PHENOTYPIC & TRANSCRIPTIONAL CONSEQUENCES OF SCHWACHMAN-BODIAN-DIAMOND SYNDROME (SBDS) DEFICIENCY DURING HEMATOPOIETIC DEVELOPMENT by Wing Man Stephanie Ng A thesis submitted in conformity with the requirements for the degree of Masters of Science Institute of Medical Science University of Toronto © Copyright by Wing Man Stephanie Ng 2017 Abstract Phenotypic and Transcriptional Consequences of Shwachman-Bodian-Diamond Syndrome (SBDS) Deficiency during Hematopoietic Development Wing Man Stephanie Ng Masters of Science Institute of Medical Science University of Toronto 2017 Shwachman-Diamond syndrome (SDS) is a genetic disorder characterized by exocrine pancreatic and hematological dysfunction. As hematological complications are major concerns for mortality with limited treatment options, the mechanisms underlying the SDS hematopoietic defects need to be elucidated to advance the development of effective treatments. We hypothesized that the onset of SDS hematopoietic defects occurred in a certain hematopoietic developmental stage. In this study, definitive hematopoietic differentiation of SDS compared to normal hiPSCs showed reduced CFU-GM and BFU-E formation, increased venous and endothelial potential, and reduced viability in embryoid bodies containing hemogenic and vascular endothelia. Over-representation analysis of dysregulated genes in SDS compared to normal hiPSCs showed enrichment for pathways including histones, glycosaminoglycan metabolism, and WNT signaling. This study offers insight into the onset of SDS hematopoietic defects and potentially disrupted pathways in SDS hiPSCs, and provides patient hematopoietic cell resources for drug screening and the potential to improve patient outcome. ii Acknowledgments I would like to take this opportunity to thank everyone who has helped me through my degree. Most importantly, I would like to express my heartfelt gratitude to my supervisor Dr. Yigal Dror for giving me the opportunity to study under his supervision, guiding me through experimental design, and supporting me through my degree. I am also appreciative of my committee members, Dr. Freda Miller, Dr. Johanna Rommens, and Dr. Ian Scott for valuable discussion and constructive criticism. I would like to express my appreciation and give credit to members of the Dror lab, including our lab manager Hongbing Li for maintaining lab operations, providing constructive criticism, and continuing with this study; our clinical research project manager Bozana Zlateska for acquiring patient samples with informed consent; our MSc alumni Alice Luca for pioneering the study of definitive hematopoiesis of SDS hiPSCs, assisting with the establishment of the protocol of definitive hematopoietic differentiation, and continuing with this study; our former research associate Chetankumar Tailor for producing lentivirus, optimizing lentiviral transduction of hiPSCs, and assisting with the establishment of transduced hiPSCs; our MSc candidate Anna Matveev for optimizing lentiviral transduction of hiPSCs; our lab technician Jake Ableser for performing Western blot and assisting with hiPSC culture and hematopoietic differentiation; our former research fellow Stephanie Heidemann for assisting in flow cytometry; our former summer undergraduate Katrina Sajewycz for assisting in the prioritization of differentially expressed genes; and our PhD candidate Santhosh Dhanraj for his assistance and constructive criticism. I would also like to express my appreciation to our collaborators. I would like to thank Andrea Ditadi, Marion Kennedy, and Dr. Gordon Keller for providing the protocol of definitive hematopoietic differentiation, assisting with troubleshooting, and providing constructive criticism. I would like to thank the lab technician Weixian Min for his assistance in hiPSC culture and the Grunebaum lab for providing OP9-DL1. I would like to thank the former lab technician Rikesh Gandhi in the Rommens lab for his assistance in real time-quantitative PCR (RT-qPCR). I would like to thank the PhD candidate Jonathon Torchia in the Huang lab for his assistance in RNA-sequencing (RNA-seq) quality assessment. I would like to thank the PhD candidate Michael Liang in the Wilson lab for his assistance in the TopHat-Cufflinks and over- representation analysis. iii I would also like to express my appreciation to the facilities that helped us with our project. I would like to thank the Centre for Commercialization of Regenerative Medicine (CCRM) for hiPSC reprogramming and characterization. I would like to thank the Sickkids Embryonic Stem Cell Facility for providing mouse embryonic fibroblasts (MEFs). I would like to thank the Sickkids Laboratory Animal Services for their assistance in irradiation. I would like to thank the Sickkids Molecular Biology Laboratory for Mycoplasma testing. I would like to thank The Centre for Applied Genomics (TCAG) for G-band karyotyping and Sanger sequencing. I would like to thank the Sickkids-University Health Network Flow Cytometry Facility for fluorescence- activated cell sorting (FACS) and purity assessment. I would like to thank the Donnelly Sequencing Centre for RNA quality assessment, complementary DNA (cDNA) library preparation, and RNA-seq. On a personal note, I would like to thank the Chau (Grandma, Grandpa, Keith, Wendy, Ho Ho, Matthew, Kelly, Justin, Max, and Ruby) and Xu (Theresa and Linus Jr.) families for their hospitality and support. I would also like to thank Kenneth, Florence, and Angus for their support and encouragement. iv Statement of Contributions My supervisor Dr. Yigal Dror and my committee members Dr. Freda Miller, Dr. Johanna Rommens, and Dr. Ian Scott contributed to the experimental design, data interpretation, and thesis revision and approval. Hongbing Li, Alice Luca, Dr. Gordon Keller, Marion Kennedy, and Andrea Ditadi contributed to the experimental design and data interpretation. Santhosh Dhanraj, Anna Matveev, Stephanie Heidemann, and Katrina Sajewycz provided critical data interpretation. Bozana Zlateska and the Canadian Inherited Marrow Failure Registry acquired patient samples with informed consent. CCRM reprogrammed and characterized hiPSCs. TCAG performed the G-band karyotyping and Sanger sequencing. The Sickkids Embryonic Stem Cell Facility provided mouse embryonic fibroblasts. The Sickkids Molecular Biology Laboratory performed the Mycoplasma testing. Dr. Eyal Grunebaum provided the OP9-DL1 cell line. The Sickkids Laboratory Animal Services provided the irradiator. Jake Ableser performed Western blotting, hiPSC culture, hematopoietic differentiation, and flow cytometry preparation. The Sickkids- University Health Network Flow Cytometry Facility performed FACS and provided flow cytometers. The Donnelly Sequencing Centre assessed RNA quality, prepared cDNA, and sequenced RNA. Chetankumar Tailor produced the lentivirus and transduced hiPSCs. I contributed to the experimental design; performed cell culture, hematopoietic differentiation, colony assays, and RT-qPCR; prepared cells for karyotyping, Mycoplasma testing, lentiviral transduction, and FACS; acquired flow cytometry data; prepared DNA for Sanger sequencing; performed statistical analysis; interpreted data; and wrote the thesis. This work was funded by Canadian Institute of Health Research, Research Training Award from Sickkids, and Nicola’s Triathlon for Kids. v Table of Contents Abstract ........................................................................................................................................... ii Table of Contents ........................................................................................................................... iii Acknowledgments.......................................................................................................................... iii Statement of Contributions ..............................................................................................................v List of Abbreviations ..................................................................................................................... ix List of Tables ............................................................................................................................... xiv List of Figures ................................................................................................................................xv Chapter 1 Introduction .....................................................................................................................1 Shwachman-Diamond Syndrome ........................................................................................1 1.1 Clinical Presentation ................................................................................................1 1.2 SBDS Gene .............................................................................................................11 1.3 SBDS Protein Function ..........................................................................................13 Induced Pluripotent Stem Cells .........................................................................................23 Hematopoiesis ....................................................................................................................27 Chapter 2 Research Aims and Hypothesis .....................................................................................36 Chapter 3 Methods .........................................................................................................................39 Cell Culture ........................................................................................................................39