Degree
Doctor of Philosophy
Program
Biochemistry
Supervisor
Dr. Gilles Lajoie
Abstract
The stem cell microenvironment contains soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to effect cellular behavior. Mass spectrometry based proteomics offers the opportunity to directly assay components of extracellular microenvironments, thereby providing a sensitive means for obtaining insight into the stem cell niche. In this study we present the generation and analysis of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) matrix microenvironments using an MS-based proteomics approach.
One of the primary limitations in the proteomics analysis of hESCs and hiPSCs is the reproducible generation of sufficient cell numbers amenable to experimentation. In Chapter 2 we develop reliable methods for the maintenance and proliferation of hESCs in metabolic labeling medium for use in stable isotope labeling with amino acids in cell culture (SILAC) experiments. Previously, the application of SILAC with hESCs was hindered by the use of medium insufficient for their reproducible culture and the problematic conversion of isotopically labeled arginine to proline. To address these issues, we demonstrate the culture of hESCs, hiPSCs, and mouse ESCs in SILAC conditions using a serum-replacement mixture and alternatively in defined medium. In these optimized conditions we show that the problematic conversion of arginine to proline can be eliminated through the simple addition of exogenous proline to the culture medium.
Within the in vitro microenvironment, the ECM plays a significant role in the maintenance and regulation of hESCs and hiPSCs. In order to better understand the communication between hESCs and hiPSCs and the extracellular matrix, we focused our investigation on this portion of the microenvironment. To facilitate the proteomic analysis of complex ECM mixtures, in Chapter 3 we describe methods for the fractionation and MS-based proteomics analysis of these matrices. Application to a variety of cell-derived and recombinant growth matrices demonstrates not only the effectiveness of our methods, but also the utility of directly assaying matrices used in hESC culture for the determination of factors beneficial for the maintenance of self-renewal and pluripotency.
Through combination of the SILAC growth (Chapter 2) and matrix analysis (Chapter 3) methods, the protein compositions of conditioned matrices from multiple pluripotent stem cell lines are determined (Chapter 4). Within these matrices, numerous antagonists of core pluripotency pathways are identified, such as soluble frizzled related protein (sFRP) 1 and 2. In Chapter 5 we focus on characterizing the functions of sFRP1 and sFRP2 in hESC self-renewal and pluripotency. Taken together, the work presented here illustrates the utility of assaying interactions between stem cells and their matrix microenvironment for the determination of pathways involved in the regulation of the pluripotent phenotype.
Recommended Citation
Hughes, Christopher, "Mass Spectrometry-Based Proteomics Analysis of the Matrix Microenvironment in Pluripotent Stem Cell Culture" (2012). Electronic Thesis and Dissertation Repository. 445.
https://ir.lib.uwo.ca/etd/445