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Characterization of Embryonic Stem Cell-Differentiated Cells As Mesenchymal Stem Cells

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Characterization of Embryonic Stem Cell-Differentiated Cells As Mesenchymal Stem Cells The University of Southern Mississippi The Aquila Digital Community Honors Theses Honors College Fall 12-2015 Characterization of Embryonic Stem Cell-Differentiated Cells as Mesenchymal Stem Cells Rachael N. Kuehn University of Southern Mississippi Follow this and additional works at: https://aquila.usm.edu/honors_theses Part of the Cell Biology Commons Recommended Citation Kuehn, Rachael N., "Characterization of Embryonic Stem Cell-Differentiated Cells as Mesenchymal Stem Cells" (2015). Honors Theses. 349. https://aquila.usm.edu/honors_theses/349 This Honors College Thesis is brought to you for free and open access by the Honors College at The Aquila Digital Community. It has been accepted for inclusion in Honors Theses by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. The University of Southern Mississippi Characterization of Embryonic Stem Cell-Differentiated Cells as Mesenchymal Stem Cells by Rachael Nicole Kuehn A Thesis Submitted to the Honors College of The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science in the Department of Biological Sciences December 2015 ii Approved by ______________________________ Yanlin Guo, Ph.D., Thesis Adviser Professor of Biological Sciences ______________________________ Shiao Y. Wang, Ph.D., Chair Department of Biological Sciences ______________________________ Ellen Weinauer, Ph.D., Dean Honors College iii ABSTRACT Embryonic stem cells (ESCs), due to their ability to differentiate into different cell types while still maintaining a high proliferation capacity, have been considered as a potential cell source in regenerative medicine. However, current ESC differentiation methods are low yielding and create heterogeneous cell populations. If transplanted in the human body, differentiated ESCs could be rejected by the immune system, form tumors, or may not function normally within the human body. On the other hand, mesenchymal stem cells (MSCs), a type of adult stem cell typically derived from bone marrow, have proved to be excellent candidates in clinical applications due to their defined differentiation capacity and immunoregulatory properties. However, MSCs lack sufficient expansion capacity and can only be derived from limited tissues. This project entails characterizing ESCs differentiated through retinoic acid induction as MSCs. It is speculated that these cells are MSCs due to the extensive similarities in behavior and differentiation capacity. To complete the characterization, the morphology of our MSCs was compared to naturally differentiated MSCs, and a cell cycle analysis was performed. The tentative MSCs were spontaneously differentiated into osteocytes, adipocytes, and chondrocytes, the three distinct cell lineages that characterize MSCs differentiation capacity. Based on the results, our cells were determined to be MSCs, thereby identifying them as ESC-MSCs. This is significant, because it allows for the formation of cells that bypass many of the challenges mentioned above. ESC-MSCs express combined advantages from both ESCs and MSCs, making them even better cell sources for future therapeutic applications. Key Words: mESCs, transcription factors, MSCs, differentiation, regenerative medicine iv ACKNOWLEGEMENTS I would first like to thank my thesis adviser, Dr. Yan-Lin Guo. Without his knowledge and support, this project would have never been possible. Due to his high standards, I have achieved and learned more than I thought possible during this experience. Also, I would like to thank Dr. Guo’s graduate students, William D’Angelo and Chandan Gurung, and undergraduate student, Natayla Ortolano, for their unlimited guidance and assistance throughout my research experience. Thank you to the University of Southern Mississippi’s Honors College, Dr. Dave Davies, Dr. Fei Xue, and Paula Mathis for providing me with this opportunity to conduct research and offering support along the way. Also, an extra thank you to the Eagle Scholars Program for Undergraduate Research for providing funding. I would like to acknowledge the Luckyday Citizenship Scholarship Program for funding my education, and enabling me to attend the University of Southern Mississippi. Last but not least, thank you to my family and friends for getting me though each day and always encouraging me to be the best I can be. v TABLE OF CONTENTS LIST OF FIGURES ....................................................................................................... viii LIST OF ABBREVIATIONS ......................................................................................... ix INTRODUCTION..............................................................................................................1 Embryonic Stem Cells ...........................................................................................1 Background ..................................................................................................1 Characteristics ..............................................................................................4 In Vitro Maintenance of Pluripotency .........................................................5 In Vitro Differentiation of ESCs ..................................................................6 Biomedical Applications ..............................................................................8 Similarities and Differences between mESCs and hESCs ...........................9 Ethical/Social Concerns and Alternatives to hESCs ..................................10 Adult Stem Cells ...................................................................................................12 Mesenchymal Stem Cells .....................................................................................13 Background ................................................................................................13 Characteristics ............................................................................................13 In Vitro Growth and Tri-Lineage Differentiation Potential .......................15 Biomedical Applications ............................................................................16 ESC-derived MSCs ....................................................................................18 RATIONAL, HYPOTHESIS, AND OBJECTIVES .....................................................20 EXPERIMENTAL DESIGN AND METHODS ...........................................................21 Experimental Design ............................................................................................21 Cell Culture Techniques ......................................................................................21 Background ................................................................................................21 ESC Culture ...............................................................................................23 In Vitro Differentiation of mESCs .....................................................................24 Retinoic Acid Induction .............................................................................24 MSC Culture ..............................................................................................24 Toluidine Blue Staining .......................................................................................25 Principle of TB Staining ............................................................................25 vi TB Staining Protocol..................................................................................25 Cell Cycle Analysis by Flow Cytometry.............................................................26 Principle of Flow Cytometry .....................................................................26 Cell Cycle Analysis Protocol .....................................................................27 Tri-lineage Differentiation Techniques ..............................................................28 Principle of Spontaneous Differentiation ...................................................28 Differentiation Protocol .............................................................................29 Osteocyte Staining .....................................................................................29 Adipocyte Staining.....................................................................................29 Chondrocyte Staining.................................................................................30 RNA Extraction and RT-qPCR ..........................................................................30 Principle of PCR-based Gene Expression ..................................................30 RNA Extraction .........................................................................................32 Reverse Transcription Protocol..................................................................33 Semi-quantitative PCR Protocol ................................................................33 RESULTS .........................................................................................................................34 Comparison of D3-ESCs, D3-MSCs, 10T1/2 Cell Morphology ...........................34 Cell Proliferation and Cell Cycle Profile ...............................................................35 Osteogenic Differentiation .....................................................................................36 Adipogenic Differentiation ....................................................................................38
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