The Role of Mediator in Maintaining and Differentiating Human Mesenchymal Stem Cell State
The maintenance of healthy cell state is required to prevent a number of human diseases, including developmental defects, autoimmune disease, and cancer. Small changes in the ability of a cell to properly regulate transcription and gene expression can result in large phenotypic changes. As medicine looks to novel therapies to treat human disease, adult stem cells and their therapeutic potential are being heavily investigated. The interest in stem cells is in their inherent cellular properties of self-renewal and their ability to be directed toward a number of terminally differentiated cell fates. To optimize the use of these cells in the clinic, it is first important that we gain a better understanding of what maintains their cell state as a healthy, multipotent cell, and what is necessary for them to properly differentiate down a specific lineage. Regulation of gene expression relies on coordinated interation of environmental stimuli, internal signaling cascades, transcription factors, chromatin modifiers, microRNAs (miRNAs), and the transcriptional machinery. Here we propose exploring the role of a large multisubunit protein complex in maintaining the unique properties of mesenchymal stem cells (MSCs), a population of adult stem cells found in human bone marrow, adipose tissue, and umbilical cord blood, recently identified to have tremendous therapeutic potential. Earlier work in this field demonstrated a critical role for Mediator in maintaining embryonic stem cell state by facilitating long-range cell-type specific interactions that promoted gene expression profiles necessary to maintain the pluripotency of embryonic stem cells (Kagey et al. 2010). The goal of the project proposed here is to determine the role that specific Mediator subunits have in adult mesenchymal stem cell state. Specifically we are interested in identifying the genes regulated by the Mediator, how expression of those genes is altered following the loss of specific Mediator subunits, how these changes in gene expression affect cell fate commitments, and finally how we can use this information to better understand the use of MSCs in clinical setting. The growing interest in adult stem cells and their use in the clinic demands more in-depth study of the mechanisms that control their distinct cell properties. Aim 1: How does Mediator regulate self-renewal and differentiation of hMSCs? Several mediator subunits have been implicated in regulating development, including Med1, Med12, and Med31. The multipotent state of adult stem cells makes them a valuable model system to study the role of these subunits by investigating how the loss of a given subunit impacts the ability of MSCs to continue to self-renew or be effectively directed down a particular developmental lineage. We will use siRNA knockdowns to study the role that Med1, Med12, and Med 31 each have in maintaining MSC cell state and differentiation potential. Aim 2: How does Mediator regulate gene expression of hMSCs? In order to understand how the loss of Mediator leads to changes in mesenchymal stem cell properties it will be necessary to investigate the genes whose expression is impacted following the loss of a given subunit. We will use microarray studies at several time points following efficient knockdown of a subunit to gain a better understanding of what genes and pathways are affected, resulting in the observed changes in cell state. We will work closely with a Dr. Prerna Dua, a colleague at Louisiana Tech University, to analyze the data and gain a deeper understanding for the role of Mediator in human mesenchymal stem cell biology. In addition we will validate direct regulation of a set of genes using chromatin immunoprecipitations and quantitative PCR. The goals outlined in the proposal will contribute to the understanding of transcriptional control in human mesenchymal stem cells. As we seek to understand the role of MSCs in human health and their potential in the clinic as a therapeutic tool, it becomes increasingly important to understand the functional properties of these cells and how they are regulated so that we may identify healthy cells for use in areas of regenerative medicine.
Principal Investigator: Newman, Jamie -- Biological Sciences
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