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Adipose Derived Stem Cell Osteogenic Differentiation: a Study

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Adipose Derived Stem Cell Osteogenic Differentiation: a Study ADIPOSE DERIVED STEM CELL OSTEOGENIC DIFFERENTIATION: A STUDY OF THE INFLUENCE OF EXTRACELLULAR MATRIX ON THE DIFFERENTIATION PROCESS BY Heather Adeline Bradbury Coan A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERISTY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in the Molecular Genetics and Genomics Program May 2011 Winston-Salem, North Carolina Approved By Mark Van Dyke, Ph.D., Advisor Stephen Walker, Ph.D., Chair Barbara D. Boyan, Ph.D. Mark O. Lively, Ph.D. Shay Soker, Ph.D. ACKNOWLEDGMENTS I first want to thank my advisor Dr. Mark Van Dyke. As a mentor and friend, Mark has provided me with guidance and support throughout my entire graduate student career. His unwavering support and confidence in my abilities allowed me to pursue my degree with the knowledge that I could learn to do anything in the lab with time and practice. This project, although largely outside of Mark’s area of expertise, took shape through his guidance and vision as well as his willingness to entrust the details to me. My future success can be attributed to Mark’s dedication to me as a student. Additionally, I owe thanks to my program director Dr. Mark Lively. As an ―old school‖ scientist, his attention to detail has taught me to pay attention to things which might seem minor however often play the biggest role in the success of an experiment. The dedication he has shown me throughout this process is greatly appreciated. Also, thanks to my committee members Dr. Shay Soker, Dr. Steve Walker, and Dr. Barbara Boyan. The suggestions and input each of them provided in experimental design and concepts were vital to this project. Others, too many to mention, who have helped throughout this project include many past and present lab members. Chris Booth, a friend and student researcher has helped me with experiments for the past four years. Emily Moorefield, Dr. David Mack, Dr. Thaleia Teli, Drs. Amber and Matt Stern, as well as the Van Dyke research group, have all provided vital input into the project design and goals. Additionally however, I want to thank several past teachers and professors whose excitement and enthusiasm for science led me down this career path. Mr. Mike Smith ―Smitty‖, my high school biology professor was first to spark my interest in the sciences. ii I give him credit for my choice to pursue science as a college degree path. Two professors at Appalachian State University, Dr. Jeffrey Butts, and Dr. Mary Connell, were also a large influence on me as a student. Dr. Butts, my advisor, was always a source of support and aid throughout my college career. Dr. Mary Connell, my molecular biology professor however, gave me my first taste of success in a scientific lab setting as well as provided support and advice while I was applying for graduate school programs. Finally, I want to thank my family. My parents, brothers, and grandparents have all provided support and love throughout this process. My parents, Kevin and Barb Bradbury, instilled in me my work ethic and the confidence to pursue anything I wanted to do. I owe them everything for providing me with a childhood full of learning experiences and unending opportunities. I could not ask for two more perfect people to model my life after. I also want to thank my in-laws Martha and Chuck Saffer, as well as Jim Coan and Suzanna Blanchard for providing me with love and support. And last of all, my wonderful husband, Beau Coan and my sweet daughter Adeline Coan. Each has provided their own brand of support throughout the process. Beau has been my rock and my support to lean on when I thought this process would never end. He has supported me and pushed me forward every step of the way. Adeline, my darling girl, who has made the process fun and brought perspective to what otherwise can be a long and arduous journey. I hope one day, she will pursue whatever passions she has, as I’ve been lucky to be able to pursue mine. Finally, to the other two family members ―the boys‖ Doc and Wyatt my two border collies who have forced me to get off my butt while writing and take them on walks and runs! iii TABLE OF CONTENTS PAGE LIST OF FIGURES ………………………………………………….......... v LIST OF TABLES ……………………………………………………….. ix LIST OF ABBREVIATIONS ……………………………………….. xi ABSTRACT ……………………………………………………….. xiv INTRODUCTION ……………………………………………………….. 1 MATERIALS AND METHODS ……………………………………….. 12 RESULTS: Chapter 1. Creating an Osteogenic Differentiation Expression Profile for Human Adipose Derived Stem Cells using Whole Transcriptome Profiling and ECM-Related Gene Expression Profiling ……………………….. 34 Chapter 2. Cell-Secreted Matrices Modulate Osteogenic Differentiation of Adipose Derived Stem Cells ……. 61 Chapter 3. Dermatopontin in the Extracellular Matrix may Enhance Osteogenic Differentiation of Adipose Derived Stem Cells ……………………………….. 117 DISCUSSION AND CONCLUSIONS ………………………………... 138 REFERENCES ………………………………………………………... 166 APPENDIX 1 ADSC Isolation ………………………………... 185 APPENDIX 2 ADSC Flow Cytometry Characterization ………… 186 APPENDIX 3 ECM: Characterization of Day 11 and Day 16 Differential Protein Content using 2D Gel Electrophoresis, LC/MS/MS, and Mascot Searches …. 188 APPENDIX 4 QPCR Confirmation of Genes Identified Through Gene Analysis ………………………………… 194 SCHOLASTIC VITA ………………………………………………… 201 iv LIST OF FIGURES PAGE MATERIALS AND METHODS Figure 1. Gene array experimental design. 21 Figure 2. Gene array analysis workflow. 22 Figure 3. Histogram comparing raw data to RMA-normalized data. 23 Figure 4. Design and construction of Lenti_DPT construct. 29 CHAPTER 1 Figure 1. Osteogenic transcription factor RUNX2 mRNA expression increases with time in culture during BM induced osteogenesis. 36 Figure 2. Alkaline phosphatase enzyme activity and mRNA levels increase with time in culture during BM induced osteogenic differentiation. 37 Figure 3. ADSC induced to differentiate deposit calcium in the form of a mineralized matrix; ADSC in maintenance medium do not. 39 Figure 4. Mature osteoblast marker osteocalcin mRNA and protein levels are significantly increased by day 21 in differentiation. 40 Figure 5. Hierarchical gene clustering of time points in differentiation. 44 Figure 6. Differentiation factors important in activating adipogenesis. 54 Figure 7. Adipose terminal differentiation markers (FABP4 and FAS) and adipokines (Leptin and Adipsin). 55 Figure 8. Hierarchical gene clustering of ECM-rg at time points in osteogenesis. 57 CHAPTER 2 Figure 1. ECM deposited on culture dish during osteogenic differentiation. 62 Figure 2. Increased calcium deposition on ECM coated dishes visualized with alizarin red. 63 v Figure 3. RUNX2 mRNA expression increases for cells induced to differentiate on day 16 and day 24 ECM. 64 Figure 4. mRNA expression of bone related genes osteopontin (OPN) and osteoprotegerin (OPG) increases when cells are induced to differentiate on day 16 ECM. 66 Figure 5. mRNA expression of bone related genes transcription factor 4 (ATF4), and alkaline phosphatase (ALP) increases when cells are induced to differentiate on day 16 ECM. 67 Figure 6. Significant increases in mRNA and protein levels of osteocalcin were observed in cells induced to differentiate on day 16 ECM. 68 Figure 7. Gene arrays grouped by overall similarity in expression profiles. 71 Figure 8. Heat map indicating high (red) or low (green) expression of genes clustered on the bases of similar expression in Experimental 1. 73 Figure 9. Heat map indicating high (red) or low (green) expression of genes clustered on the bases of similar expression in Experimental 2. 74 Figure 10. Venn diagrams for each time point with each of the groups (Control in blue, Experimental 1 in yellow, and Experimental 2 in green). 94 Figure 11. Gene expression of adipose differentiation factors, PPARγ and C/EBPα important in activating adipogenesis, shows differential expression among the groups. 104 Figure 12. Gene expression of adipose differentiation factors, C/EBPβ and C/EBPδ important in activating adipogenesis, shows differentiation expression among the groups. 105 Figure 13. Gene expression of adipose terminal differentiation markers fatty acid binding protein 4 and fatty acid synthase varies between groups. 106 Figure 14. Gene expression of adipokines leptin and adipsin varies between groups. 107 vi Figure 15. Heat map indicating high (red) or low (green) expression of ECM-rgs clustered on the basis similar expression patterns. 109 Figure 16. Heat map indicating high (red) or low (green) expression of ECM-rg clustered on the bases of similar expression in Experimental 1. 112 Figure 17. Heat map indicating high (red) or low (green) expression of ECM-rg clustered on the bases of similar expression in Experimental 2. 113 CHAPTER 3 Figure 1. Dermatopontin mRNA expression increases during osteogenic differentiation in Control and Experimental 1 groups. 119 Figure 2. Dermatopontin protein levels increase dramatically between days 10 and 16 in the Controls as observed through western blotting. 120 Figure 3. HEK293T cells transfected with Lenti_DPT construct. 121 Figure 4. Cell sorting of ADSC exposed to Lenti_DPT viral supernatant showed low infection efficiency. 122 Figure 5. Cell sorting enriched Lenti_DPT infected ADSC population. 123 Figure 6. ADSC infected with Lenti_DPT and sorted using ZsGreen express high levels of DPT mRNA. 124 Figure 7. Dermatopontin protein in ADSC_DPT cells increases corresponding to an increase in DPT gene overexpression driven by the Lentiviral infection. 125 Figure 8. Cell sorting enriched Lenti_DPT infected ADSC population cultured for 30 days maintain fluorescence. 126 Figure 9. Gene expression of RUNX2 and osteocalcin is downregulated at day 21 in ADSC_DPT cells compared to Controls and Lenti_vector alone cells induced to differentiate down the osteogenic lineage. 129 vii Figure 10. Gene expression of TAZ and Wnt5A is downregulated at day 21 in ADSC_DPT cells compared to Controls. 130 Figure 11. Gene expression of RUNX2 increases in DPT_ECM seeded cells at day 21 compared to Day 21 Control and Experimental 1.
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