CELL REPROGRAMMING TECHNOLOGIES FOR TREATMENT AND UNDERSTANDING OF GENETIC DISORDERS OF MYELIN by ANGELA MARIE LAGER Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis advisor: Paul J Tesar, PhD Department of Genetics and Genome Sciences CASE WESTERN RESERVE UNIVERSITY May 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Angela Marie Lager Candidate for the Doctor of Philosophy degree*. (signed) Ronald A Conlon, PhD (Committee Chair) Paul J Tesar, PhD (Advisor) Craig A Hodges, PhD Warren J Alilain, PhD (date) 31 March 2015 *We also certify that written approval has been obtained from any proprietary material contained therein. TABLE OF CONTENTS Table of Contents……………………………………………………………………….1 List of Figures……………………………………………………………………………4 Acknowledgements……………………………………………………………………..7 Abstract…………………………………………………………………………………..8 Chapter 1: Introduction and Background………………………………………..11 1.1 Overview of mammalian oligodendrocyte development in the spinal cord and myelination of the central nervous system…………………..11 1.1.1 Introduction……………………………………………………..11 1.1.2 The establishment of the neuroectoderm and ventral formation of the neural tube…………………………………..12 1.1.3 Ventral patterning of the neural tube and specification of the pMN domain in the spinal cord……………………………….15 1.1.4 Oligodendrocyte progenitor cell production through the process of gliogenesis ………………………………………..16 1.1.5 Oligodendrocyte progenitor cell to oligodendrocyte differentiation…………………………………………………...22 1.1.6 Oligodendrocytes and their role in myelinating the central nervous system………………………………………………...24 1.1.7 Summary………………………………………………………..29 1.2 Leukodystrophies: progressive, heritable disorders of myelin………..31 1.2.1 Introduction……………………………………………………..31 1.2.2 Pelizaeus-Merzbacher disease………………………………33 1 1.2.3 Krabbe disease………………………………………………...33 1.2.4 Summary………………………………………………………..39 1.3 Mouse models of human myelin-related diseases…………………….40 1.3.1 Introduction……………………………………………………..40 1.3.2 Shiverer: mouse model of hypomyelination………………...41 1.3.3 Twitcher: mouse model of Krabbe disease…………………43 1.3.4 Jimpy: mouse model of Pelizaeus-Merzbacher disease…..46 1.3.5 Summary………………………………………………………..48 1.4 Current cell reprograming and differentiation technologies…………..49 1.5 Summary and research aims…………………………………………….56 Chapter 2: Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells………………..58 2.1 Abstract…………………………………………………………………….59 2.2 Introduction………………………………………………………………...59 2.3 Methods…………………………………………………………………….61 2.4 Results……………………………………………………………………...72 2.5 Discussion………………………………………………………………….94 Chapter 3: Rapid screening platform for elucidating the role of candidate genes in the oligodendrocyte lineage……………………………………………96 3.1 Introduction………………………………………………………………...96 3.2 Methods…………………………………………………………………….98 3.3 Results…………………………………………………………………….108 3.4 Discussion………………………………………………………………..129 2 Chapter 4: Discussion of future directions…………………………………….131 4.1 Summary………………………………………………………………….131 4.2 Generation of autologous gene-corrected cell based therapies for human disorders of myelin……………………………………………...134 4.3 Oligodendrocyte in vitro differentiation method as a first tier platform for mouse model generation……………………………………………137 4.4 Identifying the genetic basis of oligodendrocyte identity by high- throughput in vitro screening……………………………………………140 4.5 Elucidating gene-regulatory networks of the oligodendrocyte lineage…………………………………………………………………….145 4.5.1 Understanding the role of oligodendrocyte enhancers in the diagnosis of rare myelin diseases………………………….145 4.5.2 Delineating enhancers that contribute to oligodendrocyte development………………………………………………….148 References…………………………………………………………………………....150 3 LIST OF FIGURES Chapter 1 Figure 1.1 - TGFβ and BMP signaling pathway……………………………………14 Figure 1.2 - Patterning of the ventral neural tube………………………………….17 Figure 1.3 - Gliogenesis in the ventral spinal cord………………………………...20 Figure 1.4 - Oligodendrocyte progenitor cell proliferation and oligodendrocyte differentiation and maturation………………………………………………………...27 Figure 1.5 - Myelin of the central nervous system…………………………………30 Chapter 2 Figure 2.1 - Eight transcription factors can reprogram mouse embryonic fibroblasts to induced oligodendrocyte progenitor cells……………………………82 Figure 2.2 - Characterization of the selected eight transcription factor pool……83 Figure 2.3 - Characterization of MEFs utilized for reprogramming………………84 Figure 2.4 - Eight transcription factor induced MEFs exhibit properties of bona fide OPCs……………………………………………………………………………….85 Figure 2.5 - Properties of 8TF-induced MEFs……………………………………..86 Figure 2.6 - Eight transcription factor induced MEFs function to generate compact myelin………………………………………………………………………...87 Figure 2.7 - A2B5 immunosorting allows for the prospective enrichment of iOPCs…………………………………………………………………………………...88 Figure 2.8 - Narrowing down the 8TF pool…………………………………………89 4 Figure 2.9 - Sox10, Olig2, and Nkx6.2 are sufficient to reprogram fibroblasts to iOPCs…………………………………………………………………………………...90 Figure 2.10 - Properties of 3TF-induced MEFs……………………………………92 Figure 2.11 - Three transcription factors are able to induce iOPCs from an additional somatic cell source………………………………………………………..93 Chapter 3 Figure 3.1 - Mouse embryonic stem cells express canonical markers of pluripotency…………………………………………………………………………...117 Figure 3.2 - Generation of a robust population of OPCs from patterned mESCs………………………………………………………………………………...118 Figure 3.3 - mESC patterned cells express markers of the neuroectoderm…..119 Figure 3.4 - mESC patterned cells give rise to neurons…………………………120 Figure 3.5 - mESC derived OPCs robustly express cell surface markers NG2 and PDGFRa………………………………………………………………………….121 Figure 3.6 - mESC derived OPCs differentiate into myelin protein expressing oligodendrocytes……………………………………………………………………..122 Figure 3.7 - Shiverer induced pluripotent stem cells express canonical markers of pluripotency………………………………………………………………………..123 Figure 3.8 - Shiverer iPSC derived OPCs generate MBP negative oligodendrocytes……………………………………………………………………..124 Figure 3.9 - Shiverer iPSC derived OPCs robustly express cell surface markers NG2 and PDGFRa…………………………………………………………………...125 5 Figure 3.10 - Gene correction of shiverer MBP deletion………………………...126 Figure 3.11 - CRISPR-Cas9 mediated gene perturbation………………………127 Figure 3.12 - Perturbations of known oligodendrocyte lineage genes cause oligodendrocytes to display deficits in myelin protein expression………………128 6 ACKNOWLEDGEMENTS I would like to thank Jared M Cregg for his critical comments and edits of this document. 7 Cell Reprogramming Technologies for Treatment and Understanding of Genetic Disorders of Myelin Abstract by ANGELA MARIE LAGER The oligodendrocyte lineage is essential for high-fidelity information transfer in neural circuits of the central nervous system. Oligodendrocytes arise from a pool of migratory progenitor cells that populate the brain and spinal cord shortly before birth. These oligodendrocyte progenitor cells undergo subsequent differentiation into mature oligodendrocytes, a cell whose primary function is to generate a multilayer protein-lipid membrane around axons termed myelin. Myelin segments allow saltatory conduction of action potentials down the axon, increasing impulse velocity by as much as 100-fold. Therefore, oligodendrocytes are thought to contribute to efficient signal processing in local microcircuits and are required for long-distance propagation of action potentials by projection neurons. The importance of oligodendrocytes in central nervous system function is underscored by the prevalence of neurological diseases characterized by abnormal myelination. These diseases, collectively termed leukodystrophies, encompass a spectrum of disorders associated with mutations in over 40 different 8 oligodendrocyte lineage-specific genes. Although the genetic etiology for a majority of these disorders is well understood, less is known about how genetic abnormalities underlie cellular dysfunction and overt disease pathology. As there are currently no standard treatments for patients suffering from leukodystrophies, addressing this gap is of fundamental importance. Recent use of mouse genetic models and cell reprogramming technologies has dramatically improved our ability to understand how genetic mutations underlie disease at the molecular, cellular, and systems level. We sought to adapt these technologies to develop a method for obtaining oligodendrocyte progenitor cells— a previously inaccessible cell type. Herein, I describe our identification of oligodendrocyte lineage-specific transcription factors and their subsequent use in direct reprogramming of mouse fibroblasts to induced oligodendrocyte progenitor cells (iOPCs). iOPCs exhibit morphology and gene expression profiles similar to bona fide oligodendrocyte progenitors, can be expanded in vitro in a progenitor state capable of differentiating into mature multiprocessed oligodendrocytes, and form compact myelin when grafted into the mouse central nervous system. We have also developed a second method that allows us to direct the differentiation of oligodendrocyte progenitor cells from wild type and mutant mouse pluripotent stem cell populations. By systematically treating mouse pluripotent stem cells with small molecules and growth factors that mimic growth factor conditions