Making Stem Cell Therapies Safer: Targeting ES Cells with Cell Cycle and miRNA Antagonists

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01530
ICOC Funds Committed: 
$0
Stem Cell Use: 
iPS Cell
Embryonic Stem Cell
Public Abstract: 
Stem cell therapies have the potential to result in entirely new treatments for a vast variety of currently incurable diseases. The goal of these therapies is to regenerate lost cells or tissues through the use of established human embryonic stem cell lines or even from the patient's own cells. One major limitation of current stem cell therapy approaches is that they may give rise to cancer. Embryonic stem cells have a limitless potential to reproduce and if they remain in an undifferentiated state could give rise to tumors. This proposal seeks to make stem cell therapies safer by using two novel targets that can selectively kill undifferentiated stem cells while sparing differentiated cells or normal cells. We propose to use small molecule inhibitors of the cell cycle and inhibitors of specific small RNAs to kill embryonic stem cells that have remained in an undifferentiated state. If validated, these therapeutic approaches could be developed to make stem cell therapies safer.
Statement of Benefit to California: 
The State of California is committed to developing new approaches to treat currently incurable diseases through the application of stem cell biology technologies. While there is much excitement about the prospects for these new treatments, there is also potential concern, that current strategies may also increase the risk of developing cancer. This proposal seeks to use new strategies to block the growth of embryonic stem cells that pose the risk of developing into cancer. It is hoped that such approaches will make stem cell-based therapies safer for us all.
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.

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