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Title:A chemical biology study of human embryonic stem cell pluripotency and differentiation
Author(s):Geng, Yijie
Director of Research:Chen, Jie
Doctoral Committee Chair(s):Belmont, Andrew S.
Doctoral Committee Member(s):Freeman, Brian C.; Yang, Jing
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):human embryonic stem cell
Human Pluripotent Stem Cells
chemical biology
high-throughput screening
small molecules
pluripotency
differentiation
Abstract:Human embryonic stem cell (hESC) research provides promising prospects for the future of regenerative medicine. To fully realize its potentials, we not only need to further our understandings toward the molecular mechanisms governing pluripotency and differentiation, but also to find new tools to manipulate the processes of directed hESC differentiations. One of the best tools for the purpose of probing and manipulating biological pathways at the molecular and cellular level is small molecules. High-throughput screening (HTS) of combinatorial chemical libraries is widely used for the search of such bioactive small molecules. My dissertation has focused on using HTS to identify novel small molecules that regulate hESC pluripotency and differentiation. Using a HTS system based on a novel design, I have screened over 170,000 small molecules (Chapter 2), and have studied three hit molecules in detail (Chapters 2, 3, and 4, respectively). The first molecule was named Displurigen (Displg for short) for its ability to disrupt hESC pluripotency. Using a biotinylated version of Displg as a probe, I pulled down and identified HSPA8 (also known as HSC70, the constitutively expressed member of the 70 kd heat-shock protein family) as its biological target, and confirmed this finding with both loss-of-function and gain-of-function assays. I showed that HSPA8 helps maintain hESC pluripotency by interacting with OCT4 protein and facilitating its binding to DNA. This study identified HSPA8 as a novel regulatory component of the hESC pluripotency network, and was the first study to demonstrate the direct involvement of a chaperone protein in pluripotency regulation. I describe this work in Chapter 2 of my dissertation. The second molecule was named Mesendogen (MEG for short) for its ability to dramatically improve the efficiencies of hESC differentiation towards the mesoderm and definitive endoderm (DE) lineages. Cell-replacement therapies require differentiated cells of high purity, yet current human pluripotent stem cell differentiation protocols remain to be improved for such purposes. I attempted to enhance the commonly used growth factor-driven mesoderm and DE differentiation protocols using hit molecules acquired from the HTS, and identified MEG as a potent enhancer of growth factor-induced mesoderm and DE differentiations. Addition of MEG to mesoderm and DE differentiation cultures dramatically enhanced both their efficiencies to near homogeneity (≥ 85%). Using target identification techniques I identified transient receptor potential cation channel, subfamily M, member 6 (TRPM6) as the biological target of MEG. I then showed that MEG most likely functions by inhibiting the magnesium import activity of the TRPM6/TRPM7 channel complex, the major cellular channel of magnesium import. This study describes a robust method for the highly dependable productions of nearly homogeneous mesoderm and DE progenitors, and also reveals for the first time that TRPM6/TRPM7 channel activity and magnesium homeostasis may be involved in the regulation of early mesoderm and definitive endoderm specifications. This work is described in Chapter 3 of my dissertation. The third molecule was named Lymphgen 1 for its activity in inducing the self-organization of a putative primitive lymphatic plexus-like structure, in the shape of a balloon, out of 2-D hESC cultures and without the addition of any growth factors. Its structural analog, Lymphgen 2, was analyzed in parallel. Recently, several studies reported self-organization events of hESCs that give rise to three dimensional (3-D) organoid structures, all of which required pre-designed programs of external guidance such as growth factors during their differentiations. Inspired by these findings, I hypothesized that by applying an appropriate stimulus such as a small molecule inhibitor at the onset of differentiation, it may be possible to trigger an intrinsic developmental program embedded in hESCs that does not require any external guidance to progress into a self-organizing structure. To find such a stimulus, I systemically examined my hit compounds for their abilities to trigger self-organizing events, and found compounds Lymphgen 1 and 2 (collectively referred to as Lymphgens hereafter) that trigger an unguided and spontaneous self-organizing event which gives rise to a balloon-like organoid structure. Gene expression analyses, functional assays, and morphological studies demonstrated that this self-organizing event may have recapitulated the in vivo human lymphatic morphogenesis program. Using this system, I unveiled the unguided emergence of a DiI-Ac-LDL+VE-cadherin+CD31+CD34+KDR+CD43- endothelial progenitor population, and identified a previously unknown VEGFR3+ progenitor population which may be responsible for human embryonic lymphatic development. This system provides a rare opportunity to visualize a truly spontaneous human developmental program in vitro. This work is described in Chapter 4 of my dissertation. In summary, my dissertation described the development of a human pluripotent stem cell-based HTS platform that was built upon a unique and novel approach which was eventually proven to be highly effective. It also described the discoveries and detailed studies of three hit molecules found in the HTS. Through comprehensive studies of these molecules, I unveiled novel regulatory mechanisms at the molecular level that govern hESC pluripotency and differentiation. Moreover, using these small molecules as tools, I significantly improved upon current protocols for both guided lineage-specific differentiations and spontaneous organoid self-organizations of hESCs. My thesis work has been dedicated to advancing the field of hESC research through the use of chemical biology.
Issue Date:2015-07-09
Type:Thesis
URI:http://hdl.handle.net/2142/88267
Rights Information:Copyright 2015 Yijie Geng
Date Available in IDEALS:2015-09-29
Date Deposited:August 201


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