INVESTIGATING PRENATAL STRESS IN A STEM CELL MODEL OF HUMAN NEURONAL DEVELOPMENT Lilia Papst Dissertation at the Graduate School of Systemic Neurosciences Ludwig‐Maximilians‐Universität München May, 2019 L. Papst Investigating prenatal stress in a stem cell model of human neuronal development Supervisor Prof. Dr. Dr. med. Elisabeth Binder Translational Research in Psychiatry Max Planck Institute of Psychiatry First Reviewer: Prof. Dr. Dr. med. Elisabeth Binder Second Reviewer: Prof. Dr. Moritz Rossner Third Reviewer: Prof. Dr. Edna Grünblatt Date of Submission: May 27th 2019 Date of Defense: September 23rd 2019 [2] L. Papst Investigating prenatal stress in a stem cell model of human neuronal development CONTENTS Abstract ...................................................................................................................................... 7 List of Abbreviations ............................................................................................................. 8 1. Introduction .................................................................................................................... 9 1.1 Theoretical Background ..................................................................................... 9 1.1.1 Epidemiology of Psychiatric Disorders ...................................................... 9 1.1.2 Prenatal Stress in Psychiatric Disorders .................................................... 9 1.1.2.1 Developmental Origins of Health and Disease ........................................... 9 1.1.2.2 Definition of Prenatal Stress ...........................................................................10 1.1.2.3 Prevalence of Prenatal Stress .........................................................................11 1.1.2.4 Impact on cognition and behaviour .............................................................12 1.1.2.5 Organisational effects on brain structure ..................................................15 1.1.2.6 Hypothalamic-Pituitary-Adrenal axis programming ............................17 1.1.2.7 Altered gene transcription and signaling pathways ..............................20 1.1.3 Prenatal brain development in vivo and in vitro .................................. 22 1.1.3.1 In vivo neurogenesis ...........................................................................................22 1.1.3.2 In vitro neurogenesis .........................................................................................25 1.1.3.3 The glucocorticoid receptor in embryogenesis .......................................27 1.2 Technical Background ...................................................................................... 28 1.2.1 hIPSC and hESC as model system ............................................................... 28 1.2.2 Illumina MethylationEPIC BeadChip Design .......................................... 29 1.2.3 Transcriptome analysis by 3’ RNA-Seq .................................................... 32 1.3 Confounders in High-Throughput Experiments ....................................... 35 1.4 Aim and scope of study ...................................................................................... 36 2. Pilot Studies ................................................................................................................... 37 2.1 Establishment of GR Expression and Translocation Assay ................... 37 [3] L. Papst Investigating prenatal stress in a stem cell model of human neuronal development 2.1.1 Background ................................................................................................................37 2.1.2 Methods .......................................................................................................................38 2.1.3 Results ..........................................................................................................................39 2.1.4 Discussion ...................................................................................................................42 2.2 Establishment Of Differentiation Protocol ................................................. 43 2.2.1 Background ................................................................................................................43 2.2.2 Methods .......................................................................................................................43 2.2.3 Results ..........................................................................................................................46 2.2.4 Discussion ...................................................................................................................51 2.3 Establishment of Dexamethasone Experiments ....................................... 52 2.3.1 Background ................................................................................................................52 2.3.2 Methods .......................................................................................................................52 2.3.3 Results ..........................................................................................................................53 2.3.4 Discussion ...................................................................................................................59 2.4 Conclusions ............................................................................................................ 62 3. Methods........................................................................................................................... 63 3.1 Study Design .......................................................................................................... 63 3.2 Cell lines .................................................................................................................. 64 3.3 Cell Culture and hESC/ hiPSC Differentiation ............................................ 65 3.4 Dexamethasone Stimulation Experiments ................................................. 67 3.5 RNA extraction and library preparation ..................................................... 68 3.6 DNA extraction and EPIC methylation array ............................................. 68 3.7 Immunofluorescence assays ........................................................................... 68 3.8 Quantification of Glucocorticoid Receptor Translocation .................... 69 3.9 Western Blot.......................................................................................................... 70 3.10 qPCR ..................................................................................................................... 71 [4] L. Papst Investigating prenatal stress in a stem cell model of human neuronal development 3.11 Biostatistical Analyses of Differential Methylation ............................. 72 3.12 Biostatistical Analyses of Differential Gene Transcription .............. 88 4. Results ............................................................................................................................. 96 4.1 In vitro differentiating cells recapitulate embryonic neurogenesis.. 96 4.2 Endogenous and dexamethasone-induced glucocorticoid receptor action in neurogenesis ............................................................................................... 123 4.3 Effects of dexamethasone exposure on the methylome ..................... 128 4.4 Effects of dexamethasone exposure on the transcriptome ............... 143 5. Discussion .................................................................................................................... 154 5.1 Technical Aspects ............................................................................................. 156 5.1.1 Induced pluripotent stem cells as model system. .................................... 156 5.1.2 Microarray and sequencing-based techniques ......................................... 159 5.1.3 Statistical Considerations .................................................................................. 160 5.2 Theoretical Aspects ......................................................................................... 162 5.2.1 Intrinsic glucocorticoid receptor action in neurogenesis ..................... 162 5.2.2 Dexamethasone-induced differential methylation .................................. 164 5.2.3 Dexamethasone-induced differential gene expression.......................... 170 5.3 Limitations .......................................................................................................... 171 5.4 Conclusions ......................................................................................................... 173 5.5 Outlook ................................................................................................................. 173 6. References .................................................................................................................... 176 7. Supplementary Information ................................................................................. 233 7.1 Supplementary Analyses ............................................................................... 233 7.2 Reagents and Resources ................................................................................ 242 7.3 Supplementary Tables .................................................................................... 246 7.4 Supplementary Figures .................................................................................. 247 Disclosure of author contributions