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Proteomic Characterisation of Rat Bone Marrow-Derived Mesenchymal Stromal Cells Cultured in ‘Stemness’ Promoting Conditions

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Proteomic Characterisation of Rat Bone Marrow-Derived Mesenchymal Stromal Cells Cultured in ‘Stemness’ Promoting Conditions PROTEOMIC CHARACTERISATION OF RAT BONE MARROW-DERIVED MESENCHYMAL STROMAL CELLS CULTURED IN ‘STEMNESS’ PROMOTING CONDITIONS. Morgan Madeline Carlton Bachelor of Biomedical Science, QUT 2015 Tissue Repair and Translational Physiology Program Institute of Health and Biomedical Innovation School of Biomedical Sciences Faculty of Health Queensland University of Technology Submitted in fulfilment of the requirements for the degree of Master of Philosophy (Health) 2018 Keywords Biological processes Bone marrow-derived mesenchymal stromal cells Bone morphogenetic protein 4 Data independent acquisition Differentiation Fibroblast growth factor 2 Fibronectin Foetal calf serum Gene Ontology Mass spectrometry Mesenchymal stromal cells MetaboAnalyst OFFGEL electrophoresis Protein profiling Proteomics Rat Serum concentration Stemness SWATH acquisition Proteomic Characterisation of Rat Bone Marrow-Derived Mesenchymal Stromal Cells Cultured in ‘Stemness’ Promoting Conditions. i Abstract Mesenchymal stromal cells (MSCs) have the potential to be powerful medical tools for utilisation in clinical practice, for the treatment of numerous ailments including wound healing, tissue regeneration and potentially even spinal injury repair. To maximise the use of MSCs, access to an off-the-shelf MSC therapeutic is highly desirable, however, achieving this outcome is challenging, as MSCs cannot survive, undifferentiated in culture for extended periods of time. Currently, research in this field aims to determine an optimal culture environment for the growth of MSCs, that maintains their stromal phenotype. Evaluation of MSC differentiation and stemness have been investigated using methods of proteomics previously however, advanced protein profiling methods, such as SWATH-MS, have yet to be utilised for this purpose. Therefore, the purpose of this study was to investigate the effect that specific growth factors had on MSCs during culture through the utilisation of SWATH-MS analysis, with specific interest in the maintenance of their undifferentiated state. Initially, bone marrow-derived MSCs (BM-MSC) were cultured in different concentrations of serum to determine the most suitable concentration to maintain BM- MSC growth without significant interference with the subsequent proteomic analysis methods. Following this, MSCs were cultured, on either fibronectin (FN) coated or uncoated flasks, in condition media supplemented with fibroblast growth factor 2 (FGF2) and/or bone morphogenetic protein 4 (BMP4). The cellular protein was collected and processed for analysis by qualitative and quantitative SWATH-MS analysis. From this research, a rat BM-MSC protein library containing a total of 943 proteins was developed. Each of these proteins was quantified in each of the treatment conditions and evaluated to determine the most predominant biological processes occurring with regards to differentiation status of the cells. Individual proteins were evaluated to further investigate the effect that the treatments had on the biology of the BM-MSCs. Interestingly, eight proteins were identified to have a significant difference in abundance between the treatment groups. The biological role of these proteins in BM-MSCs were assessed. ii Proteomic Characterisation of Rat Bone Marrow-Derived Mesenchymal Stromal Cells Cultured in ‘Stemness’ Promoting Conditions. Characterisation of the rat BM-MSC proteome provides insight into the molecular processes that are occurring in the cells when they are stimulated with FGF2 and BMP4. Understanding the effect that these treatments have on the protein profiles of BM-MSCs provides important knowledge required to develop an optimal culture environment for BM-MSCs. Proteomic Characterisation of Rat Bone Marrow-Derived Mesenchymal Stromal Cells Cultured in ‘Stemness’ Promoting Conditions. iii Table of Contents Keywords .................................................................................................................................. i Abstract .................................................................................................................................... ii Table of Contents .................................................................................................................... iv List of Figures ........................................................................................................................ vii List of Tables ........................................................................................................................... xi List of Abbreviations .............................................................................................................. xii Statement of Original Authorship ......................................................................................... xiii Acknowledgements ............................................................................................................... xiv Chapter 1: Literature Review ........................................................................... 17 1.1 Background/Introduction ............................................................................................. 17 1.2 MSCs ............................................................................................................................ 17 1.2.1 Stemness ............................................................................................................ 19 1.2.2 Differentiation .................................................................................................... 20 1.3 Current Culture Conditions .......................................................................................... 21 1.4 Advances in Culture Conditions .................................................................................. 23 1.5 Proteomics in MSC research ........................................................................................ 26 1.6 Conclusion, perspective and Study Rationale .............................................................. 28 1.6.1 Summary and Implications / Conclusions ......................................................... 28 1.6.2 Perspective and Research Problem .................................................................... 28 1.6.3 Purpose .............................................................................................................. 29 1.6.4 Significance, Scope and Definitions .................................................................. 29 1.6.5 Thesis Outline .................................................................................................... 29 1.6.6 Hypothesis and Aims ......................................................................................... 30 Chapter 2: Materials and Methodology ........................................................... 31 2.1 Methodology and Research design .............................................................................. 31 2.1.1 Methodology ...................................................................................................... 31 2.1.2 Research design ................................................................................................. 31 2.2 Materials....................................................................................................................... 32 2.2.1 General Reagents ............................................................................................... 32 2.2.2 General consumables ......................................................................................... 32 2.2.3 Growth Factors .................................................................................................. 33 2.2.4 Instrumentation .................................................................................................. 33 2.2.5 Software ............................................................................................................. 33 2.3 Procedures .................................................................................................................... 34 2.3.1 Cell Culture ........................................................................................................ 34 2.3.1.1 Ethics ............................................................................................................... 34 2.3.1.2 Sample Population .......................................................................................... 34 2.3.1.3 Cell Collection ................................................................................................ 34 2.3.1.4 Protein Collection ............................................................................................ 34 2.3.2 Protein Processing.............................................................................................. 35 2.3.2.1 Acetone Precipitation ...................................................................................... 35 iv Proteomic Characterisation of Rat Bone Marrow-Derived Mesenchymal Stromal Cells Cultured in ‘Stemness’ Promoting Conditions. 2.3.2.2 Protein Quantification ......................................................................................35 2.3.2.3 Pooled Samples ................................................................................................35 2.3.2.4 FASP ................................................................................................................35 2.3.2.5 LDS-PAGE ......................................................................................................36
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