La lecture en ligne est gratuite
Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres
Télécharger Lire

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

De
119 pages
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.
Voir plus Voir moins




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 neurons ............................................74
5.2. Efficient cryopreservation of purified human neurons...........76
5.3. Enhanced migration of human neurons as pure population ..................................77
5.3.1. Migration of DCX-EGFP positive neurons as pure population and within a neural
stem/progenitor cell containing population ............................................................77
5.4. Mechanisms causing core formation of neural stem/progenitor cell containing
transplants ...................................................................................................................79
5.4.1. Analysis of chemoattractive factors and associated receptors in neural
stem/progenitor cells and immature neurons.........................80
5.4.2. Interference with the chemoattractive mechanisms between neural
stem/progenitor cells and immature neurons in vitro .............................................81
5.5. Perspective ..................................................................................82
6. ABSTRACT.........................................84
7. ZUSAMMENFASSUNG ......................................................................................86
8. REFERENCES ....................................................................................................88
9. ACKNOWLEDGMENT......................................................................................105
10. ERKLÄRUNG....................................................................................................106
11. CURRICULUM VITAE.......................................................................................107
IV Abbreviations

ABBREVIATIONS

°C Degree Celsius
ApoER2 ApoE receptor type 2
BDNF Brain-derived neurotrophic factor
BMP Bone morphogenetic protein
bp Base pair
BrdU Brom-desoxyuridine
cAMP Cyclic adenosine monophosphate
CC Corpus callosum
CAM Cell adhesion molecule
Cdk5 Cyclin-dependent kinase 5
cDNA Complementary DNA
Chat Choline acetyltransferase
CNS Central nervous system
Cx Connexin
CXCR4 Chemokine (C-X-C motif) receptor 4
Dab1 Disabled-1
DAPI 4ʼ,6-diamidino-2-phenylindole
DCX Doublecortin
DG Dentate gyrus
DMEM Dulbeccoʼs Modified Eagle Medium
DMSO Dimethyl sulfoxide
DNA Desoxyribonucleic acid
dNTP Desoxynucleotidtriphosphate
DsRED2 Red fluorescent protein 2
EB Embryoid body
EGF Epidermal growth factor
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid
EGFP Enhanced green fluorescence protein
ES cells Embryonic stem cells
FACS Fluorescence-activated cell sorting
FCS Fetal calf serum
FGF Fibroblast growth factor
V Abbreviations

Flk/KDR Vascular endothelial growth factor receptor 2
Flt1 Fms-related tyrosine kinase 1
Flt-4 Fms-related tyrosine kinase 4
g Gram
GABA γ-Aminobutyric acid
GAD Glutamic acid decarboxylase
GC Granular cell
GCL Granular cell layer
GDNF Glial derived neurotrophic factor
GFAP Glial fibrillary acidic protein
h Hour
HC Hippocampus
HB-EGF Heparin-binding EGF
hES cell Human embryonic stem cell
Hz Hertz
ICM Inner cell mass
iPS cell Induced pluripotent stem cell
kg Kilogram
KO-SR Medium containing KO-DMEM and serum replacement
LIS1 Lissencephalic 1
Lt-hESNSC Long-term self-renewing hES cell derived neural stem cells
M Molarity
MAP Microtubule associated protein
MEF Mouse embryonic fibroblasts
mg Milligram
min Minute
mM Millimolar
mOsm Milliosmolar
ms Millisecond
MZ Marginal zone
Neo Neomycin
nM Nanomolar
NSC Neural stem cell
OB Olfactory bulb
P Postnatal day
VI Abbreviations

PBS Phosphate-buffered saline
PCR Polymerase chain reaction
PDGF Platelet-derived growth factor
PFA Paraformaldehyde
PNS Peripheral nervous system
PO Poly-l-ornithine
PSA-NCAM Polysialylated neural cell adhesion molecule
PSC Postsynaptic current
PTB Phosphotyrosine-binding
RMS Rostral migratory stream
RNA Ribonucleic acid
rpm Revolutions per minute
RT Reverse transcriptase
sec Second
SDF-1 Stromal cell-derived factor-1
SHH Sonic hedgehog
SGZ Subgranular zone
SOX2 Sex determining region Y box 2
SR Serum replacement
ST Striatum
SVZ Subventricular zone
TGF Transforming growth factor
TH Tyrosine hydroxylase
TX Transplantation
VEGF Vascular endothelial growth factor
VLDLR Very low-density lipoprotein receptor
VZ Ventricular zone
µm Micrometer
µM Micromolar

VII

Un pour Un
Permettre à tous d'accéder à la lecture
Pour chaque accès à la bibliothèque, YouScribe donne un accès à une personne dans le besoin