Lineage selection and enhanced tissue integration of functional and cryopreservable human embryonic cell-derived neurons [Elektronische Ressource] / vorgelegt von Julia Ladewig

Julia Ladewig

Sujets

• Gesundheit

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2009
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Lineage Selection and Enhanced Tissue Integration
of Functional and Cryopreservable Human
Embryonic Stem Cell-Derived Neurons



Dissertation


Zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-
Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität
Bonn


Vorgelegt von

Julia Ladewig

aus Lippstadt

Bonn, 2008



Anfertigung mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der
Rheinischen Friedrich-Wilhelms-Universitiät Bonn























1. Referent: Prof. Dr. Oliver Brüstle
2. Referent: Prof. Dr. Michael Hoch

Tag der Prüfung: 20.04.2009


Diese Dissertation ist auf dem Hochschulschriftenserver der ULB Bonn unter
http://hss.ulb.uni-bonn.de/diss_online elektronisch publiziert.
Erscheinungsjahr: 2009


2



























DAS SCHÖNSTE, WAS WIR
ENTDECKEN KÖNNEN, IST DAS
GEHEIMNISVOLLE

(ALBERT EINSTEIN)


IN GEDENKEN AN
MEINEN VATER
3 Contents

CONTENTS

ABBREVIATIONS……………………………………………………………………………...……....IV
1. INTRODUCTION ...................................................................................................1
1.1. Stem cells and their neurogenic potential..1
1.1.1. Generation of pluripotent stem cells........2
1.1.2. Strategies for the differentiation of pluripotent stem cells ........................................4
1.1.3. Long-term self-renewing neuroepithelial stem cells from hES cells ........................6
1.1.4. Potential therapeutic use of stem cells in CNS disorders........6
1.2. Cell migration in the vertebrate CNS.........................................................................10
1.2.1. Migration in early CNS development.....10
1.2.2. Mechanisms of neuronal migration in the CNS .....................................................11
1.2.3. Factors regulating neuronal migration in the CNS.................14
1.2.4. Neuronal migration defects in the CNS..................................................................17
1.3. Objectives of this study..............................................................20
2. MATERIALS........................................................................................................21
2.1. Technical equipment...................................21
2.2. Chemicals and reagents.............................................................23
2.3. Cell lines and animal stocks.......................................................27
2.4. Plasmids................................................................27
2.5. Cell culture reagents...................................................................27
2.5.1. Cell culture stock solutions ....................................................................................27
2.5.2. Cell culture media..28
2.5.3. Cell dissociation reagents......................29
2.5.4. Coating materials...................................................................................................30
2.5.5. FACS solutions......30
2.6. Reagents for immunohistochemistry........................................................................31
2.6.1. Primary antibodies.................................32
I Contents

2.6.2. Secondary antibodies ............................................................................................32
2.7. Reagents for molecular biology.................33
2.7.1. Primers ..................................................................................................................33
2.7.2. Kits.........................34
2.8. Software .......................................................................................................................35
3. METHODS...........36
3.1. Cultivation of pluripotent hES cells...........................................................................36
3.1.1. Generation, cultivation and mitotic inactivation of murine fetal fibroblasts ............36
3.1.2. Cultivation of hES cells ..........................................................................................36
3.2. In vitro differentiation of hES cells into lt-hESNSC..................36
3.3. Stable nucleofection of lt-hESNSC............................................................................38
3.4. Fluorescence activated cell sorting..........38
3.5. Preparation of primary astrocytes.............................................................................39
3.5.1. Direct-/ in-direct shared media culture with primary astrocytes .............................39
3.6. Cryopreservation of purified human neurons ..........................................................39
3.7. In vitro migration assays ............................................................40
3.7.1. Transwell migration assay.....................................................40
3.7.2. Matrigel migration assay........................41
3.8. Transplantation............................................................................................................41
3.8.1. Transplantation onto rat hippocampal slice cultures..............41
3.8.2. Transplantation into the rodent brain.....41
3.8.3. Transplantation into the neonatal rodent brain ......................................................42
3.9. Immunocytochemistry and immunohistochemistry................42
3.9.1. Immunocytochemistry............................................................................................42
3.9.2. Immunohistochemistry...........................43
3.10. RT-PCR .........................................................................................................................43
3.11. Electrophysiological recordings of purified neurons..............45
II Contents

4. RESULTS............................................................................................................46
4.1. Generation and validation of a lineage selection protocol to derive pure
cultures of immature neurons from hES cells..........................................................46
4.1.1. Expression profile of doublecortin at different stages of neural differentiation in lt-
hESNSC ................................................................................................................46
4.1.2. Lt-hESNSC stably expressing a doublecortin reporter/selection marker...............47
4.1.3. Purification of DCX-EGFP-positive neurons by FACS...........48
4.1.4. Functional maturation of purified hES cell-derived neurons ..................................52
4.2. Generation of an efficient cryopreservation protocol for human neurons ...........54
4.2.1. Cryopreservation of purified hES cell-derived neurons..........................................54
4.2.2. Transplantation of purified and cryopreserved neurons into the neonatal rodent
brain.......................................................................................................................56
4.3. Enhanced migration of purified human neurons.....................57
4.3.1. In vitro migration of purified human neurons .........................................................57
4.3.2. Migration of purified human neurons on hippocampal rat slice cultures................58
4.3.3. In vivo migration of purified human neurons in the CNS of adult rats....................60
4.4. Interaction between neural stem/progenitor cells and immature neurons ...........62
4.4.1. Chemoattraction between neural stem/progenitor cells and immature neurons....62
4.4.2. Migration of immature neurons in a cell mixture with neural stem/progenitor cells
on hippocampal rat slice cultures and in the CNS of adult rats .............................63
4.4.3. Soluble factors with chemoattractive effect on immature neurons.........................65
4.4.4. Expression profile of chemoattractants and their receptors in neural
stem/progenitor cells and immature neurons ........................................................66
4.4.5. Interaction with chemoattractants expressed by neural stem/progenitor cells in
vitro........................................................................................67
4.4.6. Interaction with chemoattractants expressed by neural stem/progenitor cells on
hippocampal rat slice cultures...............................................69
5. DISCUSSION ......................................................................71
5.1. Genetic lineage selection of hES cell derived neurons ..........................................71
5.1.1. Surface bound versus genetic lineage selection...................71
5.1.2. Doublecortin as candidate marker for the selection of immature neurons.............72
5.1.3. Establishment of a DCX-EGFP lineage selection system .....................................73
III Contents

5.1.4. Characterization of the DCX-EGFP purified neu