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Characteristics of Dental Follicle Stem Cells and Their Potential Application for Treatment of Craniofacial Defects

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Characteristics of Dental Follicle Stem Cells and Their Potential Application for Treatment of Craniofacial Defects Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2014 Characteristics of Dental Follicle Stem Cells and Their otP ential Application for Treatment of Craniofacial Defects Maryam Rezai Rad Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Medicine and Health Sciences Commons Recommended Citation Rezai Rad, Maryam, "Characteristics of Dental Follicle Stem Cells and Their otP ential Application for Treatment of Craniofacial Defects" (2014). LSU Doctoral Dissertations. 2982. https://digitalcommons.lsu.edu/gradschool_dissertations/2982 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. CHARACTERISTICS OF DENTAL FOLLICLE STEM CELLS AND THEIR POTENTIAL APPLICATION FOR TREATMENT OF CRANIOFACIAL DEFECTS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Interdepartmental Program in Veterinary Medical Science through The Department of Comparative Biomedical Sciences by Maryam Rezai Rad D.D.S., Tehran University of Medical Sciences, 2007 August 2014 To my beloved husband, Mahdi, and our lovely daughter, Rose. ii ACKNOWLEDGMENTS Thank God for giving me the opportunity to explore the world. First and foremost, I would like to express my sincerest gratitude to my supervisor, Dr. Shaomian Yao, for his invaluable and insightful guidance throughout the course of my Ph.D. Study at Louisiana State University. He gave me the main idea of this work, and without his continuous encouragement as well as his deep knowledge of molecular biology, this dissertation could not have been accomplished. I am always thankful for his support and all I have learned from his great professional vision and scientific insight. He taught me how to face problems in my research and developed the true research spirit in me. In addition to all he has done regarding my Ph.D. studies, I will always remember his kindness concerning my life’s difficulties from the day I entered his lab. What he has done to help me through this arduous task is more than I would have expected to find in an advisor, and it will never be forgotten. I want to express my special thanks to my graduate committee members for patiently attending my general and final exams and providing me with their thoughtful comments and suggestions. Their help has always been constructive, beneficial, and very valuable to me. Thanks to Dr. Henrique Cheng for wonderful and unforgettable lectures regarding intracellular pathways and to Dr. Ji-Ming Feng for his comprehensive advice in immunocytochemistry. Also, thank you to Dr. Jeffery Gimble for his wonderful guidance through my graduate program. His deep knowledge and valuable vision of stem cell therapy greatly impacted my entire dissertation research. I also need to thank the people who helped me with my project: Dr. Arthur Penn for his course, Critical Analysis in Molecular Biology/Medicine, Dr. Denial Hayes and Dr. Ammer Qurashi for providing the scaffolds, Dr. Xiaochu Wu for his tireless assistance in electron iii microscopy, Dr. Jonathan Bova and Dr. Jenny Pepping for helping with the animal studies, Ms. Sherry Ring and Dr. Fabio Del Piero for histological analysis, and Dr. Kip Matthews and Dr. Mahdi Orooji for micro-CT analysis. I would like to express my thanks to my parents, Dr. Gholamali Rezai Rad and Dr. Mansoureh Nikravesh, who taught me to be self-motivated, have a strong work ethic, and take pride in my work. Their support and prayers have always been invaluable. Most certainly, this dissertation would not have been possible without the love, encouragement and support of my husband, Dr. Mahdi Orooji. His sacrifice for this project was much more than anticipated and far more than fair. I also would like to acknowledge my colleagues, Dr. Mike Flanagan and Ms. Chunhong Li for their warm friendship and the support they provided in a variety of ways. And finally, I appreciate all my instructors in the department of Comparative Biomedical Sciences and the department of Biological Sciences at Louisiana State University. Their classes have had an immense impact on my knowledge. iv TABLE OF CONTENTS ACKNOWLEDGMENTS ....................................................................................................... iii LIST OF TABLES ............................................................................................................... viii LIST OF FIGURES ................................................................................................................ ix ABSTRACT ............................................................................................................................ xi CHAPTER 1: STEM CELLS AND DENTAL FOLLICLE STEM CELLS IN REGENERATIVE MEDICINE .................................................................................................1 1.1 OVERVIEW ............................................................................................................1 1.2 STEM CELLS ........................................................................................................2 1.2.1 Embryonic stem cells (ESCs) ................................................................. 2 1.2.2 Adult stem cells (AdSCs) ........................................................................ 3 1.2.3 Induced pluripotent stem cells (iPSCs) ................................................... 4 1.3 DENTAL STEM CELLS .......................................................................................4 1.4 DENTAL FOLLICLE AND DENTAL FOLLICLE STEM CELLS .................... 5 1.5 IN VIVO HARD TISSUE-FORMING POTENTIAL OF DFSCS .........................9 1.6 USE OF SCAFFOLDS IN TISSUE ENGINEERING .........................................10 1.7 SPECIFIC AIMS AND SIGNIFICANCE OF THIS RESEARCH ......................10 1.8 REFERENCES ......................................................................................................12 CHAPTER 2: CHARACTERIZATION AND OSTEOGENIC DIFFERENTIATION OF DENTAL FOLLICLE STEM CELLS .....................................................................................18 2.1 INTRODUCTION .................................................................................................18 2.2 MATERIALS AND METHODS ..........................................................................19 2.2.1 Establishment of cell cultures ................................................................19 2.2.2 Evaluation of osteogenic capability of the DFSCs and DFCs .............. 22 2.2.3 Evaluation of osteogenic capability of different passages of DFSCs ....23 2.2.4 Expression of marker genes in the DFSCs and DFCs ...........................23 2.2.5 Methods for real-time RT-PCR..............................................................25 2.2.6 Expression of DMP1 protein in the DFSCs and DFCs ..........................26 2.2.7 Statistical analysis ..................................................................................26 2.3 RESULTS ..............................................................................................................27 2.3.1 Osteogenic differentiation of DF derived cells ......................................27 2.3.2 Expression of differentiation marker genes in DFSCs in response to osteogenic induction .............................................................................27 2.3.3 Osteogenic capability of DFSCs during in vitro expansion ..................29 2.3.4 Expression of stem cell-related genes in the DFSCs and DFCs ............32 2.4 DISCUSSION .......................................................................................................35 2.5 REFERENCES ......................................................................................................38 v CHAPTER 3: THE ROLE OF DENTIN MATRIX PROTEIN 1 (DMP1) IN REGULATION OF OSTEOGENIC DIFFERENTIATION OF DENTAL FOLLICLE STEM CELLS (DFSCS) ......................................................................................42 3.1 INTRODUCTION .................................................................................................42 3.2 MATERIALS AND METHODS ..........................................................................43 3.2.1 Cell cultures ...........................................................................................43 3.2.2 Correlation of DMP1 expression and osteogenic differentiation in different passages of DFSCs .................................................................44 3.2.3 Transfection of DFSCs with DMP1-siRNA ..........................................44 3.2.4 Effect of exogenous DMP1 on osteogenic differentiation of DFSCs ....45 3.2.5 Statistical analysis ..................................................................................47 3.3 RESULTS ..............................................................................................................47 3.3.1 Reduction in DMP1 expression during in vitro culture of DFSCs ........47 3.3.2 Correlation between DMP1 expression and osteogenesis of DFSCs.....47 3.3.3 Inhibition of osteogenesis by knockdown of DMP1
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