Epigenetic mechanisms of human cell reprogramming

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01530
ICOC Funds Committed: 
$0
Stem Cell Use: 
iPS Cell
Embryonic Stem Cell
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.
Progress Report: 
  • Our laboratory is known for its discovery of the family of nuclear receptors (NHRs) that use hormones to control genes and thereby regulate embryonic development, cell growth, physiology and metabolism. The goal of this project is to explore how NHRs activate gene networks to produce human induced pluripotent stem cells (hiPSCs). We will determine the specific sites on the genome where NHRs and the reprogramming factor (Oct4) bind and determine how binding results in “epigenetic” modifications. Epigenetic modifications are the result of enzymatic action on chromatin which is a combination of DNA and histones. The first goal is a massive project to establish all the genome wide DNA methylation changes in adipose derived human induced pluripotent stem cells and embryonic stem cells. DNA methylation is considered a silencing signal in the genome and marks genes that are inactive in a particular cell. Using state-of-the-art technology we discovered the first differences in the methylation patterns between these two cell types. These important differences between ES and iPSC cell types may influence their differentiation capabilities. We are currently performing experiments to map the sites of histone modifications and will correlate these sites with the identified DNA methylation sites. We have also used high resolution RNA sequencing technology to determine the global collection of all genes that are expressed (termed the “transcriptome”) in human iPS cells. A comparison of this transcriptome with an ES cell (ES H1) demonstrated that these 2 cell types are very similar at the gene level. We are currently on track to complete the stated milestones and goals of the funded project.
  • Our laboratory is known for its discovery of the family of nuclear hormone receptors (NHRs) that use hormones to control genes and thereby regulate embryonic development, cell growth, physiology and metabolism. Our goal is to explore how NHRs activate gene networks to produce human induced pluripotent stem cells (hiPSCs). We will determine the specific sites on the genome where NHRs and the reprogramming factor (Oct4) bind and determine how binding results in “epigenetic” modifications. One of our main goals is a massive project to compile all of the gene expression changes in adipose- and keratinocyte-derived hiPSCs, embryonic stem cells, and parental somatic cells. Gene expression differences between somatic, embryonic stem and hiPSC cell types may influence their differentiation capabilities. We are currently performing experiments to map the sites of histone modifications and will correlate these sites with the previously identified DNA methylation sites and the gene expression changes. We are currently on track to complete the stated milestones and goals of the funded project.
  • Generation of induced pluripotent stem cells (iPSCs) from somatic cells through cellular reprogramming offers tremendous potential for personalized medicine, the study of disease states, and the elucidation of developmental processes. Our laboratory is known for its discovery of the large family of nuclear hormone receptors that use hormones to control gene expression and thereby regulate embryonic development, cell growth, physiology and metabolism. Thus, our goal has been to explore how nuclear hormone receptors activate specific gene networks required for the production and maintenance of human induced pluripotent stem cells.
  • Using our highly efficient protocol for generating iPSCs from readily-available human adipose (fat) tissue, we have determined the changes in gene expression induced by reprogramming parental adipose cells into adipose-derived human iPSCs, as well as compared the gene expression pattern of our adipose-derived human iPSCs with embryonic stem cells. The determined gene expression profiles highlighted the differences between the reprogrammed iPSCs and the fully differentiated somatic adipocyte, as well as underscored their similarity to embryonic stem cells, providing insight into their relative differentiation capabilities. Notably, these studies identified the transient expression of the nuclear hormone receptor estrogen related receptor alpha (ERRα) during reprogramming. Consistent with the established roles of ERRs in regulating cellular metabolism, we observed transient increases in both lipid and glucose metabolism coincident with the increased expression of ERRα. Furthermore, we found that this transient increase in metabolism was essential for the generation of iPSCs, and was dependent on ERRα expression.
  • To understand the role of the transient increase in ERRα and the associated increase in cellular metabolism during iPSC generation, we are determining the specific sites on the genome where ERRα binds. In addition, we are mapping genome-wide epigenetic changes, in particular, changes in the location and/or identity of histone acetylation/methylation, that occur during the generation of iPSCs. The sites of histone modifications linked to gene activation/repression will be correlated with the identified ERRα binding sites, as well as with the previously characterized DNA methylation sites, to understand the molecular requirements for ERRα during “epigenetic” reprogramming.

© 2013 California Institute for Regenerative Medicine