UC Riverside Electronic Theses and Dissertations
Total Page：16
File Type：pdf, Size：1020Kb
UC Riverside UC Riverside Electronic Theses and Dissertations Title The Embryotoxic Effects of Harm Reduction Tobacco Products on Osteoblasts Developing from Human Embryonic Stem Cells Permalink https://escholarship.org/uc/item/32z2s123 Author Sparks, Nicole Renee Publication Date 2018 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA RIVERSIDE The Embryotoxic Effects of Harm Reduction Tobacco Products on Osteoblasts Developing from Human Embryonic Stem Cells A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Environmental Toxicology by Nicole Renee Sparks June 2018 Dissertation Committee: Dr. Nicole zur Nieden, Chairperson Dr. David Eastmond Dr. Yinsheng Wang Copyright by Nicole Renee Sparks 2018 v The Dissertation of Nicole Renee Sparks is approved: Committee Chairperson University of California, Riverside vi ACKNOWLEDGEMENTS I am beyond appreciative to have many people in my life that have made this dissertation possible. I am grateful for the advice and the guidance I received from my principal investigator Nicole I. zur Nieden and the many cups of coffee we enjoyed together. Her knowledge and dedication to stem cell research and toxicology has been inspiring. She has been instrumental in my doctoral career and beyond. For some of the work presented herein I had technical assistance from Lauren M. Walker (mouse work), Ivann Martinez, Joseph Madrid (in vivo injections), Riley Bottom, and Cristina Soto. Thank you, Lauren, for lending your ear and pastries. Thank you to all my lab mates from the zur Nieden lab and friends made in the Riccomagno lab. We are like family and I have enjoyed my time shared with you all. Thank you to the Environmental Toxicology doctoral program and my dissertation committee members Dr. David Eastmond and Dr. Yinsheng Wang. A special, thank you to Dawn Loyola who has helped me every quarter of my doctoral career. I would also like to acknowledge my funding sources: The Dean’s fellowship, fellowships from the graduate research mentorship program, a Cornelius Hopper Diversity and pre‐doctoral fellowship as well as a doctoral fellowship from the Tobacco Related Disease Research Program and a fellowship from the International Foundation for Ethical Research. Research funding came from the Tobacco Related Disease Research Program, the John Hopkins Center for Alternatives to Animal Testing as well as the National Institutes for Dental and Craniofacial Research. iv I would like to acknowledge Dr. Feodor Price (Harvard University), Dr. Derrick Rancourt (University of Calgary), and Dr. Amander Clark (University of California, Los Angeles) for kindly sharing genomic DNA of hESC and hiPSC lines analyzed in Appendix Fig 1.2.2. I would like to thank Dr. Martín García‐Castro for kindly donating SOX10 and PAX7 antibodies. I would like to thank Dr. Prue Talbot (University of California, Riverside) for generously providing our lab with the mainstream and sidestream tobacco smoke solutions used for some of my work. I would also like to thank to Dr. Martin Riccomagno (University of California, Riverside) for kindly providing the pBK-Flex shRNA plasmid and Eric Wong for technical assistance. Finally, I am forever thankful to my husband Jason. His understanding, encouragement, and amazing patience has been my foundation. Without his support I could have not completed my dissertation. My amazing children, Gavin and Austen Sparks, who have shown patience and understanding and have kept me full of joy and laughter. v ABSTRACT OF THE DISSERTATION The Embryotoxic Effects of Harm Reduction Tobacco Products on Osteoblasts Developing from Human Embryonic Stem Cells by Nicole Renee Sparks Doctor of Philosophy, Graduate Program in Environmental Toxicology University of California, Riverside, June 2018 Dr. Nicole zur Nieden, Chairperson Cigarette smoke is a mixture of over 7000 toxic components with at least 69 smoke chemicals producing cancers, suggesting that smoking is harmful to smokers. More importantly, the World Health Organization has estimated that 10% of the world’s population is involuntarily exposed to environmental tobacco smoke, a number that does not include developing embryos. However, tobacco exposure during embryonic development is one of the risk factors for developing a congenital birth defect. An understudied, yet important abnormality caused by maternal smoking is the improper development of the embryonic skeleton, which can result in long-term burdens on the affected child and their family, creating a need for investigation that is addressed in this thesis. Due to the difficulty in quitting smoking while pregnant, women often look to use harm reduction tobacco products (HRTPs) because they are perceived as a safer alternative in comparison to conventional cigarettes. These products include ultra-filtered cigarettes with reduced carcinogen content, and chewing tobacco, which omits exposure to harmful vi combustion products. However, there is a lack of investigation to reveal the molecular mechanisms involved in skeletal embryotoxicity induced by conventional and harm- reduction tobacco, the unraveling of which is another objective of this thesis. In the process of this investigation, an in vitro model based on human embryonic stem cells was developed. These cells are the only currently available cell culture system, which mimics the development of the human embryo. As this thesis shows, their differentiation into osteoblasts in vitro can be used to study chemical toxicity and the molecular mechanisms thereof. vii TABLE OF CONTENTS Acknowledgements ................................................................................................... iv Abstract ..................................................................................................................... vi List of figures ............................................................................................................ ix List of tables .............................................................................................................. xii Chapter 1: Introduction to stem cells ........................................................................ 1 Chapter 2: Low osteogenic yield in human pluripotent stem cells associates with differential neural crest promoter methylation ................................ 14 Chapter 3: Pluripotent stem cells as tools to assess developmental toxicity: diversity instead of consolidation ............................................................... 50 Chapter 4: Differential predictivity of human pluripotent stem cell lines in skeletogenic developmental toxicity assays correlates with their innate differentiation bias towards neural crest or mesodermal osteogenesis .............................................................................................................. 73 Chapter 5: Tobacco-induced oxidative stress disrupts osteogenic differentiation in human embryonic stem cells .......................................................... 119 Chapter 6: Harm-reduction tobacco alters bone development through tobacco sensitive transcriptional regulators ............................................................. 155 Conclusion ................................................................................................................ 191 APPENDIX 1: Supplemental information ................................................................ 199 APPENDIX 2: List of differentially expressed genes .............................................. 208 viii LIST OF FIGURES Figure 1.1. Human blastocyst ................................................................................... 1 Figure 1.2. Pluripotent stem cell differentiation ....................................................... 2 Figure 1.3. Stem cell division and differentiation ..................................................... 3 Figure 1.4. Adult stem cell ........................................................................................ 4 Figure 1.5. Generation and application of induced pluripotent stem cells (iPSCs) ..................................................................................................... 6 Figure 1.6. Contributions of neural crest, paraxial mesoderm, and lateral plate mesoderm to bone development............................................................. 7 Figure 2.1. Cytogenetic, pluripotent, and genetic characterization of human induced pluripotent stem cell (hiPSC) lines ............................................. 24 Figure 2.2. Osteogenic differentiation of human embryonic stem cells ................... 25 Figure 2.3. Intermediate differentiation in H9 cells is through the neural crest lineage .............................................................................................................. 27 Figure 2.4. Osteogenic differentiation in RIV9 and RIV4 human induced pluripotent stem cells (hiPSCs) ................................................................... 30 Figure 2.5. Passage‐specific analysis of bone parameters and gene expression ................................................................................................................. 32 Figure 2.6. Osteoblast origin correlates with methylation of the TWIST1 and PAX7 promoters ................................................................................. 34 Figure 2.7. Toxicity of