Unique properties of neuron-restricted progenitors derived from human stem cells

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01530
ICOC Funds Committed: 
$0
Stem Cell Use: 
iPS Cell
Embryonic Stem Cell
Public Abstract: 
Neural stem cells are self-renewing cells that generate progenitors capable of further differentiation into functional cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. During brain formation, the stem cells of the embryonic cortex generate neurons early during the developmental process but form astrocytes at later stages, suggesting that progenitor fate potential shifts over time. Despite evidence for neuron-restricted and astrocyte-restricted progenitors, little is known about the cellular characteristics that critically differentiate these two cell types from each other. In part, this lack of understanding is due to the shortage of specific markers that will distinguish these cell types. Specific progenitors can be used for transplantation to form a particular type of final, functional cell. Analysis of neuron- and astrocyte-forming neural stem/progenitor cells (NSPCs) by a technique termed dielectrophoresis (DEP) demonstrates that cell behavior in DEP correlates with potential to form neurons. DEP is a label-free, non-toxic and unbiased method for analyzing cells that detects formation of frequency-induced dipoles in cells. The dielectric properties of neuron- and astrocyte-forming mouse and human NSPCs significantly differ from each other and reflect their fate biases such that neuron-forming progenitors become more similar to neurons and astrocyte-forming progenitors to astrocytes. Furthermore, the specific dielectric properties of human NSPCs isolated from brain represent differences in the membrane compartments of those cells. The goal of this proposal is to utilize DEP to determine the cellular properties that discriminate neuron-forming progenitors from other cells. We hypothesize that stem cell fate potential revealed by dielectric signature is due to the cell membrane and that modifications altering the effective membrane thickness or surface area, such as modification of certain membrane components, are the main contributors to the cell fate-specific dielectric signatures. We will test this hypothesis with neural lineage cells derived from human ES cells, iPS cells, and brain. Experiments to test this hypothesis will identify the contribution of membrane and cytoplasmic cellular compartments to cell lineage-specific dielectric properties and test the involvement of membrane modifications in progenitor cell dielectric properties. We expect that NSPC dielectric properties will be a biophysical measure of their fate potential, providing a novel approach for investigating lineage-committed NSPCs and a way to use these cells to obtain specific cell types, such as neurons, after transplantation.
Statement of Benefit to California: 
The goal of this project is to determine whether a novel strategy using DEP can serve to identify specific characteristics of stem cell subpopulations. In the course of these studies, we expect to learn more about human progenitor cells that specifically generate neurons, a cell population of interest for basic biological studies, therapeutic approaches, and as a source of human neurons for drug testing. Our hope is that this label-free method for investigating stem cell subpopulations will greatly increase the speed of stem cell research in California and improve our understanding of how to control the composition of cells used in therapeutics.
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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