An investigation into the characteristics and potential therapeutic application of human neural progenitor cell-derived astrocytes in experimental spinal cord injury [Elektronische Ressource] / Tobias Führmann

An Investigation into the Characteristics and Potential
Therapeutic Application of Human Neural Progenitor
Cell-Derived Astrocytes in Experimental
Spinal Cord Injury
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen
University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften
genehmigte Dissertation
vorgelegt von
Diplom-Biologe Tobias Führmann
aus Essen
Berichter:
Privatdozent Dr. Gary Anthony Brook
Universitätsprofessor Dr. Hermann Wagner
Tag der mündlichen Prüfung: 3. November 2011
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.2
Summary
English version
Damage to the spinal cord leads to severe, and often permanent, motor, sensory and
autonomic disturbances. A multitude of mechanism may contribute to spinal cord injury
(SCI), including apoptotic and necrotic death of neurons, astrocytes and oligodendrocytes,
axonal injury, demyelination, excitotoxicity, ischemia, oxidative damage, and inflammation.
The lack of axonal regeneration is not primarily due to an inherent lack of capacity for
axonal growth potential, but rather to the overall balance of axon-growth promoting (e.g.
migration of Schwann cells into the lesion site and the local presentation of growth promoting
factors and extracellular matrix proteins) and axonal growth inhibitory/repulsive molecules
(e.g. myelin-associated proteins, the presence of a physical barrier presented by the glial
scar and the development of fluid-filled cystic cavities) at- and around the lesion site. Over
recent years, increasingly more detailed knowledge has been gained that demonstrates the
potential for axonal regeneration within the nervous system of both experimental animals
and humans. While the strongest regenerative capacity has been identified in the lesioned
peripheral nervous system (PNS), it could also be demonstrated that axonal regeneration
and compensatory sprouting takes place in the injured central nervous system (CNS). Such
findings have led to the development of a number of interventional approaches to support
or enhance the regenerative capacity of the CNS. Within the CNS, the main thrust of
these approaches has focused either on new surgical methods, novel medications (including
the use of blocking/neutralising antibodies and peptides), cell-based intervention strategies
(including stem/progenitor cells) or, most recently, the application of tissue engineering
strategies using biomaterials.
The aim of the present study was based on the two latter approaches, combining the
growth promoting properties of human neural progenitor-derived astrocytes and a highly3
orientated three dimensional collagen matrix in a tissue engineering strategy to bridge acute
spinal cord lesions in adult rats.
The present investigation is separated into two parts:
The first part of the thesis addresses the issue of cell characterization, cell-cell and cell-
substrate interactions in vitro. Human neural progenitor cells (hNPC) are characterized
by their expression of certain stem/progenitor markers as well as for their ability to show
multi-potency. Furthermore their ability to generate highly enriched populations of human
neural progenitor-derived astrocytes (hNP-AC) is demonstrated. The axon growth promot-
ing effects of the hNP-AC are shown in a simple 2D- as well as in a more complex 3D-culture
system. After seeding onto orientated electrospun nanofibres hNP-AC show the ability to
adhere, extend processes and migrate. Following seeding onto 3D collagen scaffolds, hNP-
AC survive, proliferate and form columns of cells along the orientated pores of the scaffold.
The highly orientated, porous microstructure of the scaffold also supports substantial inter-
mixing of hNP-AC and migrating Schwann cells / fibroblasts from the DRG explant, cell
populations that are normally mutually repulsive. This suggests that the topography of
3D scaffolds may not only influence cell-substrate interactions but also cell-cell interactions
within the scaffold.
The second part of the thesis addresses the integration and functional benefits of trans-
planting hNP-AC into spinal cord injured rats in vivo. Two different types of experimental
spinal cord injuries are investigated. In the first in vivo SCI model, hNP-AC are trans-
planted one week after a balloon compression injury and animals are subjected to standard
behavioural tests (i.e. BBB, open field and gridwalk scores, as well as the Hargreaves’ heat
sensitivity test) with correlative morphological investigations (i.e. amount of spared tissue,
survival of transplanted cells, glial scarring, microglia activation, Schwann cell migration and
axonal regeneration).
TransplantationofhNP-ACpromotesabetterfunctionalrecoverycomparedtothecontrol,
but not the control + immunosuppression and the hFbl + immunosuppression (cell control
group). hNP-AC integrate well into the host spinal cord in 90% of host animals, where they
reduce glial scarring and promote a greater extent of axonal regeneration into the lesion site.
Furthermore, Schwann cell migration into the lesion site is enhanced in the presents of hFbl
and microglia activation is enhanced by immunosuppression.4
In thesecond in vivo SCI model, hNP-AC seeded collagen scaffolds are implanted into
a partly transected spinal cord directly after lesion. Since the lesioned animals show a faster
recovery with this lesion model (compared to the compression model), more sophisticated
behavioural analyses are possible (CatWalk, gridwalk (footfalls) and von Frey filaments).
Morphological analysis includes survival of the transplanted cells, integration of the scaffold,
neurite regeneration into the scaffold, glial scarring and microglia activation.
Survival of the cells within the implanted collagen scaffold is surprisingly low, with only
half of the animals demonstrating morphological signs of viable donor hNP-AC, and only
10% for the donor hFbl. Although the hNP-AC fail to promote a greater functional recovery
than the other investigated control groups (i.e. scaffold + immunosuppression (IS) and hFbl-
seeded scaffold + IS), they lead to a greater graft-host integration and reduced host glial
scarring. Furthermore microglia activity is reduced by hNP-AC compared to the control +
IS, indicating a modulated immune response.5
German version / deutsche Version
VerletzungendesRückenmarksführenzuschwerwiegendenundmeistpermanentenSchädi-
gungen motorischer und sensorischer Nerven sowie des autonomen Nervensystems. Eine
Vielzahl von Mechanismen können zu Rückenmarksverletzungen beitragen, dazu zählen
apoptotischerundnekrotischerTodvonNeuronen,AstrozytenundOligodendrozyten,Schädi-
gungendesAxons,Demyelinisierung,Excitotoxizität,Ischämie,oxidativerStressundEntzün-
dungen.
Die ausbleibende axonale Regeneration liegt nicht an einem fehlenden inhärenten Po-
tential für axonales Wachstum, sondern eher an einem generellen (Un-)Gleichgewicht von
wachstumsfördernden (z.B. der Migration von Schwann-Zellen in die Läsionsstelle und die
lokale Bereitstellung von Wachstumsfaktoren und extrazellulärer Matrix) und wachstumsin-
hibierenden, -repulsiven Molekülen (z.B. Myelin-assoziierte Proteine, der Ausbildung einer
physikalischen Barriere in Form der glialen Narbe und die Entwicklung von mit Flüssigkeit
gefüllter Hohlräume) an der Läsionsstelle und um sie herum.
Im Laufe der letzten Jahre wurde ein immer detaillierteres Wissen über das regenerative
Potential von Axonen im Nervensystem von Tieren und Menschen gewonnen. Das periph-
ere Nervensystem weist dabei ein großes regeneratives Potential auf, jedoch konnte darüber
hinaus gezeigt werden, dass axonale Regeneration und kompensatorisches Nervenwachstum
auch im verletzten zentralen Nervensystem (ZNS) selbst stattfindet. Diese Erkenntnisse
führten zur Entwicklung einer großen Anzahl von verschiedenen Strategien, um die Regen-
eration im ZNS zu unterstützen und zu fördern. Im Bereich des ZNS fokussieren sich diese
Strategien vor allem auf neue operative Methoden, neue Medikamente (zu denen auch block-
ende/neutralisierendeAntikörperundPeptidegehören), zellbasierteStrategien(u.a. Stamm-
und Vorläuferzellen) und neuerdings die Anwendung von Biomaterialien als eine Zell- und
Gewebeersatz-Strategie.
DievorliegendeStudiebasiertdabeiaufdenbeidenletztgenanntenAnsätzenundbeschreibt
dieNutzungderwachstumsförderndenEigenschaftenvonaushumanenneuralenVorläuferzellen
gewonnenen Astrozyten (hNV-AZ) alleine oder in Kombination mit einer orientierten drei-
dimensionalen Kollagenmatrix in einer Zell- und Gewebeersatz-Strategie, um funktionelle
Regeneration nach akuten Rückenmarksverletzungen in erwachsenen Ratten zu fördern.
Die Dissertation gliedert sich in zwei Teile:6
Der erste Teil der Dissertation befasst sich mit Zellcharakterisierung, Zell-Zell- und Zell-
Substrat-Interaktionen in vitro. Humane neurale Vorläuferzellen werden sowohl anhand der
Expression verschiedener Stamm- und Vorläuferzellenmarker charakterisiert als auch an-
hand ihrer Fähigkeit, Multipotenz zu zeigen. Zudem wird ihre Fähigkeit, sich in nahezu
reine Populationen von hNV-AZ zu differenzieren, dargestellt. Die wachstumsfördernde
Eigen