Molecular Characterization and Functional Exploration of Nuclear Receptors in hiPSCs

Molecular Characterization and Functional Exploration of Nuclear Receptors in hiPSCs

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01530
Approved funds: 
$1,712,880
Stem Cell Use: 
iPS Cell
Embryonic Stem Cell
Public Abstract: 
Our lab is known for its discovery of the family of nuclear hormone receptors (NHRs) that use vitamins/hormones to control genes and thereby regulate embryonic development, cell growth, physiology and metabolism. Of 48 known NHRs, we discovered that a unique subset of 38 receptors are expressed in adipose-derived human induced pluripotent stem cells (hiPSCs). The process of converting adult cell types like skin or fat into stem cells literally occurs in the nucleus by a process known as epigenetic reprogramming. A unique property of NHRs that distinguishes them from other classes of receptors is their ability to directly interact with and control the expression of genomic DNA. Consequently, NHRs play key roles in both the etiology and the treatment of disease by controlling genes. Drugs targeting NHRs are among the most widely prescribed in the world. While adipose-derived iPSCs express 38 NHRs, virtually nothing is known about their function in controlling stem cell renewal and differentiation into specific cell types (cell fate). How the extensive family of hormonal ligands can be used to control iPSC generation, maintenance and cell fate has profound implications for regenerative medicine. We wish to take advantage of our lab's expertise to understand, at the molecular and hormonal level, how nuclear receptors can be exploited to accelerate the use of iPSCs in regenerative medicine.
Statement of Benefit to California: 
Our lab 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. This work was all done in California and has brought in more than $100,000,000 of private and federal funding to my group over the last 30 years. It has led to my employment of 150+ people and the publication of more than 300 research papers. Three biotech companies were founded from this work that in aggregate raised more than $1B in research and development support. Several FDA approved drugs for cancer, diabetes, osteoporosis and low white blood cells (leukopenia) were developed with this technology. Of 48 known NHRs, we discovered that a unique subset of 38 receptors are expressed in adipose-derived human induced pluripotent stem cells (hiPSCs). Drugs to nuclear receptors are among the most widely prescribed in the world. While adipose-derived iPSCs express 38 NHRs virtually nothing is known about their function in controlling stem cell renewal and differentiation into specific cell types (cell fate). How the extensive family of hormonal ligands can be used to control iPSC generation, maintenance and cell fate has profound implications for regenerative medicine. We wish to take advantage of our lab's expertise to understand, at the molecular and hormonal level, how nuclear receptors can be exploited to accelerate the use of iPSCs in regenerative medicine. Thus, our proposed study should be beneficial to the State of California in several ways: 1) by maintaining a unique training environment for students, postdoctoral fellows and academic and clinical physicians; 2) discovering how to better and more efficiently generate and use human iPSCs; 3) decipher the molecular genetic logic of nuclear reprogramming; 4) determine how a pharmacopeia of hormones and drugs can be brought to play on directing stem cell renewal, differentiation and therapy.
Progress Report: 

Year 1

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.

Year 2

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.

Year 3

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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