Regionalisation of human ES cell derived neural precursors DISSERTATION zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Johanna Viola Driehaus aus Lüneburg Bonn, 2008 Anfertigung mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. Oliver Brüstle 2. Gutachter: Prof. Dr. Dieter Fürst Tag der Promotion: 27. Februar 2009 Diese Dissertation wurde durch ein Graduiertenstipendium der Konrad-Adenauer- Stiftung e.V gefördert. Sie ist auf dem Hochschulschriftenserver der ULB Bonn http://hss.ulb.uni-bonn.de/diss_online elektronisch publiziert. Erscheinungsjahr: 2009 Für meine Eltern Table of Contents Abbreviations..................................................................................................................... 8 1 Introduction................................................................................................................... 11 1.1 Stem cells ............................................................................................................................................................... 11 1.1.1 Embryonic stem cells..................................................................................................................................... 13 1.1.1.1 Derivation and characteristics of human embryonic stem cells.......................................................... 13 1.1.1.2 Developments in hES derivation .......................................................................................................... 15 1.1.1.3 New developments in pluripotent cell derivation................................................................................ 16 1.1.1.4 In vitro differentiation of hES cells ...................................................................................................... 18 1.1.2 Neural stem cells............................................................................................................................................ 20 1.1.2.1 Endogenous neural stem cells ............................................................................................................... 20 1.1.2.2 ES cell derived neural cells ................................................................................................................... 22 1.2 Development and specification of the nervous system ................................................................................... 24 1.2.1 Regionalisation and patterning...................................................................................................................... 25 1.2.2 Plasticity and regional gene expression in neural progenitors.................................................................... 27 1.2.2.1 Endogenous neural progenitors............................................................................................................. 27 1.2.2.2 ES cell-derived neural progenitors ....................................................................................................... 28 1.3 Epigenetic regulation of developmental potential........................................................................................... 29 1.4 Aim ......................................................................................................................................................................... 30 2 Materials and Methods ................................................................................................ 32 2.1 General................................................................................................................................................................... 32 2.2 Cell types ............................................................................................................................................................... 32 2.3 Human embryonic stem cells ............................................................................................................................. 32 2.3.1 Culture of murine embryonic fibroblasts ..................................................................................................... 32 2.3.2 Mitotic inactivation of murine embryonic fibroblasts via γ-irradiation ..................................................... 33 2.3.3 Thawing of mitotically inactivated murine embryonic fibroblasts............................................................. 33 2.3.4 Thawing of human embryonic stem cells..................................................................................................... 33 2.3.5 Passaging of human ES cells......................................................................................................................... 34 2.3.6 Freezing of human ES cells........................................................................................................................... 34 2.3.7 Induction of multi-lineage differentiation from hES in vitro...................................................................... 34 2.4 HES cell derived neural precursors .................................................................................................................. 35 2.4.1 Derivation and culture ................................................................................................................................... 35 2.4.2 Propagation..................................................................................................................................................... 36 2.4.2.1 Preparation of polyornithine/laminin coated dishes ............................................................................ 36 2.4.2.2 Passaging of hESNSCs.......................................................................................................................... 36 2.4.3 Induction of differentiation from hESNSCs................................................................................................. 36 2.4.3.1 Preparation of Matrigel coated dishes .................................................................................................. 36 2.4.3.2 Induction of differentiation ................................................................................................................... 36 2.4.4 Co-cultures and transplantation of hESNSCs .............................................................................................. 37 2.4.4.1 Shared medium co-culture .................................................................................................................... 37 2.4.4.2 Physical contact co-culture.................................................................................................................... 38 2.4.4.3 Re-aggregation co-culture ..................................................................................................................... 39 2.4.4.4 Transplantation of neural precursors onto organotypic slice cultures................................................ 39 2.4.4.5 Transplantation into neonatal rats......................................................................................................... 40 2.4.5 DNA demethylation/ histone hyperacetylation experiments ..................................................................... 40 2.4.5.1 VPA/AzaC treatment............................................................................................................................. 40 2.4.5.2 Methylation status analysis ................................................................................................................... 40 2.4.5.3 Western Blot........................................................................................................................................... 41 2.4.5.3 Karyotypic stability ............................................................................................................................... 41 2.4.5.4 Regional expression profile and neurogenic potential after treatment............................................... 42 2.4.5.5 Co-culture with VPA/AzaC pre-treated cells....................................................................................... 42 2.4.5.6 Treatment with BMBP4/ RA following VPA/AzaC administration.................................................. 42 2.5 Analytical Methods .............................................................................................................................................. 42 2.5.1 Determination of cell number ....................................................................................................................... 42 2.5.2 Proliferation assay.......................................................................................................................................... 43 2.5.3 RNA extraction and quantitative RT-PCR analysis .................................................................................... 43 2.5.3.1 RNA extraction ...................................................................................................................................... 43 2.5.3.2 Reverse transcription ............................................................................................................................. 43 2.5.3.3 Design of human-specific primers........................................................................................................ 44 2.5.3.4 Quantitative RT-PCR ...........................................................................................................................