Embryonic Diapause as a Model for Pluripotent Stem Cell Quiescence Lilyana Margaretha A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2012 Reading Committee: C. Anthony Blau, Chair Carol Ware Hannele Ruohola-Baker Program Authorized to Offer Degree: Molecular and Cellular Biology University of Washington Abstract Embryonic Diapause as a Model for Pluripotent Stem Cell Quiescence Lilyana Margaretha Chairperson of the Supervisory Committee: Professor C. Anthony Blau Department of Hematology Pluripotent stem cells inhabit, and can transition between, distinct metastable states. Embryonic stem cells (ESC), derived from the pre-implantation inner cell mass (ICM) of mouse embryos, retain a relatively more primitive (or “naïve”) phenotypic and functional profile compared to “primed” pluripotent cells derived from the post-implantation epiblast. Based on the hypothesis that a physiological state of stem cell dormancy, known as diapause, might represent a novel pluripotent state, I characterized the transcriptional programs of pluripotent stem cells from: i) the ICM of pre-implantation embryos; ii) the epiblast of post-implantation embryos; and iii) the ICM of embryos induced to enter diapause. The results presented in this dissertation demonstrate significant differences in the molecular signatures of these states and define diapause as a distinct “quiescence-associated” pluripotent state. Using this information I constructed a “developmental yardstick” for pluripotent stem cells and found that the quiescence-associated pluripotent state can be modeled in vitro. My findings also point to Runx1 as a likely regulator of the quiescence-associated state and provide a roadmap for dissecting the molecular basis of stem cell quiescence. ii Table of Contents List of Figures ................................................................................................................. iv List of Tables ................................................................................................................... v Chapter 1 : Introduction .............................................................................................................. 1 Aims ............................................................................................................................... 1 Different pluripotent stem cell states ............................................................................... 1 Embryonic diapause: Overview ...................................................................................... 3 Embryonic diapause is evolutionarily conserved across mammals ................................. 4 Embryonic diapause as a model of stem cell quiescence ............................................... 5 Figures ........................................................................................................................... 8 Chapter 2 : A Developmental Yardstick for Pluripotent Stem Cells ............................................10 Summary .......................................................................................................................10 Introduction ...................................................................................................................10 Results ..........................................................................................................................11 Discussion .....................................................................................................................16 Methods ........................................................................................................................20 Figures ..........................................................................................................................28 Tables ...........................................................................................................................36 Chapter 3 : An Evolutionarily Conserved Program for Stem Cell Dormancy ..............................39 Summary .......................................................................................................................39 Introduction ...................................................................................................................39 Results ..........................................................................................................................41 Discussion .....................................................................................................................47 Methods ........................................................................................................................51 Figures ..........................................................................................................................55 Tables ...........................................................................................................................66 Chapter 4 : Perspectives ...........................................................................................................69 Figures ..........................................................................................................................75 Bibliography ..................................................................................................................77 iii List of Figures Figure 1.1 Butyrate effect on gene expression profiles of mouse and human ESC .................... 8 Figure 1.2.Hypothesis: butyrate (HDACi) exposure mimics embryonic diapause ...................... 9 Figure 2.1 Schematic diagram of experimental procedures. ......................................................28 Figure 2.2 Quality control of RNA-Seq samples ........................................................................29 Figure 2.3 Comparisons to prior knowledge ..............................................................................31 Figure 2.4 Comparisons to cultured pluripotent stem cells ........................................................33 Figure 2.5 Developmental yardstick ..........................................................................................35 Figure 3.1 RUNX1 and OCT4 correlation in diapause embryos ................................................55 Figure 3.2 Study of Runx1 using 2-HDACi model in vitro ..........................................................57 Figure 3.3 Regulation of AMPKα2 (Prkaa2) in diapause embryos. ............................................61 Figure 3.4 Regulation of the short isoform of LKB1 (LKB1Short) in diapause embryos ................62 Figure 3.5 Introduction of LKB1 short isoform into mouse ESC .................................................64 Figure 4.1 Graphical abstract of the use of developmental yardstick .........................................75 Figure 4.2 A proposed mechanism of stem cell quiescence .....................................................76 iv List of Tables Table 2.1 Summary of RNA-Seq reads mapped to mm9 genome .............................................36 Table 2.2 Description of samples used in Hunter study .............................................................37 List of Supplementary Tables for Chapter 2 ............................................................................. 38 Table 3.1 Summary of GSEA result using c3 (motif) database. .................................................66 Table 3.2 Summary of GSEA result using c2 (curated) database. .............................................67 List of Supplementary Tables for Chapter 3 ............................................................................. 68 v Acknowledgements I wish to express my sincere gratitude to my two advisors, Tony Blau and Carol Ware, for all of their invaluable input, support and excellent mentorship throughout my graduate work. I also greatly appreciate the advice and guidance from the other members of my other supervisory committee: Hannele Ruohola-Baker, Karol Bomsztyk, and Denny Liggitt. I am grateful for collaborating with Brig Mecham, Larry Ruzzo, Daniel Jones, Jay Shendure, Ruolan Qiu, Charlie Lee, John Scott and Jennifer Whiting. I would also like to acknowledge all of the past and present members of the Blau laboratory and the UW ISCRM ESC core staff, with a special thanks to Chris Cavanaugh for tremendous help in the mouse work. I would like to thank Ron Seifert from the UW ISCRM Garvey Imaging Lab for assistance in confocal imaging. I greatly appreciate the administrative help from the UW Molecular and Cellular Biology program and the UW Institue for Stem Cell and Regenerative Medicine. This thesis would never have been completed without the immense support and encouragement from my family and friends. vi Dedication This doctoral thesis is dedicated to my parents, Alvin and Anita, for their love, support, encouragement throughout my life To my husband Erwin for his love and support throughout my graduate career To my daughter Vanessa for brightening my days. vii Chapter 1 : Introduction Aims The goal of my work has been to study stem cell quiescence in pluripotent stem cells. My dissertation addresses four main questions: (1) How distinct are the transcriptional profiles of pluripotent stem cells obtained directly from embryos at various stages of development? (2) What are the defining transcriptional features of embryonic diapause, a quiescent-associated state of development? (3) What are the mechanisms underlying diapause? (4) Do ESC treated with