Progenitors from the postnatal and adult mammalian retina-neurogenic competence and plasticity [Elektronische Ressource] / vorgelegt von Maren Engelhardt

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• Publié par: universitat_regensburg
• Publié le: 01 janvier 2005
• Nombre de lectures: 63
• Langue: English
• Poids de l'ouvrage: 57 Mo

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PROGENITORS FROM THE POSTNATAL AND ADULT
MAMMALIAN RETINA - NEUROGENIC COMPETENCE AND
PLASTICITY
DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER
NATURWISSENSCHAFTEN (DR. RER. NAT.) DER
NATURWISSENSCHAFTLICHEN FAKULTÄT III-BIOLOGIE UND
VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG
vorgelegt von
Maren Engelhardt
aus Ulm
2004Promotionsgesuch eingereicht am: 21.12.2004
Die Arbeit wurde angeleitet von: Prof. Dr. rer. nat. Inga Neumann und Prof. Dr. med.
Jürgen Winkler in der Klinik und Poliklinik für Neurologie der Universität Regensburg
Prüfungsausschuss:
Vositzender: Prof. Dr. C. Förster
1. Prüfer (Erstgutachten): Prof. Dr. I. Neumann
2. Prüfer (Zweitgutachter): Prof. Dr. J. Winkler
3. Prüfer: Prof. Dr. S. Schneuwly-The eye to this day gives me a cold shudder-
Charles DarwinTABLE OF CONTENTS I
Table of Contents
I. Introduction ............................................................................................ 1
I.1. The eye........................................................................................................................................ 1
I.1.1. Anatomical features of the mammalian eye ...................................................................................... 1
I.1.2. Embryogenesis of the mammalian retina .......................................................................................... 4
I.2. Stem cells.................................................................................................................................... 5
I.2.1 Somatic stem cells during development ............................................................................................. 6
I.2.2. Adult neurogenesis - Somatic stem cells in the adult........................................................................ 7
I.2.3. Adult somatic neural stem cells in vitro .......................................................................................... 11
I.2.4. Retinal stem cells during development............................................................................................ 12
I.2.5. Retinal stem cells in the adult .......................................................................................................... 12
I.2.5.1. Pigmented cells in the ciliary body: multipotent somatic neural stem cells .......................... 13
I.2.5.2. A possible role as neural stem cell for Müller glia in the sensory retina ............................... 14
I.2.5.3. Retinal pigment epithelium cells: somatic neural stem cells in lower vertebrates and their
possible role in mammals...................................................................................................................... 15
I.2.6. Fate determination of retinal stem cells: the molecular basis......................................................... 17
I.2.6.1. The role of the Notch/Delta system during retinal development............................................ 17
I.2.6.2. Pax6, a homeobox gene regulating cell fate in the retina ....................................................... 18
I.2.6.3. Basic helix-loop-helix transcription factors during retinal development............................... 19
II. Aim of study .........................................................................................21
III. Material and Methods ........................................................................23
III.1. Material................................................................................................................................. 23
III.1.1. Chemicals....................................................................................................................................... 23
III.1.1.1. Cell culture............................................................................................................................. 23
III.1.1.2. Immunodetection ................................................................................................................... 23
III.1.1.3. Antibodies .............................................................................................................................. 24
III.1.1.4. Viral infections....................................................................................................................... 25
III.1.1.5. HPLC...................................................................................................................................... 25
III.1.2. Solutions......................................................................................................................................... 25
III.1.3. RT-PCR primers ............................................................................................................................ 27
III.1.3.1. Rat primers............................................................................................................................. 27
III.1.3.2. Human primers....................................................................................................................... 28
III.1.4. Equipment and Instruments........................................................................................................... 28
III.2. Methods................................................................................................................................. 28
III.2.1. Preparation and cell culture........................................................................................................... 28
III.2.1.1. Postnatal and adult derived cells from the sensory retina .................................................... 28
III.2.1.2. Adult derived cells from the ciliary body ............................................................................. 29
III.2.1.3. Adult derived cells from the retinal pigment epithelium ..................................................... 30
III.2.1.4. Adult derived stem cells from the subventricular zone and hippocampus.......................... 31
III.2.1.5. Passaging of NSCs and retinal progenitor cells.................................................................... 31
III.2.1.6. Clonal assays.......................................................................................................................... 32
III.2.1.7. Differentiation of cells........................................................................................................... 32
III.2.2. Immunocytochemistry ................................................................................................................... 33
III.2.3. Quantitative analysis...................................................................................................................... 34
III.2.4. RT-PCR.......................................................................................................................................... 34
III.2.5. HPLC.............................................................................................................................................. 35
III.2.6. Retroviral vector design and production....................................................................................... 35TABLE OF CONTENTS II
III.2.7. Retroviral infection of SVZ and HC cells in vitro........................................................................ 36
III.2.8. Detection of the myc tag in retrovirally infected cells by Western Blot ..................................... 37
III.2.8.1. Gel preparation....................................................................................................................... 37
III.2.8.2. Sample preparation ................................................................................................................ 37
III.2.8.3. Blotting procedure ................................................................................................................. 37
IV. Results .................................................................................................39
IV.1. Adult derived neural stem cells from the subventricular zone and hippocampus of
the rat.............................................................................................................................................. 39
IV.1.1. Optimization and standardization of cell culture conditions for NSCs from adult rats ........................ 39
IV.1.1.1. Comparison of Accutase‰ and trypsin treatment for neurosphere passaging .................... 40
IV.1.1.2. Effects of different culture media and supplements on NSC expansion and survival................... 43
IV.1.1.3. Comparison of different media for establishing primary neurospheres .............................. 45
IV.1.1.4. Comparison of different media and supplements for single cell cloning............................ 45
IV.1.2. Immunocytochemical analysis of expression patterns for progenitor and adult markers in
NSCs........................................................................................................................................................... 46
IV.1.2.1. Detection of proliferation markers in NSC derived Error! neurospheres Bookmark not
defined.
IV.1.2.2. Detection of the progenitor marker nestin in NSCs ............................................................. 47
IV.1.2.3. Analysis of differ