University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2016 Regulating p21 Expression to Increase Chondrogenic Potential in Human Mesenchymal Progenitor Cells Bertram, Karri Bertram, K. (2016). Regulating p21 Expression to Increase Chondrogenic Potential in Human Mesenchymal Progenitor Cells (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/27580 http://hdl.handle.net/11023/3397 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Regulating p21 Expression to Increase Chondrogenic Potential in Human Mesenchymal Progenitor Cells by Karri Bertram A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOMEDICAL ENGINEERING CALGARY, ALBERTA SEPTEMBER, 2016 © Karri Bertram 2016 Abstract Cartilage does not regenerate in humans, and therefore cartilage degeneration is a problem that affects a significant percentage of the population, including those with diseases such as Osteoarthritis (OA). The p21 knockout (p21-/-) mouse contains the only known single mutation in mammals that can induce a cartilage regenerative phenotype. Work in this thesis aims to identify p21 expression inhibitors for use in humans and to characterize their effects on human synovial mesenchymal progenitor cells (MPCs) during culture and chondrogenesis. I have identified one putative p21 expression inhibitor (acting through HSP90), that induces human synovial MPCs to display phenotypic properties similar to fibroblasts from p21-/- mice. Additionally, this inhibitor promotes cartilage formation in a mouse cartilage injury model. These results indicate that p21 inhibition through HSP90 may be a potential pharmaceutical target for stimulating chondrogenic regeneration for the treatment of cartilage defects or in cartilage degenerating diseases such as OA. ii Acknowledgements First and foremost, I would like to thank my supervisor, Dr. Roman Krawetz for his tremendous guidance through four summer studentships and especially through my graduate work. The individual time that you spend on each one of your students is remarkable and you have made this journey a truly fun and rewarding process. I would also like to thank the members of my supervising committee, Dr. Tina Rinker and Dr. Jeff Biernaskie, thank you for your expertise and guidance. I also want to thank all of the members of the Krawetz lab, my fellow graduate students (Asmaa, Christina, Nedaa, and Guomin), our post docs (Priya and Saleem), our lab techs (Catherine and Pankaj) and summer students (Nadia and Ted). Thank you all for your technical assistance throughout this project as well as moral support. I would like to thank my BMEG peers for giving me a sense of community as we all struggle through the same milestones. I would also like to thank Dr. Jim Powell and the Southern Alberta Organ and Tissue Donation Program for tissue that made this project possible. I would like to acknowledge the funding that made this work possible from The Stem Cell Network and the Biomedical Engineering Graduate Program. Finally, I would like to thank all of my friends and family that have encouraged me throughout this journey. iii Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iii List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii List of Symbols, Abbreviations, and Nomenclature ............................................................x Chapter One: Background ....................................................................................................1 1.1 OSTEOARTHRITIS ......................................................................................................1 1.2 ROLES OF CELLS AND SIGNALLING PATHWAYS IN OA .................................3 1.3 MESENCHYMAL STEM CELLS ................................................................................5 1.4 CURRENT TREATMENTS .........................................................................................6 1.5 CURRENT AREAS OF RESEARCH IN MSC CLINICAL TRIALS .........................9 1.6 CARTILAGE REGENERATION ...............................................................................12 1.7 CELL CYCLE .............................................................................................................18 1.8 HYPOTHESIS AND SPECIFIC AIMS ......................................................................23 1.8.1 Hypothesis ............................................................................................................23 1.8.2 Specific Aims ........................................................................................................23 1.8.3 Experimental design .............................................................................................24 Chapter Two: Methods ......................................................................................................26 2.1 ETHICS STATEMENTS.............................................................................................26 2.1.1 Human Ethics Statement .......................................................................................26 2.1.2 Animal Ethics Statement ......................................................................................26 2.2 CELL STRAINS ..........................................................................................................26 2.3 DRUG SCREENING ...................................................................................................28 2.4 DRUG LIBRARY ........................................................................................................28 2.5 DRUG INDUCED METABOLIC TOXICITY ...........................................................29 2.6 CELL CYCLE ANALYSIS .........................................................................................29 2.6.1 EDU Analysis of Proliferation ..............................................................................29 2.6.2 PI Analysis of Cell Cycle ......................................................................................30 2.7 DRUG KINETICS .......................................................................................................30 2.7.1 p21 protein nuclear localization ............................................................................30 2.7.2 p21 mRNA (qPCR) ...............................................................................................32 iv 2.8 CHONDROGENESIS .................................................................................................32 2.8.1 Culture ..................................................................................................................32 2.8.2 qPCR (mRNA) ......................................................................................................33 2.8.3 Histology (proteoglycan)/ Pellet Size ...................................................................34 2.9 IN VIVO MOUSE WOUND HEALING MODEL ......................................................34 2.9.1 Mice ......................................................................................................................34 2.9.2 Histology ...............................................................................................................35 2.10 STATISTICS .............................................................................................................36 Chapter Three: Results .......................................................................................................37 3.1 IDENTIFICATION OF P21 EXPRESSION INHIBITORS .......................................37 3.1.1 Drug screening ......................................................................................................37 3.1.2 Metabolic toxicity of drugs on XMAN cells ........................................................40 3.2 CHARACTERIZATION OF CHOSEN COMPOUNDS ON MPCS DURING CULTURE ................................................................................................................42 3.2.1 Metabolic toxicity on MPCs .................................................................................42 3.2.2 Cell Cycle .............................................................................................................44 3.2.3 mRNA p21 ............................................................................................................47 3.2.4 p21 nuclear localization ........................................................................................49 3.3 CHARACTERIZATION OF CHOSEN COMPOUNDS ON THE CHONDROGENESIS OF HUMAN MPCS ............................................................51