Regionalisation of human ES cell derived neural precursors [Elektronische Ressource] / vorgelegt von Johanna Viola Driehaus

Johanna Viola Driehaus

Sujets

• Gesundheit

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2008
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

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 ............................................................ 44
2.5.4 Karyotype analysis......................................................................... 44
2.5.4.1 G-Banding .............................................................................. 45
2.5.4.2 Fluorescence in situ hybridisation (FISH) ........................................................... 45
2.5.5 Agarose gel electrophoresis................................................................ 45
2.5.6 Fluorescence – activated cell sorting............................................................................................................ 46
2.5.7 Immunocytochemistry ................................... 46
2.5.7.1 Detection of surface markers in cells grown as monolayer ................................................................ 47
2.5.7.2 Detection of nuclear markers in cells grown as monolayer 47
2.5.7.3 Immunohistochemistry of organotypic slices cultures ........................................................................ 47
2.5.7.4 Immunochistochemistry using tyramide amplification....... 48
2.5.7.5 Detection of BrdU.................................................................. 48
2.5.8 Extraction of total protein.............................................................. 48
2.5.9 Statistical Analysis......................................... 48

3 Results............................................................................................................................ 49
3.1 HESNSCs adopt a specific regional fate under standard culture co