A comparative analysis of human adult mesenchymal and fetal neuronal stem cells with regard to their neurogenic potential [Elektronische Ressource] : an electrophysiological and immuncytochemical analysis of the differentiation potential of human mesenchymal and human fetal neural stem cells towards a mature neuronal phenotype / vorgelegt von Guilherme Lepski

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A COMPARATIVE ANALYSIS OF HUMAN ADULT MESENCHYMAL AND FETAL NEURONAL STEM CELLS WITH REGARD TO THEIR NEUROGENIC POTENTIAL An electrophysiological and immuncytochemical analysis of the differentiation potential of human mesenchymal and human fetal neural stem cells towards a mature neuronal phenotypeGUILHERME LEPSKIINAUGURAL-DISSERTATIONZur Erlangung des Medizinischen édition scientifique Doktorgrades der VVB LAUFERSWEILER VERLAGFakultät der VVB LAUFERSWEILER VERLAG ISBN 3-8359-5439-3STAUFENBERGRING 15 Albert-Ludwigs-Universität Freiburg i. Br.D-35396 GIESSENTel: 0641-5599888 Fax: -5599890redaktion@doktorverlag.dewww.doktorverlag.de 9 7 8 3 8 3 5 9 5 4 3 9 7édition scientifiqueVVB LAUFERSWEILER VERLAGVVBGUILHERME LEPSKI NEURONAL DIFFERENTIATION OF MSC AND NSCDas Werk ist in allen seinen Teilen urheberrechtlich geschützt. Jede Verwertung ist ohne schriftliche Zustimmung des Autors oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme.1. Auflage 2009All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Author or the Publishers.
Publié le : jeudi 1 janvier 2009
Lecture(s) : 41
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Source : WWW.FREIDOK.UNI-FREIBURG.DE/VOLLTEXTE/7106/PDF/LEPSKI_2009.PDF
Nombre de pages : 98
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A COMPARATIVE ANALYSIS OF HUMAN ADULT
MESENCHYMAL AND FETAL NEURONAL STEM
CELLS WITH REGARD TO THEIR NEUROGENIC
POTENTIAL
An electrophysiological and immuncytochemical analysis
of the differentiation potential of human mesenchymal
and human fetal neural stem cells towards a mature
neuronal phenotype
GUILHERME LEPSKI
INAUGURAL-DISSERTATION
Zur Erlangung des Medizinischen
édition scientifique Doktorgrades der VVB LAUFERSWEILER VERLAG
Fakultät der
VVB LAUFERSWEILER VERLAG ISBN 3-8359-5439-3
STAUFENBERGRING 15 Albert-Ludwigs-Universität Freiburg i. Br.
D-35396 GIESSEN
Tel: 0641-5599888 Fax: -5599890
redaktion@doktorverlag.de
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 4 3 9 7
édition scientifique
VVB LAUFERSWEILER VERLAGVVB
GUILHERME LEPSKI NEURONAL DIFFERENTIATION OF MSC AND NSCDas Werk ist in allen seinen Teilen urheberrechtlich geschützt.
Jede Verwertung ist ohne schriftliche Zustimmung des Autors
oder des Verlages unzulässig. Das gilt insbesondere für
Vervielfältigungen, Übersetzungen, Mikroverfilmungen
und die Einspeicherung in und Verarbeitung durch
elektronische Systeme.
1. Auflage 2009
All rights reserved. No part of this publication may be
reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical,
photocopying, recording, or otherwise, without the prior
written permission of the Author or the Publishers.
st1 Edition 2009
© 2009 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
édition scientifique
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: 0641-5599888 Fax: 0641-5599890
email: redaktion@doktorverlag.de
www.doktorverlag.deAus der Abteilung Stereotaktische Neurochirurgie der Neurochirurgischen Universitätsklinik
der Albert-Ludwigs-Universität Freiburg i.Br.




A COMPARATIVE ANALYSIS OF HUMAN ADULT
MESENCHYMAL AND FETAL NEURONAL STEM
CELLS WITH REGARD TO THEIR NEUROGENIC
POTENTIAL


An electrophysiological and immuncytochemical analysis of the
differentiation potential of human mesenchymal and human fetal neural
stem cells towards a mature neuronal phenotype









INAUGURAL DISSERTATION
zur Erlangung des Medizinischen Doktorgrades
der Medizinischen Fakultät der Albert-Ludwigs-Universität
Freiburg i. Br.


vorgelegt 2009
von Guilherme Lepski
geboren in São Paulo/Brasilien
Dekan: Prof. Dr. med. Christoph Peters
1. Gutachter: Prof. Dr. med. Guido Nikkhah
2. Gutachter: Prof. Dr. rer. nat. Josef Bischofberger
Jahr der Promotion: 2009







































Erklärung nach § 2 Abs. 2 Nr. 5 und 6


Ich erkläre, dass ich die der Medizinischen Fakultät der Albert-Ludwigs-Universität
Freiburg i. Br. Zur Promotion eingereichte Dissertation mit dem Titel


A COMPARATIVE ANALYSIS OF HUMAN ADULT
MESENCHYMAL AND FETAL NEURONAL STEM
CELLS WITH REGARD TO THEIR NEUROGENIC
POTENTIAL


An electrophysiological and immuncytochemical analysis of the
differentiation potential of human mesenchymal and human fetal neural
stem cells towards a mature neuronal phenotype



in der Abteilung Stereotaktische Neurochirurgie der Neurochirurgischen Klinik des
Universitätsklinikums Freiburg unter der Betreuung von Prof. Dr. med. Guido Nikkhah ohne
sonstige Hilfe durchgeführt und bei der Abfassung der Dissertation keine anderen als die dort
aufgeführten Hilfsmittel benutzt habe.

Ich habe diese Dissertation bisher an keiner in- oder ausländischen Hochschule zur
Promotion eingereicht. Weiterhin versichere ich, dass in den beantragten Titel noch nicht
erworben habe.



Freiburg, den 19.02.2009


Guilherme Lepski







To my inseparable companion Cinthia Elim, for having taken
up my dreams as if they were her own, and remaining forever by
my side striving for the same goals, and making our successes
sweeter and our failures more palatable.

To our son Julian, for showing us what is truly important in
life.

To my parents Roberto and Sônia for their love, affection and
efforts invested in my growth both inside and outside the home,
enabling me to realize all my dreams.

To my grandparents Zilda and Alberto (posthumously) for
having guided my ethical and moral values that will remain with
me for the rest of my life.








































“Aufklärung ist der Ausgang des Menschen aus seiner selbst-
verschuldeten Unmündigkeit“



Immanuel Kant (1724-1804)























TABLE OF CONTENTS

ABREVIATIONS...................................................................................................................................................I
LIST OF FIGURES.............III
LIST OF TABLES.................V
I. INTRODUCTION ................................................................................................. 1
I.1. Historical background on neural transplants.......................................................................................... 5
I.2. Clinical trials on transplantation in Parkinson’s disease ....................................................................... 8
I.3. Potential of cellular therapy for treating other neurological disorders................................................ 12
I.4. Search for alternative sources of stem-cells 14
I.4.1. Neural stem cells.............................................................................................................................. 15
I.4.1.1. Neurogenesis in vitro ............................................................................................................... 15
I.4.1.2 Neurogenesis in vivo ................................................................................................................. 17
I.4.2. Embryonic stem cells ....................................................................................................................... 18
I.4.3. Mesenchymal stem cells................................................................................................................... 20
I.4.3.1. Studies in vitro ......................................................................................................................... 20
I.4.3.2. Studies in vivo .......................................................................................................................... 25
I.5. Objectives................................................................................................................................................ 27
II. MATERIAL AND METHODS.............................................................. 28
II.1. Sample collection... 28
II.2. MSC isolation......... 29
II.3. Fluorescence activated cell sorting (FACS) analysis .......................................................................... 29
II.4. Neuronal differentiation of MSC ......................................................................................................... 29
II.5.Co-culture of MSCs and rodent fetal hippocampal tissue.................................................................... 30
II.6. Isolation and differentiation of fetal neural stem cells (NSCs)........................................................... 31
II.7. Cell viability assay................................................................................................................................. 32
II.8. Bromodeoxyuridine (BrdU) incorporation assay................................................................................. 33
II.9. Fixation and immunostaining.............................................................................................................. 33
II.10. Confocal microscopy, quantification, and fluorescence measurement............................................. 34 II.11. Electrophysiology................................................................................................................................ 35
II.12. Statistical analysis 37
III. RESULTS............................................................................................................. 38
III.1. Isolation of stem cells derived from human bone marrow................................................................. 38
III.2. Expansion from fetal and mesenchymal neural precursor cells ....................................................... 40
III.3. Neuronal differentiation of stem cells derived from the bone marrow.............................................. 44
III.4. Mechanisms involved in neuronal differentiation ............................................................................. 50
III.5. Time course of differentiation............................................................................................................. 53
III.6 Neuronal differentiation of fetal derived human neural stem cells (NSCs)....................................... 53
III.7. Comparative electrophysiology of differentiated cells derived from MSCs and NSCs...................... 57
IV. DISCUSSION..................................................................................................... 59
V. CONCLUSIONS.................................................................................................. 66
VI. ABSTRACT .................................................................................................................................................. 67
VII. ZUSAMMENFASSUNG ............................................................................................................................ 68
REFERENCES..................... 69
ACKNOWLEDGEMENTS............................................................................................................82













ABREVIATIONS

ΔF/F mean fluorescence variation (F-Fb) over background
fluorescence (Fb)
5HT 5-OH-tryptamin, serotonin
cAMP cyclic adenosine-monophosphate
ANOVA analysis of variance
B27 serum supplement
BDNF brain derived neurotrophic factor
bFGF basic fibroblast growth factor
BHA butylated hydroxyanisole
BMA β mecaptoethanol
BrdU bromodeoxyuridine
cAMP cyclic adenosine monophosphate
CD cluster of differentiation
CREB cAMP-responsive element binding protein
DAPI 4,6-diamidino-2-phenylindole dihydrochloride
Dcx doublecortin
DNA deoxyribonucleic acid
DMEM Dulbecco´s modified Eagle medium
DMSO dimethyl sulfoxide
DT doubling time
E Nernst reversal potential
EAE experimental autoimmune encephalomyelitis
E13, E14 etc embryos, 13, 14 days of intrauterine life
EGF Epidermal growth factor
F Faraday constant
F12 Ham´s supplement
FACS Fluorescence-activated cell sorting
FCS fetal calf serum
FGF 8 Fibroblast growth factor 8
FITC fluorescein
FSK forskolin
g conductance
GABA gama amino-butyric acid
GDNF glial derived neurotrophic factor
GFAP glial fibrilar acidic protein
GFP green fluorescent protein
HPLC high pressure liquid cromatography
i current
IBMX 3-isobutyl-1-methylxanthine
IgG immunoglobulin G
I hyperpolarizing current h
MAP2 microtubule-associated protein 2
MAPC multipotent adult progenitor cells
MEM minimal essencial medium
MSCs mesenchymal stem cells
MSCBM m cell basal medium
I

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