vivo), it might be easier to measure the impact of HDACs on differentiation if the investigators chose an in vitro pathway of differentiation to study -- much as they have done for their studies on murine ESCs (forcing them to differentiate into neuronal cells). A second concern is that there is a lot of weight put on the first aim. This problem might not be the most pressing issue that needs to be addressed in the first round of seed funds for hESC studies.
DISCUSSION: This work is significant in that it deals with chromatin remodeling in gene regulation. The design of the experiments is fine, but tremendous weight is placed in the results of Aim 1. The main criticism is that the experiments rely on the HDAC association assumptions from the preliminary studies in mESC. Reviewers would also like to see more relevant in-vivo assays than teratoma formation.