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

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PROGENITORS FROM THE POSTNATAL AND ADULTMAMMALIAN RETINA - NEUROGENIC COMPETENCE ANDPLASTICITYDISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DERNATURWISSENSCHAFTEN (DR. RER. NAT.) DERNATURWISSENSCHAFTLICHEN FAKULTÄT III-BIOLOGIE UNDVORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURGvorgelegt vonMaren Engelhardtaus Ulm2004Promotionsgesuch eingereicht am: 21.12.2004Die 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 RegensburgPrüfungsausschuss:Vositzender: Prof. Dr. C. Förster1. Prüfer (Erstgutachten): Prof. Dr. I. Neumann2. Prüfer (Zweitgutachter): Prof. Dr. J. Winkler3. Prüfer: Prof. Dr. S. Schneuwly-The eye to this day gives me a cold shudder-Charles DarwinTABLE OF CONTENTS ITable of ContentsI. Introduction ............................................................................................ 1I.1. The eye........................................................................................................................................ 1I.1.1. Anatomical features of the mammalian eye ...................................................................................... 1I.1.2. Embryogenesis of the mammalian retina .......................................................................................... 4I.2. Stem cells.................

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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 differentiation potential of SVZ and HC derived stem cells............................ 48
IV.1.3. Analysis of cell fate in modified NSC cultures constitutively expressing Notch1..................... 50
IV.1.3.1. Detection and sorting of NSCs constitutively expressing Notch1-eGFP............................ 50
IV.1.3.2. Neuronal and glial differentiation of NSCs constitutively expressing Notch1................... 52
IV.2. Postnatal derived progenitor cells from the sensory retina of the rat eye.................... 55
IV.2.1. Isolation, proliferation and generation of retinal neurospheres ................................................... 56
IV.2.2. Expression of progenitor markers in postnatal retinal cells......................................................... 57
IV.2.3. Differentiation potential of postnatal retinal progenitors.................................................................. 59
IV.2.3.1. Expression of early and late neuronal and glial markers in postnatal retinal cells.................... 59
IV.2.3.2. Generation of new neurons and glia from postnatal retina in vitro ..................................... 62
IV.2.3.3. Analysis of neurogenic competence and stem cell character in postnatal retinal cells....... 64
IV.2.3.4. Expression of retina-specific markers in differentiated retinal cultures.............................. 66
IV.3. Adult derived progenitor cells from the ciliary body and retinal pigment epithelium
of the rat eye................................................................................................................................... 67
IV.3.1. Isolation, proliferation and generation of neurospheres............................................................... 68
IV.3.2. Clonal expansion and growth as adherent monolayer cultures.................................................... 69
IV.3.3. Expression of progenitor markers in adult derived retinal cells: RT-PCR.................................. 71
IV.3.4. Expression of progenitor and early neuronal markers: morphological alterations........................ 72
IV.3.5. Differentiation along the glial lineage .......................................................................................... 76
IV.4. Adult derived progenitor cells from the human retinal pigment epithelium............... 77
IV.4.1. Cell culture conditions: growth as neurospheres and adherent cultures...................................... 77
IV.4.2. Differentiation potential of human RPE cells: neuronal lineage ................................................. 79
IV.4.3. Differentiation potential of human RPE cells: glial lineage ........................................................ 80
V. Discussion .............................................................................................82
V.1. Differentiation of NSCs and their phenotype plasticity after improvements of growth
conditions........................................................................................................................................ 82
V.1.1. Growth and proliferation of NSCs as neurospheres ...................................................................... 82
TMV.1.1.1. Improved passaging of NSCs using Accutase .................................................................. 83
V.1.1.2. Adult derived NSCs self-renew under clonal conditions ...................................................... 84
V.1.1.3. Media-supplements differentially affect growth rates........................................................... 85
V.1.2. NSC derived neurospheres express markers for all three major cell classes of the CNS ............ 87
V.1.3. Constitutive expression of Notch1 has different effects on the SVZ and HC derived neural
stem cell populations.................................................................................................................................. 88TABLE OF CONTENTS III
V.2. Postnatal derived progenitor cells from the sensory retina share typical
characteristics with NSCs............................................................................................................. 90
V.2.1. Proliferation and growth as neurospheres...................................................................................... 91
V.2.2. Postnatal derived retinal progenitors express markers specific for precursors and stem cells .... 93
V.2.3. Postnatal retinal progenitors differentiate along the neuronal and glial lineage .......................... 94
V.2.4. New neurons and glia can be generated from postnatal retina in vitro......................................... 96
V.2.5. The neurogenic competence of postnatal retinal progenitors is age-dependent........................... 97
V.2.6. A subset of early postnatal retinal progenitors is mitotic and expresses the retina-specific
photoreceptor marker recoverin................................................................................................................. 99
V.3. Adult derived progenitor cells from the ciliary body and retinal pigment epithelium
share characteristics with NSCs ................................................................................................ 100
V.3.1. Adult CB and RPE derived progenitor cells proliferate and self-renew in vitro........................ 100
V.3.2. Loss of pigmentation in adult derived progenitors expanded under adherent conditions..................... 102
V.3.3. Specific progenitor markers are expressed in adult derived CB and RPE cells......................... 103
V.3.4. CB and RPE derived cells expressing young neuronal and progenitor markers change their
morphology............................................................................................................................................... 104
V.3.5. Expression of neuronal and glial markers in adult derived RPE cells: de-differentiation or
trans-differentiation?................................................................................................................................ 105
V.4. Adult human derived RPE cells proliferate in vitro and express early and late
neuronal and glial markers ........................................................................................................ 110
V.5. Potential therapeutic application of retinal progenitor and stem cells ........................ 111
V.5.1. Replacement strategies for degenerating photoreceptor and RPE cells...................................... 112
V.5.2. Neuroprotection and stimulation of and endogenous regeneration potential in the diseased or
injured retina............................................................................................................................................. 114
VI. Summary...........................................................................................117
VII. Zusammenfassung...........................................................................120
VIII. Bibliography...................................................................................123
Acknowledgements..................................................................................145LIST OF ABBREVIATIONS IV
List of Abbreviations
bp Base pair
BSA Bovine Serum Albumin
BrdU Bromodesoxyuridine
CB Ciliary body
cDNA complementary DNA
CNS Central nervous system
°C centigrade
Da Dalton
DAPI 4’,6’-diamidino-2-phenylindol-dihydrochlodire-hydrate
DCX Doublecortin
DPBS Dulbeccor’s phosphate buffered saline
DMEM Dulbecco’s Modified Eagle’s Medium
DNA Desoxyribonucleotide acid
dNTP Desoxyribonucleotide-triphosphate
E Embryonal day
EGF Epidermal growth factor
eGFP Enhanced green fluorescent protein
EtOH Ethanol
FACS Fluorescence activated cell sorting
FCS Fetal calf serum
FGF-2 Basic fibroblast growth factor
Flk-1 Fetal liver kinase-1
-3 g Gram (10 g)
GAPDH Glycerinaldehyde-3-Phosphate-Dehydrogenase
h Hour(s)
HBSS Hanks Balanced Salt Solution
HC Hippocampus
HEPES N-(2-Hydroxyethyl)piperazin-N‘-(2-Ethansulfonacid)
H O Distilled water2 dest
l liter
MEM Modified Eagles Medium
mg milligram
ml milliliter
mRNA messenger RNA
-6µ micro (10 )
NMDA N-methyl-D-aspartate
NSC Neural stem cell
PBS Phosphate buffered NaCl-solution
PCR Polymerase chain reaction
+pH Negative Logarithm of the H -Ion concentration
PFA Paraform aldehyde
SDP Postnatal dayLIST OF ABBREVIATIONS V
rcf Relative centrifugal force
RPE Retinal pigment epithelium
RNA Ribonuclein acid
RT Reverse transcriptase
RT-PCR Reverse transcriptase polymerase chain reaction
SC Spinal cord
SD Standard deviation
SR Sensory retina
SVZ Subventricular zone
TBE Tris-Borate-EDTA-Elektrophoresis Buffer
TBS Tris-Borate buffered saline
TE Tris –EDTA-buffer
TH Tyrosine hydroxylase
Tris Tris-(hydroxymethyl)-aminomethane
V Volt
v/v volume per volume
w/v weight per volume
w/w weight per weightINTRODUCTION 1
I. Introduction
I.1. The eye
The eye, throughout all species, is one of the most complex and fascinating
organs of the body. The fact that it is designed so differently among various species and
yet apparently underlies the same conserved genetic program makes the developmental
process even more intriguing. In humans, the eye is one of the most important
connections to the surrounding environment. The eye basically functions like a camera,
but in comparison to its mechanical counterpart, links the visual input to a complex and
sophisticated wired network in the brain, which leads to all kinds of reactions, both of
physical and emotional nature.
A common hall mark of retinal diseases such as macular degeneration or retinitis
pigmentosa is the selective loss of retinal neurons, mostly photoreceptor cells. The
continuous loss of retinal cells points towards a therapeutic strategy to replace cellular
elements, either by applying exogenous cells or by stimulating endogenous progenitor or
stem cell populations. A key aspect is the characterization of retinal stem or progenitor
cells for this purpose.
This thesis focuses on the cues of mammalian eye development and investigates
the possibilities of finding retinal progenitor or even stem cells in the postnatal and adult
retina. Comprehension of the nature of retinal progenitor cells and their biology might
eventually lead to progress in the field of generating new neurons for a diseased retina.
I.1.1. Anatomical features of the mammalian eye
The eye is an organ that is specialized in detecting, localizing and analyzing
sources of light. As shown in Fig.I.1., the pupil is the opening that enables light to enter
the eye. The pupil is an adjustable part and its size is controlled by the iris, a circular
muscle. Both the pupil and the iris are covered by the cornea, the external surface of the
eye. The cornea does not contain blood vessels and is nourished by the fluid right behind
it, the aqueous humor. The cornea is continuous with the sclera, which forms the resistantINTRODUCTION 2
wall of the eyeball. The sclera itself is continuous with the dura. Three pairs of
extraocular muscles are inserted into the sclera and move the eye within the sockets of
the skull. The lens is located right behind the iris. It is suspended by ligaments, the zonule
fibers that are attached to the ciliary muscles. These muscles in turn attach to the sclera
and thus form a ring within the eye. The changes in the shape of the lens help the eye to
adjust the focus to different viewing distances.
Fig.I.1. Gross anatomy of the eye. Saggital section through the adult human eye. RPE=retinal
pigment epithelium; SR=sensory retina; CB=ciliary body. Adapted from W. Barry VanWinkle
(http://medic.med.uth.tmc.edu/Lecture/Main/eye.htm)
The lens also divides the interior of the eye into two distinct compartments
containing slightly different fluids. The aqueous humor is the watery fluid between the
cornea and the lens. The more viscous vitreous humor lies between the lens and the
retina, its pressure serves to keep the globe of the eye spherical. The retina serves as a
sensor for incoming light and can turn this information into impulses transmitted via
neurons, which makes it the major link to the visual system of the brain.